405
Biochimica et Biophysics @ Elsevier/North-Holland
Acta, 487 (1977) Biomedical Press
405-421
BBA 56996
PURIFICATION AND PROPERTIES FROM MYCOBACTERIUM PHLEI
JOHN L. PAZNOKAS
* and ARNOLD
Department of Microbiology, 63104 (U.S.A.) (Received
November
24th,
St. Louis
OF A TRIACYLGLYCEROL
LIPASE
KAPLAN University
School
of Medicine,
Saint Louis,
Mo.
1976)
Summary In order to study the metabolism of triacylglycerol in mycobacteria, an intracellular particulate triacylglycerol lipase (EC 3.1.1.3) was purified BOO-fold from stationary phase cells of ililycobacterium phlei. Extraction of whole cell suspensions with 5% Triton X-100, followed by ion-exchange chromatography of the extract on two successive DEAE-cellulose columns produced a preparation which was nearly homogeneous by the criterion of analytical isoelectric focusing in acrylamide gels (one band, p1. 3.8) and by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the preparation into six protein bands. Lipase activity stable to electrophoresis in sodium dodecyl sulfate was extracted from the 40 000 molecular weight region of the gels. With phosphate or maleate buffer the enzyme exhibits a broad pH optimum around 6.0 with sigmoid saturation kinetics (Hill number 2), and an apparent K, of 8.8 mM for tripalmitoylglycerol. Citrate and other carboxylic acids increase the apparent V up to 3-fold with the Hill number approaching 1.0. In a series of p-nitrophenyl esters tested (C,-C,,), p-nitrophenylmyristate was hydrolyzed most rapidly. The saturation curve for p-nitrophenylmyristate was sigmoid and unaffected by citrate. The role of this activity in the metabolism of triacylglycerols by Mycobacteria is discussed.
Introduction Mycobacterium
most rapidly
phlei contains in the late stationary
* Present address: Department Pullman, Wash. 99164, U.S.A.
of
a pool of triacylglycerol which turns over phase of growth in liquid culture. Stephen-
Bacteriology
and
Public
Health,
Washington
State
University,
406
son and Whetman [I] first observed that in late stationary phase endogenous oxygen uptake persisted and the respiratory quotient decreased from 1.72 to 0.74 as would be expected from a transition to oxidation of fatty acids. Later Asselineau [ 21 directly demonstrated that mycobacteria including M. phlei contain triacylglycerols. More recently Brennan et al. [3] have shown that both glycogen and triacylglycerols accumulate in M. phlei during growth and that glycogen is preferentially utilized when growth nutrients are depleted. This work was confirmed and extended by McCarthy [ 41. His experiments revealed that the uptake of labeled fatty acids (especially palmitic) into iM. phlei triacylglycerol was followed by a loss of the label from the triacylglycerol pool and its subsequent appearance in phospholipid, mycolic acids, and other cellular constituents. Reports of mycobacterial acyl hydrolases [5-141 do not include a description of an enzyme system capable of catalyzing the turnover of acylglycerols of long chain fatty acids. We report here on the purification and properties of an enzyme which preferentially catalyzes the hydrolysis of long change triacylglycerols and which may be responsible for the turnover of triacylglycerols in this organism. Experimental
Procedure
Materials Glyceryl-tri[ 1-14C] palmitate (tri[ “C]palmitoylglycerol) and glyceryl-tri[ 9,lO (n)-“H,] palmitate (tri[ “II] palmitoylglycerol) were obtained from Amersham/Searle (Des Plaines, Ill.). Tripalmitoylglycerol, p-nitrophenyl esters of saturated fatty acids, and octylphenoxypolyethoxyethanol (Triton X-100) were obtained from Sigma Chemical Co. (St. Louis, MO.). Microgranular diethylaminoethyl cellulose (DE-52) was obtained from Pharmacia (Piscataway, N.J.). Ampholyte mixture, pH range 3-10 was obtained from LKB (Rockville, Md). N,N’-methylenebisacrylamide and N,N,N’,N’-tetramethyleneAcrylamide, diamine were purchased from Eastman Kodak Co. (Rochester, N.Y.). The purity of tripalmitoylglycerol was confirmed by thin-layer chromatography [ 151. Molecular sieve gels were prepared for use according to the instructions of the manufacturer.
Methods Growth
and harvesting procedures. M. phlei, ATCC 345, a gift from Professor M.M. Weber, St. Louis University, was grown in liquid medium containing: 1.5% casamino acids, 0.12% K,HP04, 0.75% fumaric acid, 0.2% sorbitan monooleate polyoxyethylene (Tween 80), 0.075% MgSO, . 17HZ0, 0.0066% FeSO, . 7H:O and enough NaOH to adjust the pH to 7.6. l-1 batches were inoculated with 5 ml of a culture which had been growing 64-72 h at 37°C. Cultures were incubated with vigorous shaking at 37OC on a New Brunswick gyratory shaker for 64 h and harvested in a Sorvall RC-2 continuous flow apparatus at 4°C. Extract preparations. To prepare crude cell extracts, aliquots (25 ml) of a 50% wet weight/volume cell suspension in 5 mM sodium phosphate buffer, pH 6.5, were sonicated for 40 min in a Raytheon 10KC sonic oscillator turned to optimum frequency. To prepare Triton X-100 extracts 12-24 1 of M. phlei
407
were cultured and harvested as described above. Washed and weighed cell pellets were chilled to 4°C and resuspended with an equal weight of ice-cold extraction buffer containing 10% Triton X-100 and 10 mM sodium phosphate, pH 6.5, then incubated at 37°C for 60 min, chilled to 4°C and centrifuged at 21 000 X g for 20 min. The supernatant liquid was decanted and designated Step II enzyme preparation. DEAE-cellulose column chromatography. Typically a 200-300 ml batch of Step II material was applied to a 2.5 X 20 cm column of DEAE-cellulose (Whatman DE-52) which had been equilibrated at 4°C with 5% Triton X-100 containing 5 mM sodium phosphate buffer, pH 6.5. Activity was eluted from the column with an exponential NaCl gradient formed from 200 ml of the equilibration buffer and 100 ml 0.25 M NaCl in the equilibration buffer. The tripalmitoylglycerol hydrolase, which eluted at 0.15 M salt was pooled and designated Step III material. Step III material was diluted with four volumes of 5% Triton X-100 in 5 mM sodium phosphate, pH 6.5. This was immediately applied to a second DEAEcellulose (0.9 X 15 cm) column which had been equilibrated with the same buffer as used above. The column was then treated with 40 ml of a buffer containing 5 mM sodium phosphate, 5 mM EDTA, 1 mM dithiothreitol, pH 6.5 (minus detergent). Activity was eluted from the column with a linear 100 ml gradient of O-O.5 M NaCl in 5 mM sodium phosphate, 5 mM EDTA, 1 mM dithiothreitol, pH 6.5. Pooled activity is designated Step IV material. This procedure (1) removes Triton X-100 and (2) results in further purification of the enzyme. Acrylamide gel electrophoresis. Sodium dodecyl sulfate-acrylamide gel electrophoresis was performed according to the method described by Weber and Osborn [16] in 7.5% acrylamide gels containing 0.1% sodium dodecylsulfate, 50 mM sodium phosphate buffer, pH 7.5, 5 mM EDTA, and 1 mM dithiothreitol (electrophoresis buffer). Step IV material was dialyzed exhaustively against water, lyophilized, and dissolved in 1% sodium dodecyl sulfate, 1 mM dithiothreitol, 5 mM EDTA, and 5% sucrose. Gels (0.55 X 7 cm) were prerun in electrophoresis buffer for 2-3 h. The material (0.05 ml) was electrophoresed at 5 mA/gel in the electrophoresis buffer until the tracking dye reached the bottom of the gel. Activity was recovered from frozen, sliced (0.4 mm/slice) gels by extracting each five consecutive slices in small test tubes containing 0.5 ml 50 n-M sodium phosphate buffer, pH 7.0, 5 mM EDTA, and 1 mM dithiothreitol at room temperature for 4 h. Activity was located with both the p-nitrophenylmyristate and lipase assays as described below. Activity from several gels was pooled and designated gel elution material. The extracted activity was separated from the gel slices by filtration through Whatman No. 1 paper. Isoelectric focusing. Analytical isoelectric focusing was performed in 7.5% acrylamide gels (0.6 X 7 cm) containing 1% ampholytes, pH 3-10. Samples were dialyzed extensively against distilled water, lyophilized, and dissolved in 5% sucrose, 1% ampholytes (pH 3-10) and applied to the top of the gel (anode). The anode buffer was 0.4% triethanolamine and cathode buffer was 0.4% H,SO,. Focusing was carried out at 160 V for 6-8 h at 4°C. Sephadex G-200 chromatography. Sephadex G-200 columns (0.9 X 20 cm)
408
were packed with the swollen gel at room temperature and equilibrated for at least 24 h with either 5 mM sodium phosphate, pH 7.0 or 0.1% sodium dodecyl sulfate, 5 mM sodium phosphate, pH 7.0. Flow rates were adjusted to 8-16 ml/cm ’ per h. Molecular weights were estimated as previously described [ 171. Proteins used to calibrate the columns were: lysozyme (egg white), 13 000; cytochrome c (beef heart), 14 300; bovine serum albumin, 69 000; and malate dehydrogenase, either 160 000 in detergent-free buffer or 40 000 for the monomers in sodium dodecyl sulfate-containing buffers. Lipase assay. Triacylglycerol hydrolase activity was determined with tri[’ 4C]palmitoylglycerol dispersed in Triton X-100 as described by Kaplan and Teng [ 181. The following standard reaction conditions were used unless otherwise indicated: 25 mg/ml Triton X-100, 3 mM tri[ “C]palmitoylglycerol, and 50 mM sodium phosphate, pH 6.5, in a final volume of 1.0 ml. Enzyme was added to initiate the reaction and incubated at 37°C for 20 min. Activity is measured as the release of [ 14C]palmitic acid from tri~i4C]palmitoylglycerol. Esterase assay. Chloroform solutions of p-nitrophenyl esters were evaporated to dryness with a stream of air or nitrogen. The compounds (25 pmol/ml final volume) were then dispersed by heating to 60°C in 83.3 mg/ml Triton X-100. Esterase activity was measured spectrophotometrically at 25°C with a Hitachi Model 124 split beam grating spe~trophotometer. The release of products was measured as the increase in absorbance at 412 nm relative to a control reaction mixture which contained no enzyme. Reaction mixtures contained 7.5 mM p-nitrophenyl ester, 25 mg/ml Triton X-100, 50 mM sodium phosphate buffer, pH 7.0, in a final volume of 0.4 ml. The reaction was started by the addition of enzyme. Protein. Protein was determined by the procedure of Lowry et al. f19]. Results Lipase act~uit~~ during M. phlei growth. M. phlei, grown as described in Methods, exhibited a doubling time of 4 h 20 min. The specific activity of the triacyiglycerol hydrolase increased S-fold during the stationary phase of growth and reached a maximum specific activity at 64 h (Fig. 1). No activity could be demonstrated in culture supernatants. Properties of crude extracts. While activity could be observed with whole cells, crude extracts prepared by exposure to sonic oscillations exhibited 20-30% more activity than unbroken cells. Sonication for 40 min produced an enzyme preparation which sedimented when centrifuged at 105 000 X g for 90 min and which also eluted in the void volume of a Bio-Gel A-15 agarose molecular sieve column (Fig. 2a). Treatment with a non-ionic detergent (Triton X-100) resulted in a form of enzyme which eluted in the included volume of the BioGel A-15 colum (Fig. Zb). Triton X-l 00 extruction of whole cells. Detergent treatment of crude extracts as described above resulted in at best a 3-fold purification. When unbroken cells were extracted with 5% Triton X-100 at 37°C for 60 min most of the activity was found in the supernatant liquid (Fig. 3). Efficiency of extraction varied from 40 to 80%. No activity was extracted at 0°C. The frac-
409
7-
CONTROL
_o,s
6
P loo
-
o-o-o
o-o-
3
d
0.2
2 -
5.0 -
0.1 t
; 0
_ ; 0 I5 J 1 r
c
/
2.0-
III.1 20
30 40 ELUTION
0 ‘* / ’ ‘0, 50 60 70 VOLUMEtml)
l
P A--A
1.0 -
A’
i
A
7-
/
5-
0.5
4 -
,.
/
O2 11
/
3 --
A--+’
O: 12
24 TIME
21
,
36 48 (Hours,
60
72
111 20 30 60 ELUTION
I 50
60
70
60
VOLUMEhI)
Fig. 1. Growth of M. phlei in liquid cuIture and triacylgiycerol hydroiase activity during growth. A 2-l flask containing 1 1 growth medium was inoculated with 46 ml of a 72 h culture, and incubated on a rotary shaker at 37°C. Wet weights (0 -@) were determined on 25-ml aliquots of the culture. Triacylglycerol hydrolase activity (A------a) was measured with loo-~1 aliquots of broken cell suspensions prepared by exposure to sonication as described in Methods (specific activity of tri[3Hlpalmitoylglycerol was 3.27 . lo6 dpm/pmol, incubation time 15 min). Protein was determined on disrupted cell suspensions by the following procedure: 1.0 ml aiiquots were heated to 100°C for 30 min in 10% trichloroacetic acid, cooled to 4°C for 1 h and centrifuged at 2000 X g for 10 min; the supernatants were decanted and the precipitate dissolved in 1 M NaOH, heated to 100°C for 30 min and centrifuged at 2000 Xg for 10 min Alisuots of this supernatant liquid were tested for Lowry-positive material [ 19). Fig. 2. Bio-Gel A-15 column chromatography of M. phlei triacylglycerol lipase prepared by exposure to sonic oscillations. Top: 0.3 ml aliquot of a crude enzyme preparation was applied to a 2.4 X 16 cm column of Bio-Gel A-15 which was equilibrated with 5 mM sodium phosphate buffer, pH 6.5. Bottom: the crude enzyme preparation was treated with 5% Triton X-100 prior to application to the column, In both eases the activity was e&ted with the equilibration buffer at 16 mIjcm2 per h. From 3.3~ml fractions O.l-ml aliquots were assayed for lipase activity as described in Methods supplemented with 100 mM citrate (specific activity tri[ “ClpalmitoylgIycero1 was 5.16 lo4 dpm/pmol).
tion of protein in the supernatant liquid was l--2% of the total under all conditions tested. ~~A~-ce~l~lose c~ro~ffto~~~~y. Reterg~nt-extracted material (Step II material) was adsorbed to a DEAE-cellulose column which had been equilibrated with 5% Triton X-100, 5 mM sodium phosphate, pH 6.5. The activity was eluted with an exponential NaCl gradient as described in Methods. Activity was located in the column fractions with both the triacylglycerol hydrolase assay and the ~-nitrophenylmyristate assay (Fig. 4). It can be seen that the elu-
410
0’ 0
, 50 .5 15 TRL&TYLNl TIMEhinf
50
f .
::
Fig. 3. Effect of treatment time and temperature on the efficiency of extraction of triacylglycerol hydrolase activity from M. phlei cells by Triton X-100. Into each of 14 tubes were weighed 4.0 g of a 65% (wet weight/volume) cell suspension in 5 mM sodium phosphate, pH 6.5, then 1.0 ml of a 25% Triton X-100 solution in 5 mM sodium phosphate, PH 6.5. was added to each tube. At the indicated times the tubes were placed in an ice bath. Triacylglycrroi hydrolase activity was assayed with 50-&l aliquots which were removed from each tube prior to ecntrifugation at 21 000 X e for 20 min. Extracted activity was determined with 200-&d aliquots of the supernatant liquids after centrifugation. Reaction mixtures contained 3 i.lmol tril~4ClPalmitoyl~lycerol (spec. act. 1.099. 10h dpm/pmoI) 25 mg Triton X-100. 50 .umol sodium phosphate, pH 6.5, and enzyme in 1.0 ml total volume.
m
0
IO
20
30
40
JO
60
m
FractionNurnbwf7ml) Fig. 4. DEAE-cellulose column chromatography of Step II material. A 2.5 X 20 cm column was esuiiibrated at 4°C with 5 mM sodium phosphate, pH 6.5, 5% Triton X-100. Approx. 650 mg (320 ml) of Step II material was applied to the column. The column was then treated with 80 ml of the equilibration buffer. The activity was eluted with an exponentiaf NaCl gradient formed from 300 ml equilibration buffer and 100 ml 0.25 M N&l in the equilibration buffer. The volume of the fractions was 7.0 ml. Hydrolase activity was measured with p-nitrophenylmyristate at 23% and triacylgfycerol lipase activity as (spec. act., 1.099 . described in Methods. Reaction mixtures contained 3 @mol tri[ * 4C]palmitoylglycero1 lo6 dpm/&unol), 25 mg T&ton X-100, 50 gmol sodium phosphate and enzyme in 1.0 ml total volume.
411
tion patterns of the triacylglycerol lipase and the p-nitrophenylmyristate hydrolase activities were identical and that the ratio of the two activities in each fraction was constant. Fractions 33-45 (Fig. 4) were pooled and designated Step III material. A 380-fold purification was achieved by this procedure. Step III material from another batch of cells was readsorbed to a DEAE column in 5% Triton X-100, 5 mM sodium phosphate, pH 6.5. The resin was then washed with 5 mM sodium phosphate to remove the detergent, and the activity was eluted with a linear NaCl gradient in a buffer containing 5 mM sodium phosphate, 5 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM dithiothreitol (Fig. 5). Under these conditions recovery was 30-45%, without EDTA and dithiothreitol recovery was less than 20%. Activity eluted from the first column at 0.15 M salt and from the second column at 0.3 M salt. Comments on the purification procedure. The procedures for purification from one 200 g wet weight batch of cells are presented in Table I. When Step IV material was subjected to gel electrophoresis at pH 7.5 in 7.5% polyacrylamide gels, one band of stainable protein was observed near the top of the gel with a small amount of material at the origin. A single protein-staining band was observed in analytical isoelectric focusing gels at a pH of 3.8. Step IV material chromatographed on a Sephadex G-200 eluted in the void volume (Fig. Sa). However, a 30 min exposure of Step IV material to 1% sodium dodecyl sulfate at room temperature resulted in a shift in the Sephadex G-200
1 N*CI 0.4
0.3 0.3
I-
NUYILR
FUACTION Fig.
5. Rechromatography
equilibrated (see and
Fig.
the
4 legend).
applied
1 mM glycerol
0.1~ml hydrolase
Methods. lo5
same
Step
Activity
material (20
column
on
(o-------c’
hydrolase
ml,
eluted
removed
for
), and activity
DEAE-cellulose.
X-100 40
was then
was then
were
activity
dpm//.unol).
III
phosphate/Triton The
aliquots
Triacylglyerol
Step
111 material
to the column.
dithiothreitol.
fractions
1.88
with
of
mg)
buffer was
treated by
the
a NaCl
A
used
diluted
with
40
to
ml
gradient
following with
0.9
X 15
80
formed
ml
with
sodium
hydrolase
column
of DEAE
the
first
DEAE
the
equilibration
phosphate,
in the latter
determinations: 3 mM
cm
equilibrate
5 mM
p-nitrophenylmyristate
was measured
to
protein
5 mM
buffer.
(A- - - - - -A)
tri[ 14Clpalmitoylglycerol
buffer EDTA,
From
(0 ---0).
was
column
5.5-ml triacyl-
as described (spec.
in act.,
I
II
III
IV
step
step
step
step
(ml)
Fraction
activity
19 000
18 700
9 500
1 800
204
31
70
L.
Total
380
Volume
tripalmitovl~lycrr~,1
with
FROM
3 520
with
1.7
25
68
20 600
19 000
(mz)
protein
Total
activity
368
M..
MYCOBACTERIL’M
500
_
M.
as substrate;
munits
ACTIVITY
L..
LlPASE
OF
I
PURIFICATION
TABLE
1.04 51.1 707
381
L.
Specific
activitv
p-nitrophen~lm~ristate
PffLEI
380 680
820 2 100
1 50
L.
152
iLI.
Fold
as substrate.
purification
4.5
48 13.2
95 6
100
L.
1
M.
Recoverv
(W)
6.1
37
100
M.
313
;I
A.
7 7
j 5
15\ - 1\\
20
mM PO,
0
d
0
10 3-
0
0 '0
0
,A,
‘O.o *0.,
1
20
10 M1 20_
0.
4
IO-15
5;
, 10
0.1 x SDS
6%
,:,
1
30
I
“\ O\O, .n 20
FRACTION
, 30
NUMBER
Fig. 6. Sephadex G-ZOO chromatography of Step IV material with and without exposure to sodium dodecyl sulfate. A 0.9 X 20 cm column of Sephadex G-200 was equilibrated with either 5 mM sodnun phosphate, pH 7.0 (A), or 50 mM sodium phosphate, 0.1% sodium dodecyl sulfate, pH 7.0 (B). Step IV material was incubated at room temperature with 0.1% sodium dodecyl sulfate (B) or without 0.1% sodium dodecyl sulfate (A) for 15 min prior to application to the column. Samples (0.2 ml) were chromatographed at a flow rate of 15 ml/cm2 per h. The volume of each fraction was 0.5 ml. Markers used to calibrate the columns were: Ml, Blue Dextran 2000; M2, bovine serum albumin; MJ. trypsin; Ma, cytochrome c.
column elution pattern of the activity. The new position of the activity was between bovine serum albumin and cytochrome c, corresponding to a molecular weight of approx. 40 000 (Fig. 6b). Step IV material separated into six protein bands when treated with 1% sodium dodecyl sulfate and electrophoresed in 7.5% polyacrylamide gels containing 0.1% sodium dodecyl sulfate (Fig. 7). These six bands correspond to the following molecular weights: (1) 69 000, (2) 60 000, (3) 50 000, (4) 42 000, (5) 27 000, (6) 22 000. The optimal assay temperature for triacylglycerol hydrolase activity was 37°C. Activity is stable at 4 or -17°C for several weeks. A 50% loss of activity was observed when Step IV material was exposed to 50°C for 10 min, and 90% was lost following a 10 min treatment at 70°C. Extraction of hydrolase activity from sodium dodecyl sulfate gels. As indicated above, Step IV material was resolved into six protein-staining bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Approx. 50% of the triacylglycerol hydrolase and the p-nitrophenylmyristate hydrolase applied to the gel was stable to sodium dodecyl sulfate and was recovered from the 40 000 molecular weight region of the gel (Fig. 7). The extracted activity was
0
01
0.2
03
04
0.5
0.6
0.7
06
09
10
Hf
Fig. 7. Localization and extraction of hydrolase activity after sodium dodecyl sulfate-acrylamidc gel electrophoresis of Step IV material. Lyophilized Step IV material was dissolved in 1.0% sodurn dodccyl sulfate, 1 mM dithiothrcitol. 1 mM EDTA, and 5 % sucrose. Approx. 80 pg of this material was apphed to the top of each gel and electrophorescd at 5 mA per gel as described in Methods. One of the gels was frwen, sliced, and extracted: the other was stained for protein. At the end of the extraction period. fractions were assayed for triacylglycerol (1 u ) and p-nitrophenylmyristate (*- - - - - -0) hydrolase activitv. The specific activity of the tri[ I 4Clpalmitoylglycero1 was 5.82 10 s dpm/pmol.
designated gel elution material. Because of the dilution factor in the extraction, this preparation contained less than 0.02% sodium dodecyl sulfate, and reaction mixtures contained less than 0.002%. We determined that 0.02% sodium dodecyl sulfate in reaction mixtures had no effect on the activity. In 100 mM acetate or phosphate buffers the pH optimum of the gel elution material is broad with a maximum at 5.8 (Table II). In 100 mM sodium maleate buffer the pH optimum was 6.3. Influence of organic anions on hydrolase activity. A stimulatory effect of organic anions was first noted as a discrepancy in overlapping points with two buffers in a pH profile study with crude (Step I) enzyme preparations. Activity in a citrate/phosphate buffer was 50% greater than in phosphate buffer at the same pH and ionic strength. Isocitrate, a-ketoglutarate, succinate, acetate, and formate also stimulated triacylglycerol lipase activity (Table III). The effect was not due to increased ionic strength since neither KC1 nor NaCl at concentrations up to 0.5 M had any effect on the activity. Inorganic anions such as phosphate or Cl- also had no effect. Sodium citrate stimulated gel elution material as well as the crude enzyme preparations. The saturation kinetics of gel elution material with tripalmitoylglycerol as substrate are presented in Fig. 8a. Sodium citrate at 100 and 200 mM concentrations not only stimulated activity but also altered the shape of the activity to substrate concentration curve (Fig. Sa). From double reciprocal plots of this data (Fig. Sb), it can be seen that in the absence of citrate the curve is not linear, whereas in the presence of citrate the curve is linear. Linear regression analysis of this data indicates a KTaDp of 8.9 ? 4 mM and a V of 13 860 nmol/min per mg protein at 100 mM citrate. At 200 mM citrate the Kmwp was
415
TABLE
II
EFFECT
OF
PH
AND
BUFFER
ON
TRIACYLGLYCEROL
HYDROLASE
ACTIVITY
OF
STEP
V
MATERIAL The 7.5
specific mM
activity
of
the
tri[ I 4ClpalmitoylgIycerol
was
5.68
. 10
5 pmol.
Reaction
mixtures
contained
tripalmitoylglycerol.
Buffer
PH
Specific
activity
(munits/mg
protein)
I Acetate
Sodium
phosphate
4.5
994
5.0
1491
5.3
2556
5.6
1988
5.8
3748
6.0
3497
6.1
3050
6.2
2627
6.3
2343
6.5
2340
6.8
2343
7.0
1775
7.5
1065
8.9
355
II Sodium
TABLE
The
5.4
489
5.6
2248
5.8
2619
6.0
2816
6.1
2816
6.2
3012
6.4
3013
6.6
3012
6.8
2658
7.0
2366
III
EFFECTS CRUDE
maleate
OF
ORGANIC
SONICATES
control
Compound
activity
ANIONS
OF for
ON
THE
MYCOBACTERIUM
this
preparation
Concentration
was
TRIACYLGLYCEROL
PHLEI 5.8
munitslmg
Control
(mM) Citrate
Iaocitrate
50
140
100
178
200
250
500
110
50
145 169
a-Ketoglutarate
100
Succinate
100
142
Acetate
100
156
Formate
100
112
protein
(%)
HYDROLASE
ACTIVITY
OF
b 2s -
20 -
1
lf-
V 10 -
5-
TRIPALMITIN
‘4
41 05
fmlUt
.._---_ )
8
C
i 0
e0.5
/ 110
I ‘1.5
LnlSl Fig. 8. Effect of sodium citrate on triacylglycerol hydrolase activity of Step IV material at different substrate concentrations. Reaction mixtures contained 20 pmol sodium maleate buffer, PH 6.5, 5 mg Triton (5.65. 10s dpm/Mmol), and 0 (*-----+), 20 X-300. 0.11 jog Step IV protein, tri[ 14CJpaimitoylglycero1 ) ymol sodium citrate in a total volume of 0.2 ml. (a) Activity versus subor 50 (“----o (A -------A), strate
plot of the data; (b) Lineweaver-Burk
plot of the same data, and (c) Hill plot of the data.
2.8 L 4 mM and the V was 11 730 nmol/min per mg protein. The Hill number in the absence of citrate was 2.2 (Fig. 8c), while in the presence of citrate, Hill numbers of approx. 1 were obtained. Sodium citrate at concentrations of up to 200 mM had no effect of the ~-nitrophe~ylmyristate hydrolase activity (Table IV).
Actiuity of gel elution different lengths. Activity
material
with p-nitrophenyl
esters of fatty acids of
at equimolar concentrations of p-nitrophenyl esters of fatty acids increased with the length of the fatty acyl group up to C4 (Table V). 4 times more activity was observed with p-nitrophenylmyristate as substrate than with p-nitrophenylacetate as substrate.
417 TABLE
IV
THE EFFECT
OF SODIUM
CITRATE
HYDROLASE
ACTIVITIES
OF STEP IV MATERIAL
ON TRIACYLGLYCEROL
AND p-NITROPHENYLMYRISTATE
(spec. act. = 5.68 . Triaeylglycerol hydrolase activity was assayed with 7.5 mM tril 14C]palmitoylglycero1 lo5 dpm/timol. 25 mg/ml Triton X-100, 0.11 l.(g Step IV protein and the indicated buffer, pH 6.5. in 0.2 ml reaction mixture at 37’C for 20 min. 50 mM sodium maleate buffer, pH 6.5, was included in all reaction mixtures. p-Nitrophenylmyristate hydrolase activity was assayed by standard procedures as described in Methods with a reaction mixture containing 7.5 mM substrate, 25 mg/ml Triton X-100, and 50 mM sodium phosphate buffer. pH 7.0,0.092 pg Step IV protein a 0.4 ml reaction mixture at 23OC. Addition
Concentration
Hydrolase triacylgiyceroi (munitsimg)
Activity p-nitrophenylmyristate (munitslmg)
50
1450
-
50 25 50 100 150 200
2460 2555 3154 4670 4732
5400 5400 5800 5700 5900 6200
(mM)
Sodium
maleate
Sodium Sodium
phosphate citrate
TABLE
V
ACYL HYDROLASE SATURATED FATTY
ACTIVITY OF STEP ACIDS (Cz-c, *)
Reaction mixtures contained Triton X-100, and 3.0 pmol tion of enzyme. ,,-Nitrophenyl
Acetic Propionic Butyric Valetic Caprnie Capric Laurie Myrktic Palmitic Stearic
ester
IV
MATERIAL
USING
p-NITROPHENYL
ESTERS
OF
0.15 @g Step IV protein, 40 pmol sodium phosphate buffer, pH 7.0, 10 mg substrate in a total volume of 0.4 ml. The reaction was started by the addi-
Carbon
2 3 4 5 6 IO 12 14 17 18
No.
Specific activity (munitsfmg protein) 725 752 1654 1816 IF54 2444 2756 2882 2756 2506
Discussion Our results demonstrate that M. phlei produces a triacylglycerol lipase activity. We believe this enzyme to be intracellular in nature and associated with the cell wall-membrane complex since (a) activity was not found in the culture supernatant liquid, (b) activity in sonicated preparations is an aggregate of more than 2 + lo6 daltons, and (c) treatment of the sonicated preparation with the non-ionic detergent Triton X-100 results in a reduction of the size of the activity. In addition, activity can be measured with whole cells only in the presence of detergent. While it is not possible to determine from this data whether the activity is on the inside or outside of the cell w~l-membrane com-
418
plex, it does suggest that the lipase is associated with that structure. A high decree of purification (680-fold) was achieved by taking advantage of the stability of the activity to treatment with non-ionic detergents as well as the ability of the detergent to selectively solubilize the enzyme. Whole cells treated with 5% Triton X-100 at 37°C are not lysed and only l---2% of the total cellular protein is released from the cells. The majority of the lipase activity is found in the supematant liquid following treatment at 37”C, while at 0°C the activity remains associated with the cells. The solubilized activity, which is already 50-60-fold purified, is completely stable in the extraction buffer for extended periods of time at either 4 or -17” C. Further purification (another 6-fold) was achieved by chromatography of the extracted material on DEAE-cellulose in the presence of detergent (Fig. 4). Wickner and Kennedy [ 201 employed a similar procedure in their purification of phosphotidylserine decarboxylase. A second DEAE-cellulose column (Fig. 5) served two functions: removal of the detergent and further purification. Since radioactive Triton X-100 was not available we could not be absolutely certain that all of the detergent was removed. We were certain, however, that the detergent concentration was reduced at least 500-fold by following the decrease in absorbance at 295 nm. Activity recovered from this column (Step IV material) appeared nearly pure by two criteria: polyacrylamide gel electrophoresis at pH 7.5 (there is some material at the origin) and analytical isoelectric focusing in acrylamide gels. This preparation of the lipase was, however, an aggregate which could be resolved into six protein bands when treated with 1% sodium dodecyl sulfate and then electrophoresed in polyacrylamide gels. Scandella and Kornberg [ 211 effectively utilized preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis in their purification of active phospholipase A, from Escherichia coli. We found that activity could be extracted from the 40 000 molecular weight region of sodium dodecyl sulfate-polyacrylamide gels with a 30-50% recovery (Fig. 7). Chromatography of sodium dodecyl sulfatetreated material on Sephadex G-200 indicates a molecular weight for the active component between 30 000 and 50 000. M. phlei lipase therefore has a molecular weight of approx. 40 000 and is found as an aggregate with five proteins. Whether the association of the activity with these proteins is formed de novo (i.e. self aggregation) or is an artifact resulting from the purification procedure is not known. Characterization of the activity. During the purification of M. phlei lipase, activity was monitored with two substrates, tripalmitoylglycerol and p-nitrophenylmyristate. Care must be taken when p-nitrophenyl esters are used to define lipase activity since it is known that e&erases as well as lipases will hydrolyze synthetic esters [ 221. The validity of using synthetic non-glycerol esters for the study of a highly purified lipase preparation was established by Brockerhoff [23] in his studies of substrate specificity of pancreatic lipase. During this study we determined that the ratio of triacylglycerol to p-nitrophenylmyristate hydrolase activities is constant for DE II material, isoelectric focused material, for the activity extracted from sodium dodecyl sulfateacrylamide gels and for DE II material chromatographed on Sephadex G-200. The most highly purified material produced in this study (gel elution material) exhibited a specificity for long chain fatty acids when tested with p-nitro-
419
phenyl esters of saturated fatty acids containing 2-18 carbons (Table V). The lowest activity was found with acetate and the highest activity with myristate. The high rate of hydrolysis of tripalmitoylglycerol and the specificity of the enzyme for long chain fatty acid esters support the identification of the activity described here as triacylglycerol lipase (EC 3.1.1.3). An interpretation of the kinetics of enzymes which utilize insoluble substrates is complicated by the kinetic effects of different phase structures of the substrate. The relationship of M. phlei lipase activity to tripalmitoylglycerol concentration was sigmoid with non-linear double reciprocal plots (Figs. 8a and Sb). Sodium citrate and other organic anions stimulated triacylglycerol hydrolase activity and also altered the relationship of activity to substrate concentration such that the curve became nearly hyperbolic in shape (Fig. 8a). Double reciprocal plots of this data now become linear with the Hill number close to 1; the Hill number was 2.2 in the absence of citrate. It is important to note that sodium citrate had no effect on the activity of the enzyme with p-nitrophenylmyristate as substrate (Table IV). It has been shown that pancreatic lipase works at an oil/ water interface and that the apparent V is equal to the maximum concentration of the enzyme at the phase surface multiplied by the rate constant (apparent V = E X surface area X K) 1241. The authors conclude that any material that can alter the nature of the oil/water interface can markedly influence the rate of triacylglycerol hydrolysis or the diffusion of the
2
4 SUBSTRATE
6
8
(mM)
Fig. 9. Effect of Triton X-100 on triacylglycrrol and p-nitrophenylmyristate hydrolase activity of Step IV ‘) or 5 (.-4) mg x ), 3.2 (‘1 material. (A) Triacylglycerol hydrolase activitv with 1.6 (XTriton X-100 in a 0.2 ml reaction mixture which also contained 0.09 pg Step IV protein, 20 ~mol sodium phosphate. pH 6.5. and tri[ 14Clpalmitin (spec. act. 5.69 lo5 dpm/ ~mol). (b) p-Nitrophenylmyristate or 5 (O0) mg Triton X-100 in a 0.4 ml X), 2.5 (‘J -----). hydrolasc activity with 1.25 (Xreaction mixture which also contained 0.11 fig Step IV protein. 40 /on01 sodium phosphate. pH 7.0, and /,-nitrophenylmyristatc.
420
product from the oil/water interface into the bulk phase. The effect that we see is somewhat specific in that inorganic anions (Cl-, phosphate) and inorganic cations (Na+, K’) do not mimic the effect of the organic anions. It appears that the stimulatory effect is dependent not on the specific ion but rather on the concentration of carboxyl groups added to the reaction mixture. While we cannot rule out a direct effect on the enzyme it is our feeling that the organic anions are somehow altering the oil/water interface or micelle structure of the substrate, and thereby altering the activity. Triton X-100 concentrations also affect the velocity to substrate relationship of this enzyme (Fig. 9). Dennis [ 251 reported that phospholipase A2 requires Triton X-100 or some other detergent for activity toward egg phosphatidylcholine, but that inhibition occurs at high concentrations of Triton X-100. Similar effects were observed by Kaplan and Teng [18] for beef liver lipase and by Kariya and Kaplan [ 151 for rat liver lysosomal lipase. Changing the concentration of Triton may alter the structure of the triacylglycerol/Triton micelle. A 2 : 1 ratio of Triton X-100 to phospholipid substrate is required for maximal phospholipase AZ activity [ 251. A molar ratio of 6 : 1 (Triton X-100 : triacylglycerol) is required for optimal M. phlei lipase activity. Metabolic role of M. phlci lipase. The specific activity of M. phlci lipase increases at least 3-fold late in stationary phase of growth (Fig. 1). This is consistent with the preferential utilization of glycogen and subsequent utilization of the triacylglycerol fatty acids as a source of endogenous energy. Although the lipase activity increases several fold late in the growth of M. phlei, there is activity present at minimal levels in early stages of growth. Triacylglyccrol pools at this time turn over rapidly. It appears likely that the enzyme described in this study is responsible for both the turnover of the triacylglycerols during early growth and for the breakdown of the triacylglycerol pools late in growth. Acknowledgement This General
work was supported by Public Health Research Support Grant 24-57-819.
Service
Grant
AM 13516
and by
References 1
Stephenson.
M. and
2
Assrlineau.
,I. (1966)
N’hvtman.
M.D.
(1924)
The* Ractcria
Prw.
Lipids.
2nd
It. Sec. rdn..
pp.
Lond..
Ser.
B 95,
156-l
57.
Holden-Dav.
20~-206 Inc.,
Calif. 3
Brvnnan,
4
McCarthv.
P.J.
Iioonrv.
S.A.
5
Davis,
B.D.
and
Dubos,
R.J.
(1946)
Arch.
6
Davis,
B.D.
and
Dubos,
K.J.
(1947)
.I. Ia:xp. Med.
and
Dubos.
R.J.
(1948)
.J. Bact
Biochimica et Biophysics @ Elsevier/North-Holland
Acta, 487 (1977) Biomedical Press
405-421
BBA 56996
PURIFICATION AND PROPERTIES FROM MYCOBACTERIUM PHLEI
JOHN L. PAZNOKAS
* and ARNOLD
Department of Microbiology, 63104 (U.S.A.) (Received
November
24th,
St. Louis
OF A TRIACYLGLYCEROL
LIPASE
KAPLAN University
School
of Medicine,
Saint Louis,
Mo.
1976)
Summary In order to study the metabolism of triacylglycerol in mycobacteria, an intracellular particulate triacylglycerol lipase (EC 3.1.1.3) was purified BOO-fold from stationary phase cells of ililycobacterium phlei. Extraction of whole cell suspensions with 5% Triton X-100, followed by ion-exchange chromatography of the extract on two successive DEAE-cellulose columns produced a preparation which was nearly homogeneous by the criterion of analytical isoelectric focusing in acrylamide gels (one band, p1. 3.8) and by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the preparation into six protein bands. Lipase activity stable to electrophoresis in sodium dodecyl sulfate was extracted from the 40 000 molecular weight region of the gels. With phosphate or maleate buffer the enzyme exhibits a broad pH optimum around 6.0 with sigmoid saturation kinetics (Hill number 2), and an apparent K, of 8.8 mM for tripalmitoylglycerol. Citrate and other carboxylic acids increase the apparent V up to 3-fold with the Hill number approaching 1.0. In a series of p-nitrophenyl esters tested (C,-C,,), p-nitrophenylmyristate was hydrolyzed most rapidly. The saturation curve for p-nitrophenylmyristate was sigmoid and unaffected by citrate. The role of this activity in the metabolism of triacylglycerols by Mycobacteria is discussed.
Introduction Mycobacterium
most rapidly
phlei contains in the late stationary
* Present address: Department Pullman, Wash. 99164, U.S.A.
of
a pool of triacylglycerol which turns over phase of growth in liquid culture. Stephen-
Bacteriology
and
Public
Health,
Washington
State
University,
406
son and Whetman [I] first observed that in late stationary phase endogenous oxygen uptake persisted and the respiratory quotient decreased from 1.72 to 0.74 as would be expected from a transition to oxidation of fatty acids. Later Asselineau [ 21 directly demonstrated that mycobacteria including M. phlei contain triacylglycerols. More recently Brennan et al. [3] have shown that both glycogen and triacylglycerols accumulate in M. phlei during growth and that glycogen is preferentially utilized when growth nutrients are depleted. This work was confirmed and extended by McCarthy [ 41. His experiments revealed that the uptake of labeled fatty acids (especially palmitic) into iM. phlei triacylglycerol was followed by a loss of the label from the triacylglycerol pool and its subsequent appearance in phospholipid, mycolic acids, and other cellular constituents. Reports of mycobacterial acyl hydrolases [5-141 do not include a description of an enzyme system capable of catalyzing the turnover of acylglycerols of long chain fatty acids. We report here on the purification and properties of an enzyme which preferentially catalyzes the hydrolysis of long change triacylglycerols and which may be responsible for the turnover of triacylglycerols in this organism. Experimental
Procedure
Materials Glyceryl-tri[ 1-14C] palmitate (tri[ “C]palmitoylglycerol) and glyceryl-tri[ 9,lO (n)-“H,] palmitate (tri[ “II] palmitoylglycerol) were obtained from Amersham/Searle (Des Plaines, Ill.). Tripalmitoylglycerol, p-nitrophenyl esters of saturated fatty acids, and octylphenoxypolyethoxyethanol (Triton X-100) were obtained from Sigma Chemical Co. (St. Louis, MO.). Microgranular diethylaminoethyl cellulose (DE-52) was obtained from Pharmacia (Piscataway, N.J.). Ampholyte mixture, pH range 3-10 was obtained from LKB (Rockville, Md). N,N’-methylenebisacrylamide and N,N,N’,N’-tetramethyleneAcrylamide, diamine were purchased from Eastman Kodak Co. (Rochester, N.Y.). The purity of tripalmitoylglycerol was confirmed by thin-layer chromatography [ 151. Molecular sieve gels were prepared for use according to the instructions of the manufacturer.
Methods Growth
and harvesting procedures. M. phlei, ATCC 345, a gift from Professor M.M. Weber, St. Louis University, was grown in liquid medium containing: 1.5% casamino acids, 0.12% K,HP04, 0.75% fumaric acid, 0.2% sorbitan monooleate polyoxyethylene (Tween 80), 0.075% MgSO, . 17HZ0, 0.0066% FeSO, . 7H:O and enough NaOH to adjust the pH to 7.6. l-1 batches were inoculated with 5 ml of a culture which had been growing 64-72 h at 37°C. Cultures were incubated with vigorous shaking at 37OC on a New Brunswick gyratory shaker for 64 h and harvested in a Sorvall RC-2 continuous flow apparatus at 4°C. Extract preparations. To prepare crude cell extracts, aliquots (25 ml) of a 50% wet weight/volume cell suspension in 5 mM sodium phosphate buffer, pH 6.5, were sonicated for 40 min in a Raytheon 10KC sonic oscillator turned to optimum frequency. To prepare Triton X-100 extracts 12-24 1 of M. phlei
407
were cultured and harvested as described above. Washed and weighed cell pellets were chilled to 4°C and resuspended with an equal weight of ice-cold extraction buffer containing 10% Triton X-100 and 10 mM sodium phosphate, pH 6.5, then incubated at 37°C for 60 min, chilled to 4°C and centrifuged at 21 000 X g for 20 min. The supernatant liquid was decanted and designated Step II enzyme preparation. DEAE-cellulose column chromatography. Typically a 200-300 ml batch of Step II material was applied to a 2.5 X 20 cm column of DEAE-cellulose (Whatman DE-52) which had been equilibrated at 4°C with 5% Triton X-100 containing 5 mM sodium phosphate buffer, pH 6.5. Activity was eluted from the column with an exponential NaCl gradient formed from 200 ml of the equilibration buffer and 100 ml 0.25 M NaCl in the equilibration buffer. The tripalmitoylglycerol hydrolase, which eluted at 0.15 M salt was pooled and designated Step III material. Step III material was diluted with four volumes of 5% Triton X-100 in 5 mM sodium phosphate, pH 6.5. This was immediately applied to a second DEAEcellulose (0.9 X 15 cm) column which had been equilibrated with the same buffer as used above. The column was then treated with 40 ml of a buffer containing 5 mM sodium phosphate, 5 mM EDTA, 1 mM dithiothreitol, pH 6.5 (minus detergent). Activity was eluted from the column with a linear 100 ml gradient of O-O.5 M NaCl in 5 mM sodium phosphate, 5 mM EDTA, 1 mM dithiothreitol, pH 6.5. Pooled activity is designated Step IV material. This procedure (1) removes Triton X-100 and (2) results in further purification of the enzyme. Acrylamide gel electrophoresis. Sodium dodecyl sulfate-acrylamide gel electrophoresis was performed according to the method described by Weber and Osborn [16] in 7.5% acrylamide gels containing 0.1% sodium dodecylsulfate, 50 mM sodium phosphate buffer, pH 7.5, 5 mM EDTA, and 1 mM dithiothreitol (electrophoresis buffer). Step IV material was dialyzed exhaustively against water, lyophilized, and dissolved in 1% sodium dodecyl sulfate, 1 mM dithiothreitol, 5 mM EDTA, and 5% sucrose. Gels (0.55 X 7 cm) were prerun in electrophoresis buffer for 2-3 h. The material (0.05 ml) was electrophoresed at 5 mA/gel in the electrophoresis buffer until the tracking dye reached the bottom of the gel. Activity was recovered from frozen, sliced (0.4 mm/slice) gels by extracting each five consecutive slices in small test tubes containing 0.5 ml 50 n-M sodium phosphate buffer, pH 7.0, 5 mM EDTA, and 1 mM dithiothreitol at room temperature for 4 h. Activity was located with both the p-nitrophenylmyristate and lipase assays as described below. Activity from several gels was pooled and designated gel elution material. The extracted activity was separated from the gel slices by filtration through Whatman No. 1 paper. Isoelectric focusing. Analytical isoelectric focusing was performed in 7.5% acrylamide gels (0.6 X 7 cm) containing 1% ampholytes, pH 3-10. Samples were dialyzed extensively against distilled water, lyophilized, and dissolved in 5% sucrose, 1% ampholytes (pH 3-10) and applied to the top of the gel (anode). The anode buffer was 0.4% triethanolamine and cathode buffer was 0.4% H,SO,. Focusing was carried out at 160 V for 6-8 h at 4°C. Sephadex G-200 chromatography. Sephadex G-200 columns (0.9 X 20 cm)
408
were packed with the swollen gel at room temperature and equilibrated for at least 24 h with either 5 mM sodium phosphate, pH 7.0 or 0.1% sodium dodecyl sulfate, 5 mM sodium phosphate, pH 7.0. Flow rates were adjusted to 8-16 ml/cm ’ per h. Molecular weights were estimated as previously described [ 171. Proteins used to calibrate the columns were: lysozyme (egg white), 13 000; cytochrome c (beef heart), 14 300; bovine serum albumin, 69 000; and malate dehydrogenase, either 160 000 in detergent-free buffer or 40 000 for the monomers in sodium dodecyl sulfate-containing buffers. Lipase assay. Triacylglycerol hydrolase activity was determined with tri[’ 4C]palmitoylglycerol dispersed in Triton X-100 as described by Kaplan and Teng [ 181. The following standard reaction conditions were used unless otherwise indicated: 25 mg/ml Triton X-100, 3 mM tri[ “C]palmitoylglycerol, and 50 mM sodium phosphate, pH 6.5, in a final volume of 1.0 ml. Enzyme was added to initiate the reaction and incubated at 37°C for 20 min. Activity is measured as the release of [ 14C]palmitic acid from tri~i4C]palmitoylglycerol. Esterase assay. Chloroform solutions of p-nitrophenyl esters were evaporated to dryness with a stream of air or nitrogen. The compounds (25 pmol/ml final volume) were then dispersed by heating to 60°C in 83.3 mg/ml Triton X-100. Esterase activity was measured spectrophotometrically at 25°C with a Hitachi Model 124 split beam grating spe~trophotometer. The release of products was measured as the increase in absorbance at 412 nm relative to a control reaction mixture which contained no enzyme. Reaction mixtures contained 7.5 mM p-nitrophenyl ester, 25 mg/ml Triton X-100, 50 mM sodium phosphate buffer, pH 7.0, in a final volume of 0.4 ml. The reaction was started by the addition of enzyme. Protein. Protein was determined by the procedure of Lowry et al. f19]. Results Lipase act~uit~~ during M. phlei growth. M. phlei, grown as described in Methods, exhibited a doubling time of 4 h 20 min. The specific activity of the triacyiglycerol hydrolase increased S-fold during the stationary phase of growth and reached a maximum specific activity at 64 h (Fig. 1). No activity could be demonstrated in culture supernatants. Properties of crude extracts. While activity could be observed with whole cells, crude extracts prepared by exposure to sonic oscillations exhibited 20-30% more activity than unbroken cells. Sonication for 40 min produced an enzyme preparation which sedimented when centrifuged at 105 000 X g for 90 min and which also eluted in the void volume of a Bio-Gel A-15 agarose molecular sieve column (Fig. 2a). Treatment with a non-ionic detergent (Triton X-100) resulted in a form of enzyme which eluted in the included volume of the BioGel A-15 colum (Fig. Zb). Triton X-l 00 extruction of whole cells. Detergent treatment of crude extracts as described above resulted in at best a 3-fold purification. When unbroken cells were extracted with 5% Triton X-100 at 37°C for 60 min most of the activity was found in the supernatant liquid (Fig. 3). Efficiency of extraction varied from 40 to 80%. No activity was extracted at 0°C. The frac-
409
7-
CONTROL
_o,s
6
P loo
-
o-o-o
o-o-
3
d
0.2
2 -
5.0 -
0.1 t
; 0
_ ; 0 I5 J 1 r
c
/
2.0-
III.1 20
30 40 ELUTION
0 ‘* / ’ ‘0, 50 60 70 VOLUMEtml)
l
P A--A
1.0 -
A’
i
A
7-
/
5-
0.5
4 -
,.
/
O2 11
/
3 --
A--+’
O: 12
24 TIME
21
,
36 48 (Hours,
60
72
111 20 30 60 ELUTION
I 50
60
70
60
VOLUMEhI)
Fig. 1. Growth of M. phlei in liquid cuIture and triacylgiycerol hydroiase activity during growth. A 2-l flask containing 1 1 growth medium was inoculated with 46 ml of a 72 h culture, and incubated on a rotary shaker at 37°C. Wet weights (0 -@) were determined on 25-ml aliquots of the culture. Triacylglycerol hydrolase activity (A------a) was measured with loo-~1 aliquots of broken cell suspensions prepared by exposure to sonication as described in Methods (specific activity of tri[3Hlpalmitoylglycerol was 3.27 . lo6 dpm/pmol, incubation time 15 min). Protein was determined on disrupted cell suspensions by the following procedure: 1.0 ml aiiquots were heated to 100°C for 30 min in 10% trichloroacetic acid, cooled to 4°C for 1 h and centrifuged at 2000 X g for 10 min; the supernatants were decanted and the precipitate dissolved in 1 M NaOH, heated to 100°C for 30 min and centrifuged at 2000 Xg for 10 min Alisuots of this supernatant liquid were tested for Lowry-positive material [ 19). Fig. 2. Bio-Gel A-15 column chromatography of M. phlei triacylglycerol lipase prepared by exposure to sonic oscillations. Top: 0.3 ml aliquot of a crude enzyme preparation was applied to a 2.4 X 16 cm column of Bio-Gel A-15 which was equilibrated with 5 mM sodium phosphate buffer, pH 6.5. Bottom: the crude enzyme preparation was treated with 5% Triton X-100 prior to application to the column, In both eases the activity was e&ted with the equilibration buffer at 16 mIjcm2 per h. From 3.3~ml fractions O.l-ml aliquots were assayed for lipase activity as described in Methods supplemented with 100 mM citrate (specific activity tri[ “ClpalmitoylgIycero1 was 5.16 lo4 dpm/pmol).
tion of protein in the supernatant liquid was l--2% of the total under all conditions tested. ~~A~-ce~l~lose c~ro~ffto~~~~y. Reterg~nt-extracted material (Step II material) was adsorbed to a DEAE-cellulose column which had been equilibrated with 5% Triton X-100, 5 mM sodium phosphate, pH 6.5. The activity was eluted with an exponential NaCl gradient as described in Methods. Activity was located in the column fractions with both the triacylglycerol hydrolase assay and the ~-nitrophenylmyristate assay (Fig. 4). It can be seen that the elu-
410
0’ 0
, 50 .5 15 TRL&TYLNl TIMEhinf
50
f .
::
Fig. 3. Effect of treatment time and temperature on the efficiency of extraction of triacylglycerol hydrolase activity from M. phlei cells by Triton X-100. Into each of 14 tubes were weighed 4.0 g of a 65% (wet weight/volume) cell suspension in 5 mM sodium phosphate, pH 6.5, then 1.0 ml of a 25% Triton X-100 solution in 5 mM sodium phosphate, PH 6.5. was added to each tube. At the indicated times the tubes were placed in an ice bath. Triacylglycrroi hydrolase activity was assayed with 50-&l aliquots which were removed from each tube prior to ecntrifugation at 21 000 X e for 20 min. Extracted activity was determined with 200-&d aliquots of the supernatant liquids after centrifugation. Reaction mixtures contained 3 i.lmol tril~4ClPalmitoyl~lycerol (spec. act. 1.099. 10h dpm/pmoI) 25 mg Triton X-100. 50 .umol sodium phosphate, pH 6.5, and enzyme in 1.0 ml total volume.
m
0
IO
20
30
40
JO
60
m
FractionNurnbwf7ml) Fig. 4. DEAE-cellulose column chromatography of Step II material. A 2.5 X 20 cm column was esuiiibrated at 4°C with 5 mM sodium phosphate, pH 6.5, 5% Triton X-100. Approx. 650 mg (320 ml) of Step II material was applied to the column. The column was then treated with 80 ml of the equilibration buffer. The activity was eluted with an exponentiaf NaCl gradient formed from 300 ml equilibration buffer and 100 ml 0.25 M N&l in the equilibration buffer. The volume of the fractions was 7.0 ml. Hydrolase activity was measured with p-nitrophenylmyristate at 23% and triacylgfycerol lipase activity as (spec. act., 1.099 . described in Methods. Reaction mixtures contained 3 @mol tri[ * 4C]palmitoylglycero1 lo6 dpm/&unol), 25 mg T&ton X-100, 50 gmol sodium phosphate and enzyme in 1.0 ml total volume.
411
tion patterns of the triacylglycerol lipase and the p-nitrophenylmyristate hydrolase activities were identical and that the ratio of the two activities in each fraction was constant. Fractions 33-45 (Fig. 4) were pooled and designated Step III material. A 380-fold purification was achieved by this procedure. Step III material from another batch of cells was readsorbed to a DEAE column in 5% Triton X-100, 5 mM sodium phosphate, pH 6.5. The resin was then washed with 5 mM sodium phosphate to remove the detergent, and the activity was eluted with a linear NaCl gradient in a buffer containing 5 mM sodium phosphate, 5 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM dithiothreitol (Fig. 5). Under these conditions recovery was 30-45%, without EDTA and dithiothreitol recovery was less than 20%. Activity eluted from the first column at 0.15 M salt and from the second column at 0.3 M salt. Comments on the purification procedure. The procedures for purification from one 200 g wet weight batch of cells are presented in Table I. When Step IV material was subjected to gel electrophoresis at pH 7.5 in 7.5% polyacrylamide gels, one band of stainable protein was observed near the top of the gel with a small amount of material at the origin. A single protein-staining band was observed in analytical isoelectric focusing gels at a pH of 3.8. Step IV material chromatographed on a Sephadex G-200 eluted in the void volume (Fig. Sa). However, a 30 min exposure of Step IV material to 1% sodium dodecyl sulfate at room temperature resulted in a shift in the Sephadex G-200
1 N*CI 0.4
0.3 0.3
I-
NUYILR
FUACTION Fig.
5. Rechromatography
equilibrated (see and
Fig.
the
4 legend).
applied
1 mM glycerol
0.1~ml hydrolase
Methods. lo5
same
Step
Activity
material (20
column
on
(o-------c’
hydrolase
ml,
eluted
removed
for
), and activity
DEAE-cellulose.
X-100 40
was then
was then
were
activity
dpm//.unol).
III
phosphate/Triton The
aliquots
Triacylglyerol
Step
111 material
to the column.
dithiothreitol.
fractions
1.88
with
of
mg)
buffer was
treated by
the
a NaCl
A
used
diluted
with
40
to
ml
gradient
following with
0.9
X 15
80
formed
ml
with
sodium
hydrolase
column
of DEAE
the
first
DEAE
the
equilibration
phosphate,
in the latter
determinations: 3 mM
cm
equilibrate
5 mM
p-nitrophenylmyristate
was measured
to
protein
5 mM
buffer.
(A- - - - - -A)
tri[ 14Clpalmitoylglycerol
buffer EDTA,
From
(0 ---0).
was
column
5.5-ml triacyl-
as described (spec.
in act.,
I
II
III
IV
step
step
step
step
(ml)
Fraction
activity
19 000
18 700
9 500
1 800
204
31
70
L.
Total
380
Volume
tripalmitovl~lycrr~,1
with
FROM
3 520
with
1.7
25
68
20 600
19 000
(mz)
protein
Total
activity
368
M..
MYCOBACTERIL’M
500
_
M.
as substrate;
munits
ACTIVITY
L..
LlPASE
OF
I
PURIFICATION
TABLE
1.04 51.1 707
381
L.
Specific
activitv
p-nitrophen~lm~ristate
PffLEI
380 680
820 2 100
1 50
L.
152
iLI.
Fold
as substrate.
purification
4.5
48 13.2
95 6
100
L.
1
M.
Recoverv
(W)
6.1
37
100
M.
313
;I
A.
7 7
j 5
15\ - 1\\
20
mM PO,
0
d
0
10 3-
0
0 '0
0
,A,
‘O.o *0.,
1
20
10 M1 20_
0.
4
IO-15
5;
, 10
0.1 x SDS
6%
,:,
1
30
I
“\ O\O, .n 20
FRACTION
, 30
NUMBER
Fig. 6. Sephadex G-ZOO chromatography of Step IV material with and without exposure to sodium dodecyl sulfate. A 0.9 X 20 cm column of Sephadex G-200 was equilibrated with either 5 mM sodnun phosphate, pH 7.0 (A), or 50 mM sodium phosphate, 0.1% sodium dodecyl sulfate, pH 7.0 (B). Step IV material was incubated at room temperature with 0.1% sodium dodecyl sulfate (B) or without 0.1% sodium dodecyl sulfate (A) for 15 min prior to application to the column. Samples (0.2 ml) were chromatographed at a flow rate of 15 ml/cm2 per h. The volume of each fraction was 0.5 ml. Markers used to calibrate the columns were: Ml, Blue Dextran 2000; M2, bovine serum albumin; MJ. trypsin; Ma, cytochrome c.
column elution pattern of the activity. The new position of the activity was between bovine serum albumin and cytochrome c, corresponding to a molecular weight of approx. 40 000 (Fig. 6b). Step IV material separated into six protein bands when treated with 1% sodium dodecyl sulfate and electrophoresed in 7.5% polyacrylamide gels containing 0.1% sodium dodecyl sulfate (Fig. 7). These six bands correspond to the following molecular weights: (1) 69 000, (2) 60 000, (3) 50 000, (4) 42 000, (5) 27 000, (6) 22 000. The optimal assay temperature for triacylglycerol hydrolase activity was 37°C. Activity is stable at 4 or -17°C for several weeks. A 50% loss of activity was observed when Step IV material was exposed to 50°C for 10 min, and 90% was lost following a 10 min treatment at 70°C. Extraction of hydrolase activity from sodium dodecyl sulfate gels. As indicated above, Step IV material was resolved into six protein-staining bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Approx. 50% of the triacylglycerol hydrolase and the p-nitrophenylmyristate hydrolase applied to the gel was stable to sodium dodecyl sulfate and was recovered from the 40 000 molecular weight region of the gel (Fig. 7). The extracted activity was
0
01
0.2
03
04
0.5
0.6
0.7
06
09
10
Hf
Fig. 7. Localization and extraction of hydrolase activity after sodium dodecyl sulfate-acrylamidc gel electrophoresis of Step IV material. Lyophilized Step IV material was dissolved in 1.0% sodurn dodccyl sulfate, 1 mM dithiothrcitol. 1 mM EDTA, and 5 % sucrose. Approx. 80 pg of this material was apphed to the top of each gel and electrophorescd at 5 mA per gel as described in Methods. One of the gels was frwen, sliced, and extracted: the other was stained for protein. At the end of the extraction period. fractions were assayed for triacylglycerol (1 u ) and p-nitrophenylmyristate (*- - - - - -0) hydrolase activitv. The specific activity of the tri[ I 4Clpalmitoylglycero1 was 5.82 10 s dpm/pmol.
designated gel elution material. Because of the dilution factor in the extraction, this preparation contained less than 0.02% sodium dodecyl sulfate, and reaction mixtures contained less than 0.002%. We determined that 0.02% sodium dodecyl sulfate in reaction mixtures had no effect on the activity. In 100 mM acetate or phosphate buffers the pH optimum of the gel elution material is broad with a maximum at 5.8 (Table II). In 100 mM sodium maleate buffer the pH optimum was 6.3. Influence of organic anions on hydrolase activity. A stimulatory effect of organic anions was first noted as a discrepancy in overlapping points with two buffers in a pH profile study with crude (Step I) enzyme preparations. Activity in a citrate/phosphate buffer was 50% greater than in phosphate buffer at the same pH and ionic strength. Isocitrate, a-ketoglutarate, succinate, acetate, and formate also stimulated triacylglycerol lipase activity (Table III). The effect was not due to increased ionic strength since neither KC1 nor NaCl at concentrations up to 0.5 M had any effect on the activity. Inorganic anions such as phosphate or Cl- also had no effect. Sodium citrate stimulated gel elution material as well as the crude enzyme preparations. The saturation kinetics of gel elution material with tripalmitoylglycerol as substrate are presented in Fig. 8a. Sodium citrate at 100 and 200 mM concentrations not only stimulated activity but also altered the shape of the activity to substrate concentration curve (Fig. Sa). From double reciprocal plots of this data (Fig. Sb), it can be seen that in the absence of citrate the curve is not linear, whereas in the presence of citrate the curve is linear. Linear regression analysis of this data indicates a KTaDp of 8.9 ? 4 mM and a V of 13 860 nmol/min per mg protein at 100 mM citrate. At 200 mM citrate the Kmwp was
415
TABLE
II
EFFECT
OF
PH
AND
BUFFER
ON
TRIACYLGLYCEROL
HYDROLASE
ACTIVITY
OF
STEP
V
MATERIAL The 7.5
specific mM
activity
of
the
tri[ I 4ClpalmitoylgIycerol
was
5.68
. 10
5 pmol.
Reaction
mixtures
contained
tripalmitoylglycerol.
Buffer
PH
Specific
activity
(munits/mg
protein)
I Acetate
Sodium
phosphate
4.5
994
5.0
1491
5.3
2556
5.6
1988
5.8
3748
6.0
3497
6.1
3050
6.2
2627
6.3
2343
6.5
2340
6.8
2343
7.0
1775
7.5
1065
8.9
355
II Sodium
TABLE
The
5.4
489
5.6
2248
5.8
2619
6.0
2816
6.1
2816
6.2
3012
6.4
3013
6.6
3012
6.8
2658
7.0
2366
III
EFFECTS CRUDE
maleate
OF
ORGANIC
SONICATES
control
Compound
activity
ANIONS
OF for
ON
THE
MYCOBACTERIUM
this
preparation
Concentration
was
TRIACYLGLYCEROL
PHLEI 5.8
munitslmg
Control
(mM) Citrate
Iaocitrate
50
140
100
178
200
250
500
110
50
145 169
a-Ketoglutarate
100
Succinate
100
142
Acetate
100
156
Formate
100
112
protein
(%)
HYDROLASE
ACTIVITY
OF
b 2s -
20 -
1
lf-
V 10 -
5-
TRIPALMITIN
‘4
41 05
fmlUt
.._---_ )
8
C
i 0
e0.5
/ 110
I ‘1.5
LnlSl Fig. 8. Effect of sodium citrate on triacylglycerol hydrolase activity of Step IV material at different substrate concentrations. Reaction mixtures contained 20 pmol sodium maleate buffer, PH 6.5, 5 mg Triton (5.65. 10s dpm/Mmol), and 0 (*-----+), 20 X-300. 0.11 jog Step IV protein, tri[ 14CJpaimitoylglycero1 ) ymol sodium citrate in a total volume of 0.2 ml. (a) Activity versus subor 50 (“----o (A -------A), strate
plot of the data; (b) Lineweaver-Burk
plot of the same data, and (c) Hill plot of the data.
2.8 L 4 mM and the V was 11 730 nmol/min per mg protein. The Hill number in the absence of citrate was 2.2 (Fig. 8c), while in the presence of citrate, Hill numbers of approx. 1 were obtained. Sodium citrate at concentrations of up to 200 mM had no effect of the ~-nitrophe~ylmyristate hydrolase activity (Table IV).
Actiuity of gel elution different lengths. Activity
material
with p-nitrophenyl
esters of fatty acids of
at equimolar concentrations of p-nitrophenyl esters of fatty acids increased with the length of the fatty acyl group up to C4 (Table V). 4 times more activity was observed with p-nitrophenylmyristate as substrate than with p-nitrophenylacetate as substrate.
417 TABLE
IV
THE EFFECT
OF SODIUM
CITRATE
HYDROLASE
ACTIVITIES
OF STEP IV MATERIAL
ON TRIACYLGLYCEROL
AND p-NITROPHENYLMYRISTATE
(spec. act. = 5.68 . Triaeylglycerol hydrolase activity was assayed with 7.5 mM tril 14C]palmitoylglycero1 lo5 dpm/timol. 25 mg/ml Triton X-100, 0.11 l.(g Step IV protein and the indicated buffer, pH 6.5. in 0.2 ml reaction mixture at 37’C for 20 min. 50 mM sodium maleate buffer, pH 6.5, was included in all reaction mixtures. p-Nitrophenylmyristate hydrolase activity was assayed by standard procedures as described in Methods with a reaction mixture containing 7.5 mM substrate, 25 mg/ml Triton X-100, and 50 mM sodium phosphate buffer. pH 7.0,0.092 pg Step IV protein a 0.4 ml reaction mixture at 23OC. Addition
Concentration
Hydrolase triacylgiyceroi (munitsimg)
Activity p-nitrophenylmyristate (munitslmg)
50
1450
-
50 25 50 100 150 200
2460 2555 3154 4670 4732
5400 5400 5800 5700 5900 6200
(mM)
Sodium
maleate
Sodium Sodium
phosphate citrate
TABLE
V
ACYL HYDROLASE SATURATED FATTY
ACTIVITY OF STEP ACIDS (Cz-c, *)
Reaction mixtures contained Triton X-100, and 3.0 pmol tion of enzyme. ,,-Nitrophenyl
Acetic Propionic Butyric Valetic Caprnie Capric Laurie Myrktic Palmitic Stearic
ester
IV
MATERIAL
USING
p-NITROPHENYL
ESTERS
OF
0.15 @g Step IV protein, 40 pmol sodium phosphate buffer, pH 7.0, 10 mg substrate in a total volume of 0.4 ml. The reaction was started by the addi-
Carbon
2 3 4 5 6 IO 12 14 17 18
No.
Specific activity (munitsfmg protein) 725 752 1654 1816 IF54 2444 2756 2882 2756 2506
Discussion Our results demonstrate that M. phlei produces a triacylglycerol lipase activity. We believe this enzyme to be intracellular in nature and associated with the cell wall-membrane complex since (a) activity was not found in the culture supernatant liquid, (b) activity in sonicated preparations is an aggregate of more than 2 + lo6 daltons, and (c) treatment of the sonicated preparation with the non-ionic detergent Triton X-100 results in a reduction of the size of the activity. In addition, activity can be measured with whole cells only in the presence of detergent. While it is not possible to determine from this data whether the activity is on the inside or outside of the cell w~l-membrane com-
418
plex, it does suggest that the lipase is associated with that structure. A high decree of purification (680-fold) was achieved by taking advantage of the stability of the activity to treatment with non-ionic detergents as well as the ability of the detergent to selectively solubilize the enzyme. Whole cells treated with 5% Triton X-100 at 37°C are not lysed and only l---2% of the total cellular protein is released from the cells. The majority of the lipase activity is found in the supematant liquid following treatment at 37”C, while at 0°C the activity remains associated with the cells. The solubilized activity, which is already 50-60-fold purified, is completely stable in the extraction buffer for extended periods of time at either 4 or -17” C. Further purification (another 6-fold) was achieved by chromatography of the extracted material on DEAE-cellulose in the presence of detergent (Fig. 4). Wickner and Kennedy [ 201 employed a similar procedure in their purification of phosphotidylserine decarboxylase. A second DEAE-cellulose column (Fig. 5) served two functions: removal of the detergent and further purification. Since radioactive Triton X-100 was not available we could not be absolutely certain that all of the detergent was removed. We were certain, however, that the detergent concentration was reduced at least 500-fold by following the decrease in absorbance at 295 nm. Activity recovered from this column (Step IV material) appeared nearly pure by two criteria: polyacrylamide gel electrophoresis at pH 7.5 (there is some material at the origin) and analytical isoelectric focusing in acrylamide gels. This preparation of the lipase was, however, an aggregate which could be resolved into six protein bands when treated with 1% sodium dodecyl sulfate and then electrophoresed in polyacrylamide gels. Scandella and Kornberg [ 211 effectively utilized preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis in their purification of active phospholipase A, from Escherichia coli. We found that activity could be extracted from the 40 000 molecular weight region of sodium dodecyl sulfate-polyacrylamide gels with a 30-50% recovery (Fig. 7). Chromatography of sodium dodecyl sulfatetreated material on Sephadex G-200 indicates a molecular weight for the active component between 30 000 and 50 000. M. phlei lipase therefore has a molecular weight of approx. 40 000 and is found as an aggregate with five proteins. Whether the association of the activity with these proteins is formed de novo (i.e. self aggregation) or is an artifact resulting from the purification procedure is not known. Characterization of the activity. During the purification of M. phlei lipase, activity was monitored with two substrates, tripalmitoylglycerol and p-nitrophenylmyristate. Care must be taken when p-nitrophenyl esters are used to define lipase activity since it is known that e&erases as well as lipases will hydrolyze synthetic esters [ 221. The validity of using synthetic non-glycerol esters for the study of a highly purified lipase preparation was established by Brockerhoff [23] in his studies of substrate specificity of pancreatic lipase. During this study we determined that the ratio of triacylglycerol to p-nitrophenylmyristate hydrolase activities is constant for DE II material, isoelectric focused material, for the activity extracted from sodium dodecyl sulfateacrylamide gels and for DE II material chromatographed on Sephadex G-200. The most highly purified material produced in this study (gel elution material) exhibited a specificity for long chain fatty acids when tested with p-nitro-
419
phenyl esters of saturated fatty acids containing 2-18 carbons (Table V). The lowest activity was found with acetate and the highest activity with myristate. The high rate of hydrolysis of tripalmitoylglycerol and the specificity of the enzyme for long chain fatty acid esters support the identification of the activity described here as triacylglycerol lipase (EC 3.1.1.3). An interpretation of the kinetics of enzymes which utilize insoluble substrates is complicated by the kinetic effects of different phase structures of the substrate. The relationship of M. phlei lipase activity to tripalmitoylglycerol concentration was sigmoid with non-linear double reciprocal plots (Figs. 8a and Sb). Sodium citrate and other organic anions stimulated triacylglycerol hydrolase activity and also altered the relationship of activity to substrate concentration such that the curve became nearly hyperbolic in shape (Fig. 8a). Double reciprocal plots of this data now become linear with the Hill number close to 1; the Hill number was 2.2 in the absence of citrate. It is important to note that sodium citrate had no effect on the activity of the enzyme with p-nitrophenylmyristate as substrate (Table IV). It has been shown that pancreatic lipase works at an oil/ water interface and that the apparent V is equal to the maximum concentration of the enzyme at the phase surface multiplied by the rate constant (apparent V = E X surface area X K) 1241. The authors conclude that any material that can alter the nature of the oil/water interface can markedly influence the rate of triacylglycerol hydrolysis or the diffusion of the
2
4 SUBSTRATE
6
8
(mM)
Fig. 9. Effect of Triton X-100 on triacylglycrrol and p-nitrophenylmyristate hydrolase activity of Step IV ‘) or 5 (.-4) mg x ), 3.2 (‘1 material. (A) Triacylglycerol hydrolase activitv with 1.6 (XTriton X-100 in a 0.2 ml reaction mixture which also contained 0.09 pg Step IV protein, 20 ~mol sodium phosphate. pH 6.5. and tri[ 14Clpalmitin (spec. act. 5.69 lo5 dpm/ ~mol). (b) p-Nitrophenylmyristate or 5 (O0) mg Triton X-100 in a 0.4 ml X), 2.5 (‘J -----). hydrolasc activity with 1.25 (Xreaction mixture which also contained 0.11 fig Step IV protein. 40 /on01 sodium phosphate. pH 7.0, and /,-nitrophenylmyristatc.
420
product from the oil/water interface into the bulk phase. The effect that we see is somewhat specific in that inorganic anions (Cl-, phosphate) and inorganic cations (Na+, K’) do not mimic the effect of the organic anions. It appears that the stimulatory effect is dependent not on the specific ion but rather on the concentration of carboxyl groups added to the reaction mixture. While we cannot rule out a direct effect on the enzyme it is our feeling that the organic anions are somehow altering the oil/water interface or micelle structure of the substrate, and thereby altering the activity. Triton X-100 concentrations also affect the velocity to substrate relationship of this enzyme (Fig. 9). Dennis [ 251 reported that phospholipase A2 requires Triton X-100 or some other detergent for activity toward egg phosphatidylcholine, but that inhibition occurs at high concentrations of Triton X-100. Similar effects were observed by Kaplan and Teng [18] for beef liver lipase and by Kariya and Kaplan [ 151 for rat liver lysosomal lipase. Changing the concentration of Triton may alter the structure of the triacylglycerol/Triton micelle. A 2 : 1 ratio of Triton X-100 to phospholipid substrate is required for maximal phospholipase AZ activity [ 251. A molar ratio of 6 : 1 (Triton X-100 : triacylglycerol) is required for optimal M. phlei lipase activity. Metabolic role of M. phlci lipase. The specific activity of M. phlci lipase increases at least 3-fold late in stationary phase of growth (Fig. 1). This is consistent with the preferential utilization of glycogen and subsequent utilization of the triacylglycerol fatty acids as a source of endogenous energy. Although the lipase activity increases several fold late in the growth of M. phlei, there is activity present at minimal levels in early stages of growth. Triacylglyccrol pools at this time turn over rapidly. It appears likely that the enzyme described in this study is responsible for both the turnover of the triacylglycerols during early growth and for the breakdown of the triacylglycerol pools late in growth. Acknowledgement This General
work was supported by Public Health Research Support Grant 24-57-819.
Service
Grant
AM 13516
and by
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