ARCHIVES

Vol.

OF BIOCHEMISTRY

287, No. 1, May

AND

BIOPHYSICS

15, pp. 167-174.1991

Leukotriene A, Hydrolase in the Human B-Lymphocytic Cell Line Raji; Indications of Catalytically Divergent Forms of the Enzyme Bjijrn Hans

Odlander,’ Hans-Erik Claesson, Tomas Bergman, Olof RBdmark, JGrnvall, and Jesper Z. Haeggstrijm Department of Physiological Chemistry, Karolinska Institutet, S-10401, Stockholm, Sweden

Received

October

1, 1990, and in revised

form

January

15, 1991

Leukotriene A4 hydrolase was purified 1400-fold, with an approximate yield of 25%, to apparent homogeneity from the human B-lymphocytic cell line Ruji. The purification included ammonium sulfate precipitations followed by anion exchange, hydrophobic interaction, and molecular exclusion fast protein liquid chromatography. Kinetic properties at 2°C varied between different enzyme preparations. Two patterns were observed, one with a K, of about 12 FM and V,,,,, of about 1.1 pmol LTB,/mg protein/min which correlated well with the properties of the human leukocytic LTA, hydrolase. In other enzyme preparations a higher catalytic activity was observed. These enzyme batches did not obey MichaelisMenten kinetics but were compatible with a mixture of enzymatic species. Heat treatment (60°C) led to a timedependent decline in catalytic activity. However, certain enzyme preparations contained a subfraction of enzymatic activity which was more resistant to heat treatment, yielding a biphasic inactivation pattern. It is thus suggested, on the basis of the kinetic properties and the heat-inactivation pattern, that these enzyme preparations contained an additional form of LTA, hydrolase. C 1991

Academic

Press,

Inc.

stable allylic epoxide LTA4’ (1). Lymphocytes apparently lack 5-lipoxygenase and thereby the ability to form LTAl (2-5). However, these cells possessLTAI hydrolase and thus the enzymatic capacity to metabolize LTA4 into LTB, (4, 5). LTB4 is a potent proinflammatory compound (1) which also augments several B-lymphocytic cell functions, such as B-cell activation, DNA synthesis, cell growth, and immunoglobulin production (6). It also induces natural killer cell activity and suppressor cell function (7) and affects the formation and biological activities of several cytokines (B-10). Thus, by influencing the levels of LTB4, lymphocytes can act autoregulatory. In a previous study, we found that the B-lymphocytic monoclonal cell line Ruji possesseda lo-fold higher capacity to convert LTA, into LTB4 than normal lymphocytic cells (4). In order to investigate the basis for the altered catalytic behavior, LTA4 hydrolase was purified and characterized from Raji, the cell which expressed the highest enzymatic activity among the investigated cells. It is suggested that the expression of the LTAl hydrolase is enhanced in Raji. Furthermore, kinetic data are presented which indicate the presence of catalytically diverging forms of the enzyme. EXPERIMENTAL

PROCEDURES

Cell Culture Leukotrienes are compounds with profound biological effects, which are formed from arachidonic acid (1). The formation of these compounds includes 5-lipoxygenase catalyzed introduction of molecular oxygen into the fatty acid and intramolecular rearrangements yielding the un-

’ To whom

0003.9861/91

Copyright All

rights

correspondence

should

$3.00 0 1991 by Academic Press, of reproduction in any form

be addressed.

Ruji (11) is a Burkitt lymphoma derived B-cell line. Ruji cells were cultured in spinner bottles at 37°C in an atmosphere of 5% CO,. Culture medium was RPM1 1640, supplemented with 50 IU/ml penicillin, 50

’ Abbreviations used: LTA,, leukotriene & [5(S)-trans-5,6-oxide-7,9trans-11,14-cis-eicosatetraenoic acid; LTB,, 5(S),lZ(R)-dihydroxy-6,14cis-8,10-trans-eicosatetraenoic acid, FPLC, fast protein liquid matography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide electrophoresis.

chrogel

167 Inc. reserved.

168

ODLANDER

pg/ml streptomycin, and 4% fetal calf serum (final concentrations). After harvesting, cells were washed in phosphate-buffered saline (PBS, Dulbecco’s formula, pH 7.4), and resuspended in PBS.

Enzyme Purification The purifications

were carried

ET

AL.

Protein Assay The method as standard.

of Bradford

(15) was utilized

with bovine

serum

albumin

Electrophoresis out at 2-5°C.

Step 1.

Haji cells (2-4 X lo9 cells), suspended in PBS at a concentration of 40-60 X lo6 cells/ml, were homogenized by sonication on ice (5 X 10 s, power-setting 4, Branson Sonifier S-125). Subcellular localization of the LTA, hydrolase was determined by high-speed centrifugation (105,OOOg). Step 2. The 10,OOOg supernatants (20 min) of Raji cell homogenates were collected and streptomycin (0.5% (w/v)) and ammonium sulfate precipitations were carried out. The 40.-80% ammonium sulfate precipitate was collected by centrifugation (15 min, 10,OOOg). The pellet was resuspended in 20 mM Tris-Cl, (pH 8.0) and salts were removed with a PD.10 molecular exclusion column (Pharmacia AB, Sweden) equilibrated with this buffer. Step 3. Further purification was performed by means of fast protein liquid chromatography (FPLC) utilizing a Mono-Q HR lO/lO anionexchange column (Pharmacia AB, Sweden). The column was equilibrated with 20 mM Tris-Cl (pH 8.0). After elution of nonadsorbing proteins, a linear gradient over 180 ml with increasing KC1 concentration up to 0.25 M, was started. LTA, hydrolase activity was recovered between 55 and 80 mM of KCl.

Step 4.

Active fractions from the anion-exchange chromatography were pooled and ammonium sulfate was added to a concentration of 1.7 M. The sample was then applied to a hydrophobic interaction FPLC column (alkyl-Superose 5/5, Pharmacia AB), from which enzyme activity was recovered at approximately 1.2 M (NH&SO,. Active fractions were pooled and concentrated to a volume less than 200 ~1 by ultrafiltration (5OOOg) in a Centricon microconcentrator (cut-off at M, 10,000). For activity determinations or gel electrophoresis, aliquots were diluted 2550 times with 20 mM Tris-Cl, pH 8.

Step 5. The final step of the purification was molecular exclusion chromatography, performed on FPLC with a Superose-12 column eluted with 50 mM Tris-Cl buffer supplemented with 75 mM KCl, pH 8.0. Enzyme activity was recovered at a retention volume corresponding to an apparent M, of 51,000. Fractions containing apparently homogenous protein were pooled. Stoke’s radius was determined by calculation of the effective gel pore radius of the Superose-12 column by the use of standards with known Stokes radius (12).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed on a PHAST system (Pharmacia AB, Sweden) utilizing lo-15% gradient gels. The gels were stained with Coomassie brilliant blue. Isoelectric focusing was also performed on the PHAST system utilizing gels with pH gradients between 4.0-6.5.

Amino Acid Analysis Samples were hydrolyzed with 6 M HC1/0.5% phenol (w/v) in evacuated tubes at 110°C for 24 h and analyzed on a Beckman 121 M instrument.

N-Terminal Sequence Analysis Gas-phase sequencer analysis of purified enzyme was performed on an Applied Biosystems 470A instrument. Phenylthiohydantoin derivatives were analyzed by RP-HPLC as described (16).

RESULTS

Enzyme Purification Leukotriene A, hydrolase activity was recovered in the 105,OOOgsupernatant of homogenized Raji cells (data not shown). The 10,OOOgsupernatants of homogenized Raji cells were used for further purification, which included streptomycin and ammonium sulfate precipitations, followed by FPLC utilizing anion exchange, hydrophobic interaction, and, finally, molecular exclusion columns. By this procedure, the enzyme was purified more than 1400-

Lanes

1

2

3

4

5

6

7

Assay of Enzyme Activity LTA, methyl ester, kindly provided by Dr. A. W. Ford-Hutchinson, (Merck-Frosst, Canada), was saponified as described (13). Specific activities were determined at the different steps of purification by incubation of aliquots (50-100 ~1) of the enzyme preparation at 20°C with LTA, (45 FM) for 1 min. The enzymatic reaction was terminated by the addition of l-2 vol methanol (quenching was equally efficient at either concentration). Analysis of the enzymatic product LTB, was performed by reverse-phase (RP) high-performance liquid chromatography (HPLC) essentially as described (14). In kinetic experiments, short-time incubations (10 s) were performed on ice with the enzyme dissolved in 20 mM Tris-Cl, pH 8.0. LTA, was dissolved in tetrahydrofuran or methanol (final solvent concentrations < 2%). Incubations were terminated by the addition of l-2 vol of methanol and analyzed on RP-HPLC. Heat treatment was carried out in a water bath at 60°C. Purified LTA, hydrolase was kept in a sealed vial from which aliquots (50-100 ~1) were removed after indicated periods of time and immediately placed on ice. Assay of the enzymatic activity was performed (10-s incubations on ice) using an LTAl concentration of 50 pM.

FIG. 1. SDS-polyacrylamide gel electrophoresis at various stages of purification of Raji LTA, hydrolase. Molecular mass markers (Lanes 1 and 7) were phosphorylase b (M, = 94 X 103), bovine serum albumin (n/r, a 45 X 1O3), carbonic anhydrase (MI M = 68 X 103), ovalbumin z 30 X 103), soybean trypsin inhibitor (M, z 22 X lo”), and tu-lactalof Raji bumin (M, = 14 X 10”). Lane 2 shows the 10,OOOg supernatant cells; lane 3 after precipitations; lane 4, after anion-exchange chromatography; lane 5, after hydrophobic interaction chromatography; lane 6, final step, after molecular exclusion chromatography.

LEUKOTRIENE

A,

HYDROLASE

IN

RAJI

169

CELLS

4.15 4.55

5.20

5.85

6.50 inhibitor Isoelectric focusing of Raji LTA, hydrolase. (A) Lanes 1 and 4 are pl markers; glucose oxidase (~1 = 4.15), soybean trypsin anhydrase B (pl r_ 6.50). Lane 2 shows Raji &lactoglobulin (pl r 5.2()), bovine carbonic anhydrase B (~1 = 5.85), human carbonic LTA, hydrolase and lane 3 shows dithiothreitol-treated enzyme. (B) Determination of pl by plotting data from (A). The mobility of the Raji enzyme is indicated hy arrow. FIG.

2.

(PI ^1 4.55),

fold to apparent homogeneity (Fig. l), with a yield of approximately 25% (Table I). Enzyme

Kinetics

Time course of catalysis. The time course for enzymatic conversion of LTA4 (22 PM) to LTB4, by Raji LTA, hydrolase at O”C, is shown in Fig. 3. The amounts of product increased linearly with time during approximately 20 s, then gradually leveled off, and lasted totally for about 40 s. Catalysis ceased while substrate still was available, as indicated by the continuous rise in the levels of the

TABLE Purification

Fraction 10,000g supernatant Precipitations Mono-Q” HICb Superose-12’

of LTA,

0.25 0.64 17.4 12X.4 354.8

I

Hydrolase

Specific activity (nmol LTB,/mg protein/min)

k 0.08 i 0.09 k 6.7 + 47 + 69

nonenzymatic breakdown products of LTA4. A second addition of LTA, did not restore enzymatic activity. Apparent kinetic constants. Short-time incubations (10 s) of purified enzyme on ice were performed in order to determine apparent kinetic constants. The concentra-

from

Ruji Cells

Yield (S)

72 58 35 26

100 + + k t

Purification (fold)

15 18 17 15

Note. Data are presented as mean + SD (n = 4-6). ” FPLC, anion-exchange chromatography. ’ FPLC, hydrophobic interaction chromatography. ’ FPLC, molecular exclusion chromatography.

2.6 69.6 494 1419

n

5 4 6 4 6

Time

(min)

FIG. 3. Time course for the formation of LTB, by Raji LTA, and the corresponding formation of nonenzymatic hydrolysis of LTA, at 0°C. LTA, (22 pM) was added to purified Raji LTA, in 20 mM Tris-Cl (pH 8.0) at time zero. After 8.5 min, a second of LTA, was made (indicated by the arrow). The formation (closed circles) and its nonenzymatically formed isomers, i.e., LTB, and 12-epi-~6-trans-LTB, (open circles) were analyzed HPLC.

hydrolase products hydrolase addition of LTB, ~6-trarzsby RP-

170

ODLANDER

tion of LTA4 ranged between 1.6 and 120 PM. Results obtained in 10-s incubations were extrapolated to 1 min. In three out of five enzyme batches (type A), the catalysis obeyed Michaelis-Menten kinetics, as shown in Fig. 4A. However, certain preparations of Raji LTA, hydrolase showed an altered kinetic behavior (Fig. 4B) with increased specific activity, especially at low substrate concentrations (type B). Data were plotted according to Eadie and Hofstee (17, 18), and apparent kinetic constants were determined (Table III). V,,,,, was found to be 1095 nmol/ mg/min k 199 and K,,, was 12.3 PM -t 3.7 (mean + SD of three experiments, each in triplicate) in the preparations of type A (Fig. 4C). The turnover number at 0°C was

ET

AL.

found to be 75 min-l. In Eadie-Hofstee plots of data from the preparations of B-type, linear correlation was poor. Thus, the enzymatic activity in type B batches did not obey Michaelis-Menten kinetics. However, the calculated values of the apparent kinetic constants (Km, I’,,,) are still reported in order to illustrate the difference between preparations of types A and B. Thus, in two preparations of B twe, V,,, were 1976 (mean of triplicate incubations) and 2215 (single incubations) nmol/mg/min, respectively, and K,,, was 2.16 and 2.77 PM, respectively. In these preparations the turnover number was approx 138 mini’. The specific activity was also determined from 1 min incubations at 20°C (45 PM LTAI). In accordance with

A C

2500-

2500

1 1

0 E : > 04 0

I

I

,

50

100

150

[LTA4]

pM V/IS]

(nmol

LTB4lmglminlFM

LTA4)

B D 2500,

2500,

2000

1500

l 0

I

, 100

50

[LTA4]

J.LM

150

01 0

100

V/[S]

200

(nmol

LTB4lmglminlpM

400

300

LTA4)

FIG. 4. Saturation kinetics of Raj; LTA, hydrolase. Dependency of initial catalytic velocity on substrate concentration in an A-type enzyme preparation (A) and a B-type (B). Aliquots of purified enzyme (A = 19 fig protein/ml, 50-J aliquots; B = 2.4 fig/ml, loo-p1 aliquots) in 20 mM Tris-Cl (pH 8.0) were incubated with various concentrations of LTAl for 10 s at 0°C. Data points are mean of triplicate incubations. Incubations were terminated with l-2 vol of methanol and LTBl was quantitated by RP-HPLC. (C) and (D) show Eadie-Hofstee plots of the data in (A) and (B), respectively.

LEUKOTRIENE

A,

HYDROLASE

IN

the time course of catalysis (Fig. 3), the specific activity under these conditions was significantly lower as compared to the calculated V,,,, and was found to be 355 nmol/mg/min + 69 (mean + SD, n = 6). This corresponds to 25 catalytic cycles/enzyme molecule before enzymatic activity was lost. Treatment of enzyme with dithiothreitol (2 mM, 30 min) did not significantly alter enzyme activity. Heat inactiuation. In order to further elucidate the discrepancies in catalysis, enzyme preparations were subjected to heat treatment. Figure 5A depicts data obtained from the enzyme preparation shown in Fig. 4A, possessing type A kinetics. A linear correlation between the natural logarithm of the specific activity (In V) and time of heat treatment was found, which was an inactivation pattern seen in all experiments on enzyme batches of type A. However, in two preparations of Raji LTA, hydrolase (both exhibiting one single band at M, 68,000 on SDSPAGE) a different inactivation pattern was found. The data shown in Fig. 5B were obtained by heat treatment of the B-type batch presented in Fig. 4B. The linear inactivation pattern found in type A batches is present, but also a more temperature insensitive enzymatic activity. Structural

Acid Composition

171

CELLS

A

I,

0

10I

Time

.

of

2b

3;

heat-treatment

4b

5b

(min)

8

6

Properties

The M, of the enzyme in batches of both type A and B was determined by SDS-PAGE to be 68,000. Molecular exclusion chromatography indicated an M, of 51,000. This discrepancy presumably reflects interactions between the gel filtration resin and LTA, hydrolase which led to increased retention. The value obtained by SDS-PAGE is the most accurate, since it is in accordance with the molecular weight deduced from molecular cloning of human cDNA (19, 20). Stokes radius was determined for Ruji LTA, hydrolase and was found to be 3.1 nm and isoelectric focusing of purified LTA4 hydrolase from Raji revealed a pl of 5.0 (Fig. 2), which was not altered after treatment with dithiothreitol (2 mM, 30 min). No differences were observed between enzyme preparations of types A and B regarding these structural parameters. Neither were any particular dissimilarities between different enzyme preparations observed during the purification procedure. Amino

RAJI

and N-Terminal

Sequence

Purified enzyme was hydrolyzed with HCl and the amino acid composition was determined. Results are given in Table IIA, with the composition of LTAI hydrolase from human leukocytes shown for comparison. Two batches of B type and one of A type were analyzed without revealing any significant differences, and the results are given as the mean of these determinations. The N-terminal amino acid sequence (21 residues) of a type A preparation is given in Table IIB, revealing a free N-terminus. This sequence is identical to the one reported for LTA, hydrolase cloned from human cDNA libraries (19, 20).

z 5 c

4

311 0

I IO

Time

I 20

of

1 30

heat-treatment

I 40

I 50

(min)

FIG. 5. Heat treatment of purified Raji LTA, hydrolase. LTA, hydrolase was kept at 60°C in a sealed vial from which aliguots were removed at indicated periods of time and immediately placed on ice. Enzymatic activity was assayed in 10-s incubations on ice, using an LTA, concentration of 50 pM. Data points represent mean of duplicate incubations. The experiments shown in (A) and (B) were performed with the enzyme preparations for which saturation kinetics are shown in Figs. 4A and 4B, respectively.

The first 10 residues could be determined in a preparation of B-type without any sequence deviation. LTA, Hydrolase Content in Raji Cells The total LTBl forming capacity in 10,OOOgsupernatants of homogenized Raji cells was 27.9 + 8.0 nmol LTBJ 10’ cells/min (mean f SD, n = 6). Calculations based on the specific activity (cf. Table I) and molecular weight, indicated a content of 1.2 nmol LTA, hydrolase/lOg Raji cells. The same calculations for human leukocytes based on data from Ref. (21) indicated a content of 1.3 nmol

172

ODLANDER TABLE Structural

Analysis

Residue A. Amino

of

II

Raji LTAl

Raji Acid

Composition

Human of LTA,

ASX Thr Ser GlX Pro

QY Ala Val Met Ile Leu Tyr Phe ‘I’~P Lys His A%

Hydrolase

Amino

Acid

20

from

Raji”

nd 9.8 7.0 8.2 10.1 6.3 5.5 6.7 7.0 1.6 3.9 11.8 3.4 4.8 nd 7.6 3.0 3.2 Sequence

of Raji

1 2 3 4 5 678 9 10 Pro-Glu-Ile-Val~Asp-Thr-X-Ser-Leu-Ala-Ser-Pro-Ala-Ser-Val 16 17 18 19 -X-Arg-Thr-Lys-HisLeu

leukocytes

nd 9.3 6.8 8.0 11.5 5.6 6.4 6.9 5.7 1.5 5.5 11.1 3.5 4.4 nd 6.7 2.8 4.3

CYS

B. N-Terminal

Hydrolase

11

LTA, 12

Hydrolase 13

14

15

21

’ Values are expressed as mole 5% disregarding concerning Roji are given as mean of determinations preparations. The composition of LTA, hydrolase eral leukocytes (21) is shown for comparison.

Cys and Trp. from three from human

LTA4 hydrolase/lOg cells, i.e., approximately amount of enzyme.

Results enzyme periph-

the same

DISCUSSION

Leukotriene A4 hydrolase was purified to apparent homogeneity from the monoclonal B-lymphocytic cell line Ruji, using precipitations with ammonium sulfate followed by FPLC utilizing anion-exchange, hydrophobic interaction, and molecular exclusion techniques. This protocol was rapid and reproducible. Calculations based on the mobility during molecular exclusion chromatography of Raji LTA, hydrolase indicated an M, of approximately 51,000. However, analysis on SDS-PAGE showed an M, of approximately 68,000. This discrepancy presumingly reflects a nonglobular behaviour of the enzyme during gel filtration, a property also observed in previous studies on the enzyme (13, 21, 22, 24). Calculations of the cellular content of LTA4 hydrolase show that Ruji and human peripheral leukocytes (21) contain approximately the same number of enzyme mol-

ET

AL.

ecules. This is in agreement with our previous findings, using Northern and Western blot analysis (5). The low LTA, hydrolase activity observed in normal peripheral lymphocytes (4) appears to be due to low expression of the enzyme, a hypothesis which is supported by immunohistochemical studies showing only few splenic lymphocytes staining for LTA4 hydrolase (25). Accordingly, this would suggest that the expression of LTA, hydrolase is enhanced in activated/transformed B-lymphocytes. The time course for LTB, formation at 0°C by Ruji LTA4 hydrolase is shown in Fig. 3. After a rapid burst of product formation, the enzymatic activity gradually ceased while substrate still was available and a second addition of substrate could not restore enzyme activity. These observations show that Ruji LTA, hydrolase, like enzyme from other sources (13, 21, 23, 24, 26), is subject to inactivation during catalysis. Although the details are still unclear, the inactivation process was shown to be associated with the catalytic mechanism of LTB4 formation by LTAj hydrolase in intact human erythrocytes (27). It was recently reported that LTA, hydrolase also possessesa peptidase activity, but in contrast to the epoxide hydrolase activity of LTA, hydrolase, this activity of the enzyme does not lead to inactivation (28, 29). Kinetic properties of Raji LTA, hydrolase were studied during short-time incubations on ice. This experimental setup was chosen to minimize the influence of spontaneous decomposition of LTA, and substrate-mediated inactivation of the enzyme. Two distinct catalytic patterns were observed (types A and B). In three out of five enzyme preparations, catalysis obeyed Michaelis-Menten kinetics, showing a maximal initial velocity of 1.1 pmol/mg/ min and a K,, of 12 PM (type A). These values are in agreement with data on LTA, hydrolase purified from human peripheral leukocytes (Table III). Two preparations showed a higher catalytic activity and were found not to obey saturation kinetics, but were compatible rather with a mixture of enzymatic activities. Values of V,,, (2.0

TABLE

III

Comparison of the Kinetic Properties at 0°C Hydrolase from Peripheral Leukocytes and Preparations of Types A and B from

for LTA, Enzyme

Raji

Raji

Peripheral A

B

1.1

2.1

leukocytes

(21)

V Inax

(~mol/mg/minl Turn-over number (min -i) Km (PM) Second-order rate constant (Mm1 s-l)

75 12 10”

138 2 106

1.1 75 20-30 5 x 104

LEUKOTRIENE

A,

HYDROI,ASE

and 2.2 pmol/mg/min) and K, (2.2 and 2.8 PM) determined under these conditions are obviously incorrect, but can still give a notion of the catalytic properties of the investigated protein (cf. Table III). The altered kinetics were evidently not due to partial denaturation of the enzyme, since the catalytic activities in the diverging preparations were higher than in those obeying MichaelisMenten kinetics. Apparently, it was not caused by oxidation of the enzyme, since treatment with dithiothreitol neither altered catalysis nor ~1. Heat-treatment (60°C) of type A enzyme led to a timedependent decline of In (V), when this was assayed in the rout.ine 10-s incubations on ice. However, also in this aspect certain preparations of enzyme were divergent. In accordance with the presence of isoforms of the LTA., hydrolase, a biphasic inactivation pattern was observed, with appearance of a more heat-resistant enzymatic activity. No differences between the human peripheral leukocyte enzyme or preparations of type A or B regarding M,, ~1, Stoke’s radius, amino acid composition, or N-terminal amino acid sequence were observed which could reflect structural differences leading to altered kinetics. Neither could any obvious differences between enzyme batches be observed during the purification procedure. However, this does not rule out that differences in structure exist. These could be amino acid exchanges or post-translational modifications, not reflected in the performed analysis. Recently, it was suggested that human respiratory epithelium contains an LTA, hydrolase with novel biochemical characteristics (30). Unfortunately, that study was performed on intact cells and in the presence of albumin, factors which influence catalysis and obstruct an adequate comparison with the data on purified LTA, hydrolase from other sources. Nonetheless, the observation that a fraction of the LTA, hydrolase activity in human erythrocytes remained also after repeated additions of substrate (27), which was true also for the enzyme in respiratory epithelium, does support the assumption that subspecies of the LTA, hydrolase exist. In addition, several polymorphic mRNA species coding for LTA, hydrolase were recently identified in mouse spleen total RNA prepared from various mouse strains.” Sequence comparison with certain peptidases and neutral proteases, typified by thermolysin, identified a putative zinc-binding site in LTA, hydrolase (31, 32). Accordingly, LTA, hydrolase from human leukocytes was recently shown to contain one atom of zinc per enzyme molecule, as determined by atomic absorption spectrometry (29, 33). Thermolysin has been shown to exhibit higher catalytic activity upon substitution of the intrinsic zinc atom for cobalt (34). However, no diff’erence in en-

’ Medina,

et al., submitted

for publication.

IN

RAJI

173

CELLS

eymatic activity was observed between zinc-containing and cobalt-containing LTA4 hydrolase (33). Until more is known about the complete structure of the Raji enzyme, discussions regarding the physicochemical basis for the increased catalytic activity in certain enzyme preparations remain speculative. Since the increased enzymatic activity was only observed in certain enzyme preparations, it might be due to varying properties of Raji subclones or to cell cycle specific events. Under conditions where LTA4 is provided, possibly by activated monocytes (35), this could be part of an autoregulatory mechanism for lymphocytes and, since LTB, is a growth factor for human B cells, lead to increased replication of activated/transformed B cells (6). ACKNOWLEDGMENTS We are gratefill to Docent Anders Ro&, Karolinska Institutet, for providing the Knj; cell line, to Professor Tamas Bartfai, Stockholm IJniversity, and Professor Arne Holmgren, Karolinska Institutet,, for helpful discussions, and to Ms. H&l&e Axson ,Johnson for expert technical assistance. This work was supported by grants from The Swedish Society for Medical Research, The Swedish Society of Medicine, O.E. & Edla Johanssons Foundation, Stiftelsen Lars Hiertas Minne, The Magnus Bergvall Foundation, Alex och Eva Wallstriims Foundation, The Royal Swedish Academy of Sciences, The Swedish Cancer Society (Project 2801), and the Swedish Medical Research Council (03X-217,03X-7467, 03X-3532, and 03X-7145)

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