Crinicu C/iimicu Actu, 197 (1991) 3.5-46 Elsevier Science Publishers B.V. ADONIS 0009898191000790
35
CCA 04943
Pu~fication and characte~zation of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human Skeletal muscle David Mantle, Brenda Lauffart and Alison Gibson ’ Neurochemistry
Department, Regional Neurological Centre and ’ MRC Neurochemical Newcastle General Hospital, Newcastle upon Tyne (UK)
Pathology
Unit,
(Received 29 March 1990; revision received 5 November 1990; accepted 23 November 1990) Key word.?: Protease; Peptidase; Aminopeptidase; Muscle
Snmmary
The purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle are described. The characteristics of leucyl aminopeptidase were as follows: optimum activity was at pH 9.5 in the presence of 5 mmol/l Mg2+ or 0.5 mm01 Mn2+. No activation of enzyme activity was obtained following addition of other divalent cations or sulp~yd~l reagents, Only the leucyl-AMC and methionyl-AMC derivatives were appreciably hydrolysed The mol mass was estimated as 280 kDa. Approx. 50% inhibition of activity was obtained following addition of p-hydroxymercuriphenyl sulphonate (10 ~mol/l), N-ethyl maleimide (2 mmol/l), o-phenanthroline (5 mmol/l), bacitracin (1 mmol/l), amastatin (1 pg/rnl) and bestatin (0.1 pmol/l); no inhibition of activity was obtained in the presence of phenyhnethanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 hg/rnl) or pepstatin (100 pg/ml). The following oligopeptides were hydrolysed by the enzyme: luliberin 7-10, proctolin and [Leus]enkephalin; oligopeptides not appreciably hydrolysed included neurotensin, angiotensin-I, substance-P and bradykinin. Pyroglutamyl aminopeptidase had the following characteristics: optimum activity was at pH 8.5 in the presence of 1 mmol/l dithiothreitol (an absolute requirement for maintenance of enzyme activity). Maximum activity was obtained in the absence of divalent cations. Only the pyroglutamyl-AMC derivative was appreciably hydrolysed. The mol mass of this enzyme was estimated as 22 kDa. Approximately 50% Abbreuiation: AMC, a~noacyl-7-~o~methyl~uma~n Correspondence to: Dr. D. Mantle, Neurochemical Department, Regional Neuroiogical Centre, Newcastle General Hospital, Newcastle upon Tyne, NE4 6BE, UK.
36
inhibition of activity was obtained on addition of phenanthroline (4 mmol/l) and antipain (7 pg/ml); no inhibition of activity was obtained following addition of phenyl methanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 pg/ml) or pepstatin (100 pg/rnl). Only oligopeptides with a pyroglutamyl N-terminal residue (thyroliberin, neurotensin, and luliberin) were hydrolysed by the enzyme. Introduction
The mechanism by which protein degradation occurs in skeletal muscle is poorly understood; a greater knowledge of this process would be of value both in understanding the normal physiological function of muscle, and in the design of novel therapeutic strategies aimed at halting or reversing the muscle degeneration associated with the muscular dystrophies and other muscle wasting conditions. In order to define the mechanism of protein breakdown in muscle, it is of fundamental importance to purify and characterize the proteolytic enzymes involved. Much of the previous research on muscle proteases has concentrated on protein degrading endoproteinases, the cathepsins and Ca2+ -activated proteinases [l]. The role of peptide degrading exopeptidases in protein breakdown is unknown, although these enzymes are presumably involved in the later stages of degradation. In an attempt to determine the role of these enzymes in muscle protein catabolism, we have undertaken a systematic investigation of the aminopeptidase group of enzymes (cY-aminoacyl-peptide hydrolases EC 3.4.11) in human skeletal muscle, since the possibility of a fundamental role for these enzymes in protein turnover is suggested by their ubiquitous distribution [2]. We have previously described the identification of four aminopeptidase enzyme types (following anion exchange chromatographic separation of muscle soluble fraction, with which the majority of tissue aminopeptidase activity is associated): alanyl-, arginyl-, leucyl- and pyroglutamyl aminopeptidases [3]. The purification and characterization of alanyl aminopeptidase (the major muscle tissue aminopeptidase, accounting for approximately 80% of the total soluble aminopeptidase activity [4]) and arginyl aminopeptidase [5] have been described previously. We now describe the further purification and characterization of leucyl and pyroglutamyl aminopeptidases from human post-mortem skeletal muscle tissue; previous work described in the literature on the characterization of these enzymes in other human/animal tissues has been detailed in the bibliography of exopeptidases by McDonald and Barrett [2]. Materials and methods
DEAE-Sephadex A50, Sephacryl S-300 and Sephacryl S-100 were obtained from Pharmacia, London, UK; aminoacyl-7-amino-4-methylcoumarin derivatives from Bachem, Bubendorf, Switzerland and Cambridge Biochemicals, Cambridge, UK; all other materials (including enzyme inhibitors and oligopeptide substrates) were obtained from Sigma, London, UK and BDH, Poole, UK, and were of analytical grade where available.
31
Enzyme extraction
Approx. 35 g of human quadriceps muscle (obtained at autopsy within 12 h of death) were homogenized using an Ultra-Turrax homogenizer (2 X 30 s at 1500 rpm). A 1: 4 tissue homogenate was prepared in the following extraction buffer: 50 mmol/l glycylglycine, 5 mmol/l EGTA, 1 mmol/l 2-mercaptoethanol, 1 mmol/l di~ot~eitol and 3 mmol/i NaN,, pH 7.5, at 6°C. The homogenate was centrifuged at 20 000 x g for 30 min at 6 o C and the soluble fraction (Sl) retained for enzyme separation. Enzyme puri~cation Step 1: separation of aminopeptidases by anion exchange
chromatography of Sl
Approximately 120 ml of Sl (NaCl cont. adjusted to 0.1 mol/l) were applied to a column (50 X 3 cm) of DEAE-Sephadex A50 anion exchange gel equilibrated with extraction buffer containing 0.1 mol/l NaCI. The column was washed with 2 bed volumes of equilibration buffer and then eluted with a linear NaCl gradient (0.1-0.5 mol/l, 1.5 1 total volume) in extraction buffer at a flow rate of 25 ml/h; 4 ml fractions were collected and assayed for enzyme activity. Step 2: gel filtration chromatography of leucyl- and pyroglutamyl aminopeptidases Fractions comprising the peaks of leucyl or pyroglutamyl aminopeptidase activity identified in Step 1 were further purified via gel filtration chromatography on Sephacryl S-300 or Sephacryl S-100 respectively. Samples were concentrated by ultrafiltration [Amicon cell, molecular mass exclusion membrane 100 kDa (leucyl aminopeptidase) or 2 kDa (pyroglutamyl aminopeptidase)] and applied to a gel column (90 x 1.5 cm) of Sephacryl S-300 or S-100 equilibrated with 50 mmol/l T&/acetate buffer pH 7.5 at 6’C containing 0.1 mol/l KCl, 1 mmol/l EDTA, 1 mmol/l mer~pt~th~ol, 1 mmol/l dit~ot~eitol and 3 mmol/l NaN, at a flow rate of 15 ml/h. Myoglobin, pepsin, creatine kinase, alcohol dehydrogenase, catalase and ferritin were used as molecular mass markers. The void volume of the column was determined using dextran blue, with fraction volumes (approximately 3 ml) determined by weight. Step 3: preparative polyacrylamide gel electrophoresis of leucyl- and pyroglutamyl aminopeptidases Fractions containing leucyl- or pyroglutamyl aminopeptidase ac-
tivity from Step 2 were pooled and concentrated to a volume of approximately 1 ml respectively (10 ml Amicon cell, 2000 kDa exclusion membr~e) and further purified by preparative polyacrylamide gel electrophoresis. Following addition of glycerol (30% v/v) and bromophenol blue (O.OOSW),samples were run at 25 mA/gel for 24 h at 6” C, using either 5% (leucyl aminopeptidase) or 12% (pyroglutamyl ~nopeptid~e~ slab gels (14 x 12 cm) and a continuous Tris/acetate buffer system (0.1 mol/l, pH 8, at 6 o C>. Gel strips 1 cm in width were removed from each slab and stained for protein following the method of Oakley et al. [6]; the gel slabs were then sectioned (3-mm slices) and the enzymes eluted by soaking the slices in assay
38
buffer for 24 h at 6” C. The protein-stained gel strips were scanned densitometer (Bio Rad model 1650, transmission mode).
using a
Assay of aminopeptidare activity Enzyme containing solution (0.1 ml) was incubated with the appropriate assay medium (total volume 0.3 ml) for 1 h at 37” C, and the reaction terminated by addition of 0.6 ml of ethanol. The fluorescence of the liberated aminoacyl-7-amino4-methylcoumarin (AMC) was measured by reference to a fluorescence standard (h,, 380 nm, A,, 440 nm). Assay blanks were run in which the enzyme was added to the medium immediately before the addition of ethanol. Assay conditions were modified for samples with high enzyme activity so that the extent of substrate utilization never exceeded 15%. Optimum assay conditions for leucyl- and pyroglutamyl aminopeptidase were as follows: leucyl aminopeptidase: 50 mmol/l glycine/NaOH buffer, pH 9.5 at 37 o C, 0.5 mmol/l MnCl, and 2 mmol/l leucylAMC; pyroglutamyl aminopeptidase: 50 mmol/l glycine/NaOH buffer pH 8.5 at 37” C, 1 mmol/l dithiothreitol and 0.125 mmol/l pyroglutamyl-AMC. The characteristics for each aminopeptidase type following purification were determined by substitution of the appropriate buffers, effecters and substrates in the above assay procedures. Protein concentrations were determined by the method of Sedmak and Grossberg [ 71. Analysis of oligopeptide degradation by aminopeptidases via high performance liquid chromatography The activity of leucyl aminopeptidase and pyroglutamyl aminopeptidase (following purification) on a series of oligopeptides was analysed by reverse phase high performance liquid chromatography, using a Waters automated system incorporating a Z-module radial compression unit and Nova-Pak C-18 cartridge [8]. Column elution was carried out with a linear gradient of acetonitrile [2-49% (v/v), 20 min at 1 ml/nun] in 11 mmol/l trifluoroacetic acid; product elution times were monitored by UV absorption at 214 nm. Degradation products were identified by comparison of elution times with those for appropriate standards. For each of the aminopeptidase types investigated, oligopeptides (10 pg) were incubated in the appropriate assay medium described above for l-5 h at 37 o C, and the reaction terminated by addition of trifluoroacetic acid (6 mmol/l final cont.). Results and discussion Purification of leucyl- and pyrogkwnyl aminopeptidases For both leucyl- and pyroglutamyl aminopeptidases, at least 90% of the enzyme activity measured in muscle homogenate was present in the soluble fraction (Sl). The separation of aminopeptidase activities in muscle soluble fraction by anion exchange chromatography is shown in Fig. 1. The data shown are derived from one muscle sample from a single individual (aged 40 yr, 12 h autopsy delay); experiments using muscle samples from other individuals differing in age (25-66 yr), sex or autopsy delay (6 h, 24 h) gave similar results. Leucyl aminopeptidase activity
39
ELUTION
VOLUME
(ml]
Fig. 1. Anion exchange chromatography of muscle soluble fraction. Aminopeptidase activities were separated by anion exchange chromatography of muscle soluble fraction, as described under ‘Materials and Methods’. The separation of leucyl aminopeptidase and pyroglutamyl aminopeptidase activities is shown in Fig. la (Leu-AMC substrate) and lb (pyroglutamyl-AMC substrate), respectively; the separation of these enzymes from alanyl- (Fig. lc; Ala-AMC substrate) and arginyl- (Fig. Id; Arg-AMC substrate) aminopeptidases previously described in skeletal muscle extract is also shown. The interpretation of the various peaks of activity is given under Results and Discussion. The [NaCl] gradient is shown (- - -). The protein elution profile (AZsOnm, 1 cm pathlength cell) is shown (-).
(equivalent to 1% of the total soluble aminopeptidase activity) eluted from the anion exchange column at a NaCl cont. of 0.1 mol/l (Fig. la), well separated from the other aminopeptidase enzyme types identified in muscle soluble fraction (Fig. lb-d). The peak of apparent leucyl aminopeptidase activity eluting from the gradient at a NaCl cont. of 0.27 mol/l (mol mass 102 kDa) resulted from the hydrolysis of leucyl-AMC by the major (alanyl-) aminopeptidase described previously [4] (Fig. lc). F’yroghttamyl aminopeptidase activity (equivalent to 2.5% of the total soluble aminopeptidase activity) eluted from the anion exchange column at a NaCl cont. of 0.22 mol/l (Fig. lb), partially overlapping the peak of arginyl aminopeptidase activity eluting at 0.17 mol/l NaCl described previously [5] (Fig.
ELUTION
C
VOLUME
(ml)
MIORATION DISTANCE (.rn)
E!
Fig. 2. Further purification of leucyl- and pyroglutamyl aminopeptidases via gel filtration chromatography and preparative ektrophoresis. The further pu~~~tioR of leucyl ~~tidase via gel filtration chromatography on Sephacryl S-300 and preparative polyacrykmide gel electrophoresiis(5% gel) is shown in Fig. 2a and c, respectively. The further purification of pyrogtutamyl aminopeptidase via gel filtration chromatography on Sephacryl SlOO and preparative polyacrylamide gel ektrophoresis (12% gel) are shown in Fig. 2b and d, respectively. Experimental details for the above procedures are given under and protein absorbance ( -) ‘Materials and Methods’. Enzyme activity (0 -0) are shown, respectively. For the preparative electrophoresis step, the direction of migration is from the cathode to the anode; the positions and intensities of the stained bands are shown.
Id). The second peak of apparent arginyl aminopeptidase activity eluting at a NaCl cont. of 0.27 mol/l in Fig. Id results from the hydrolysis of arginyl-AMC via alanyl aminopeptidase. Fractions comprising leucyl aminopeptidase or p~~ut~yl ~~op~ti~se activity peaks identified in Step 1 were pooled, concentrated by ultrafiltration and further purified by gel filtration chromatography on Sephacryl S-300 (Fig. 2a) or Sephacryl S-100 (Fig. 2b) respectively, as described under ‘Materials and Methods’. A single peak of activity was obtained for each enzyme type in this procedure; complete separation of arginyl- and pyroglutamyl aminopeptidases was obtained following this step. Fractions #~~on~g to leucyl- or p~o~u~yl aminopeptidase activities in Step 2 were concentrated and further purified via preparative
(Sl)
5.4
9.1
105
0.115
11.4
12.7
450
1176
47.0
0.087
0.025
0.011
Details of the enzyme extraction and purification procedures, determination Methods. Enzyme activity in Sl was determined in the presence of 1 mmol/l and pyroglutamyl aminopeptidase substrates.
Anion exchange chromatography Gel filtration chromatography Preparative polyacrylamide gel electrophoresis
Soluble extract
0.090
3
29
1176
5.9
19.9
28.5
31.7
65.6
6.63
0.980
0.027
( u mol/h/mg)
2429
243
36
_
of enzyme activity and protein concentration are given under Materials and puromycin, to inhibit the action of the major muscle aminopeptidase on leucyl-
4272
7.9
2.3
_
42
polyacrylamide gel electrophoresis (Fig. 2c and 2d, respectively), as described under ‘Materials and Methods’. For each enzyme type, a single protein staining band corresponding with the respective enzyme activity was obtained after re-electrophoresis of purified enzyme, following the above procedure; we would emphasize however that the latter is not an absolute criterion for enzyme homogeneity, but represents a minimum standard of enzyme purity required prior to undertaking the characterization work described in this paper. The results of a typical purification of leucyl- and pyroglutamyl aminopeptidase from skeletal muscle soluble extract are shown in Table I. Enzyme characterization
following purification
Following purification as outlined above, leucyl aminopeptidase and pyroglutamy1 aminopeptidase were found to have the following characteristics (determined as described under ‘Materials and Methods’): pH optimum of enzyme activity Optimal activity for leucyl aminopeptidase was at pH 9.5, with 50% of optimal activity at pH 8 and pH 11. Optimal activity for pyroglutamyl aminopeptidase was at pH 8.5, with 50% of optimal activity at pH 7 and pH 9.5. Effect of cations on enzyme activity Maximum activity for leucyl aminopeptidase was obtained in the presence of 5 mmol/l Mg*+ or 0.5 mmol/l Mn*‘. There was no significant enzyme activity in the absence of these cations, or on addition of the following (0.05-5 mmol/l): Ca*+, Co*‘, Sr*+, Ba*+ Cu*+, Zn*‘. The cations listed above were added in the form of their chloride salts. Maximum activity for pyroglutamyl aminopeptidase was obtained in the absence of divalent cations (1 mmol/l EDTA). Addition of the divalent cations listed above (0.05-5 mmol/l) resulted in partial or complete loss of enzyme activity.
For leucyl aminopeptidase, only Enzyme activity on aminoacyl-AMC derivatives the leucyl-AMC and methionyl-AMC (36% relative hydrolysis rate) derivatives were appreciably hydrolysed. With pyroglutamyl aminopeptidase, only the pyroglutamylAMC derivative was appreciably hydrolysed. For both aminopeptidases there was no appreciable hydrolysis of the following aminoacyl-AMC derivatives: alanyl-, arginyl-, glycyl, cY-glutamyl-, isoleucyl-, lysyl-, ornithyl-, phenylalanyl-, prolyl-, seryl-, tyrosyl- and valyl-. The effect of a series of potential inhibiEffect of inhibitors on enzyme activity tors on the activity of leucyl- or pyroglutamyl aminopeptidase is shown in Table II. For leucyl aminopeptidase, approximately 50% inhibition of activity was obtained with the following: o-phenanthroline (5 mM), p-hydroxymercuriphenyl sulphonate (10 pmol/l), bestatin (0.1 pmol/l), amastatin (1 pg/rnl) and bacitracin (1 mmol/l). For pyroglutamyl aminopeptidase, approximately 50% inhibition of activity was obtained with o-phenanthroline (4 rnmol/l) and antipain (7 pg/rnl). Neither enzyme
43 TABLE
II
Effect of inhibitors
on leucyl- and pyroglutamyl
aminopeptidase
activity
Compound
Cont.
Leucyl aminopeptidase
Pyroglutamyl aminopeptidase
phenylmethanesulphonyl fluoride lima bean trypsin inhibitor leupeptin chymostatin pepstatin EDTA
1 mmol/l 100 KS/ml 100 /%/ml 100 PLg/ml 100 M/ml 5 mmol/l 5 mmol/l 4 mmol/l
100 115 97 86 97 50 50
100 122 80 83 98 120
2 mmol/l IO pmol/l
50 50 88
ND ND
(
o-phenanthroline N-ethylmaleimide p-hydroxymercuriphenylsulphonate antipain elastinal arphamenine puromycin
B
100 M/ml 7 &ml 100 /%/ml 100 M/ml 2 mmol/l 100 /G/ml I k%/ml
bacitracin bestatin
1 mmol/l 100 j.tmol/l 0.1 gmol/l
50
70 97 81
50 70 95 95 85
50 50
122 90
50
Enzyme and inhibitor were preincubated at the concentrations for 2 min in ice. Assays were started by addition of substrate, followed. ND: not determined.
shown in assay medium minus substrate and the standard assay procedure then
showed inhibition of activity following addition of phenyl methanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 pg/ml) or pepstatin (100 pg/ml). Effect of sulphyldryl reagents on enzyme activity With leucyl aminopeptidase, maximum activity was obtained in the absence of sulphhydryl reagents; no increase in enzyme activity was observed following addition of 2-mercaptoethanol, dithiothreitol, cysteine or glutathione (0.05-5 mmol/l). For pyroglutamyl aminopeptidase, the presence of a sulphhydryl reagent was an absolute requirement for the maintenance of enzyme activity; optimum activity was obtained in the presence of 1 mmol/l dithiothreitol.
Estimation of molecular mass The mol mass of leucyl aminopeptidase (determined by gel filtration chromatography on S-300) was estimated as 280 kDa. The mol mass of pyroglutamyl aminopeptidase (determined via gel filtration chromatography on Sephacryl S-100) was estimated as 22 kDa.
Estimation of K,,, dase and leucyl-AMC
A value for K, for the reaction between leucyl aminopeptiwas estimated (via linear regression analysis) as 1.5 mmol/l
44
from a Lineweaver-Burk plot of l/v vs l/[S] over the substrate cont. range 0.2-4 mmol/l [correlation coefficient (r) = 0.991. The K, value for the reaction between pyroglutamyl aminopeptidase and pyroglutamyl-AMC was similarly estimated as 80 pmol/l (substrate cont. range 20-250 pmol/l; r = 0.99). Enzyme activity on oligopeptide substrates The following oligopeptides were hydrolysed by leucyl aminopeptidase (N-terminal amino acid and relative hydrolysis rate shown in parentheses): luliberin 7-10 (Leu-Arg-; 100%); [Leu’lenkephalin (Tyr-Gly; 43.8%); proctolin (Arg-Tyr-; 9.7%). Peptides which were not appreciably hydrolysed by leucyl aminopeptidase included neurotensin (pGlu-Leu-), angiotensin-1 (Asp-Arg-), substance-P (Arg-Pro-) and bradykinin (Arg-Pro-). For the hydrolysis of [Leu’]enkephalin by leucyl aminopeptidase, only the N-terminal Tyr residue was removed; in the absence of Mg2+ (5 mmol/l) or Mn” (0.5 mmol/l) enzyme activity on the latter peptide substrate was reduced to less than 1.0% of optimum. Thyroliberin (pGlu-His-, loo%), neurotensin (pGlu-Leu, 59%) and luliberin (pGlu-His, 50%) were hydrolysed by pyroglutamyl aminopeptidase (N-terminal amino acid and relative hydrolysis rate in parentheses). In each case, only the N-terminal pGlu-residue was removed. Oligopeptides with free N-termini (e.g. [Leu’]enkephalin) or N-acetylated N-termini (e.g. melanocyte stimulating hormone) were not appreciably hydrolysed by pyroglutamyl aminopeptidase. In summary, we have described the characteristics of leucyl aminopeptidase and pyroglutamyl aminopeptidase following purification (as judged by non-denaturing polyacrylamide gel electrophoresis) from human post-mortem skeletal muscle tissue. Although each of these aminopeptidase types has been previously characterized to a varying degree in several other tissues/species [2], as far as we are aware this is the first report describing the purification and characterization of these enzymes from muscle tissue, particularly with respect to their action in the degradation of oligopeptides. The data obtained suggest that leucyl- and pyroglutamyl aminopeptidases are metalloenzymes with active site thiol group participation, although these enzymes differ otherwise in biophysical and optimal assay characteristics; for leucyl aminopeptidase the relevance of the high pH optimum of activity, and activation by Mg*+ or Mn” ions, to the subcellular localization and mode of action of the enzyme in vivo remains to be established. The specificity of pyroglutamyl aminopeptidase in hydrolysing oligopeptides or AMC derivatives is very restricted, with only substrates with an N-terminal pyroglutamyl residue being recognized. The specificity of leucyl aminopeptidase in hydrolysing oligopeptides is less restricted than for the latter enzyme, although not as broad as that expressed by alanyl aminopeptidase purified from human muscle [4]. The characteristics of leucyl aminopeptidase and pyroglutamyl aminopeptidase purified from human skeletal muscle are very similar to those for corresponding enzymes purified from human brain [9] and human kidney [lo] tissues, using a similar experimental approach. The occurrence of such enzymes, present in similar proportions in each tissue, with correspondingly similar characteristics, in such functionally dissimilar tissues as skeletal muscle, brain and kidney suggests that these enzymes are of fundamental importance in the general cellular protein catabolic process. The work described in
45
this paper completes the systematic purification and characterization of the aminopeptidase group of enzymes previously identified in human skeletal muscle tissue [3]; further work will now be required to establish the role of this group of enzymes in the degradation of key structural proteins within the muscle cell. References 1 Pennington RJT. Proteinases of muscle. In: Barrett AJ, ed. Proteinases in mammalian cells and tissues. Amsterdam: North-Holland, 1977;515-545. 2 McDonald JK, Barrett AJ. Mammalian proteases: a glossary and bibliography. Vol. 2: Exopeptidases. London: Academic Press, 1986. 3 Lauffart B, Mantle D. Rationalization of aminopeptidase activities in human skeletal muscle soluble extract. Biochim Biophys Acta 1988;956:304l-306. 4 Mantle D, Hardy MF, Lauffart B, McDermott JR, Smith AI, Pennington KJT. Purification and characterization of the major aminopeptidase from human skeletal muscle. Biochem J 1983;211:567573. 5 Mantle D, Lauffart B, McDermott JR, Kidd AM, Pennington RJT. Purification and characterization of two Cl- activated aminopeptidases hydrolysing basic termini from human skeletal muscle. Eur J Biochem 1985;147:307-312. 6 Oakley BR, Kirsch DR, Morris NR. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 1980;105:361-363. 7 Sedmak JJ, Grossberg SE. A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G250. Anal B&hem 1977;79:544-552. 8 Smith AI, McDermott JR. High performance liquid chromatography of neuropeptides using radially compressed polythene cartridges. J Chromatogr 1984;306:99-108. 9 Mantle D, Lauffart B, Perry EK, Perry RH. Comparison of major cortical aminopeptidase activity in normal brain and brain from patients with Alzheimer’s disease. J Neurol Sci 1989;89:227-234. 10 Mantle D, Lauffart B, McDermott JR, Gibson AM. Characterization of aminopeptidases in human kidney soluble fraction. Clin Chim Acta 1990;187:105-114.
35
CCA 04943
Pu~fication and characte~zation of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human Skeletal muscle David Mantle, Brenda Lauffart and Alison Gibson ’ Neurochemistry
Department, Regional Neurological Centre and ’ MRC Neurochemical Newcastle General Hospital, Newcastle upon Tyne (UK)
Pathology
Unit,
(Received 29 March 1990; revision received 5 November 1990; accepted 23 November 1990) Key word.?: Protease; Peptidase; Aminopeptidase; Muscle
Snmmary
The purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle are described. The characteristics of leucyl aminopeptidase were as follows: optimum activity was at pH 9.5 in the presence of 5 mmol/l Mg2+ or 0.5 mm01 Mn2+. No activation of enzyme activity was obtained following addition of other divalent cations or sulp~yd~l reagents, Only the leucyl-AMC and methionyl-AMC derivatives were appreciably hydrolysed The mol mass was estimated as 280 kDa. Approx. 50% inhibition of activity was obtained following addition of p-hydroxymercuriphenyl sulphonate (10 ~mol/l), N-ethyl maleimide (2 mmol/l), o-phenanthroline (5 mmol/l), bacitracin (1 mmol/l), amastatin (1 pg/rnl) and bestatin (0.1 pmol/l); no inhibition of activity was obtained in the presence of phenyhnethanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 hg/rnl) or pepstatin (100 pg/ml). The following oligopeptides were hydrolysed by the enzyme: luliberin 7-10, proctolin and [Leus]enkephalin; oligopeptides not appreciably hydrolysed included neurotensin, angiotensin-I, substance-P and bradykinin. Pyroglutamyl aminopeptidase had the following characteristics: optimum activity was at pH 8.5 in the presence of 1 mmol/l dithiothreitol (an absolute requirement for maintenance of enzyme activity). Maximum activity was obtained in the absence of divalent cations. Only the pyroglutamyl-AMC derivative was appreciably hydrolysed. The mol mass of this enzyme was estimated as 22 kDa. Approximately 50% Abbreuiation: AMC, a~noacyl-7-~o~methyl~uma~n Correspondence to: Dr. D. Mantle, Neurochemical Department, Regional Neuroiogical Centre, Newcastle General Hospital, Newcastle upon Tyne, NE4 6BE, UK.
36
inhibition of activity was obtained on addition of phenanthroline (4 mmol/l) and antipain (7 pg/ml); no inhibition of activity was obtained following addition of phenyl methanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 pg/ml) or pepstatin (100 pg/rnl). Only oligopeptides with a pyroglutamyl N-terminal residue (thyroliberin, neurotensin, and luliberin) were hydrolysed by the enzyme. Introduction
The mechanism by which protein degradation occurs in skeletal muscle is poorly understood; a greater knowledge of this process would be of value both in understanding the normal physiological function of muscle, and in the design of novel therapeutic strategies aimed at halting or reversing the muscle degeneration associated with the muscular dystrophies and other muscle wasting conditions. In order to define the mechanism of protein breakdown in muscle, it is of fundamental importance to purify and characterize the proteolytic enzymes involved. Much of the previous research on muscle proteases has concentrated on protein degrading endoproteinases, the cathepsins and Ca2+ -activated proteinases [l]. The role of peptide degrading exopeptidases in protein breakdown is unknown, although these enzymes are presumably involved in the later stages of degradation. In an attempt to determine the role of these enzymes in muscle protein catabolism, we have undertaken a systematic investigation of the aminopeptidase group of enzymes (cY-aminoacyl-peptide hydrolases EC 3.4.11) in human skeletal muscle, since the possibility of a fundamental role for these enzymes in protein turnover is suggested by their ubiquitous distribution [2]. We have previously described the identification of four aminopeptidase enzyme types (following anion exchange chromatographic separation of muscle soluble fraction, with which the majority of tissue aminopeptidase activity is associated): alanyl-, arginyl-, leucyl- and pyroglutamyl aminopeptidases [3]. The purification and characterization of alanyl aminopeptidase (the major muscle tissue aminopeptidase, accounting for approximately 80% of the total soluble aminopeptidase activity [4]) and arginyl aminopeptidase [5] have been described previously. We now describe the further purification and characterization of leucyl and pyroglutamyl aminopeptidases from human post-mortem skeletal muscle tissue; previous work described in the literature on the characterization of these enzymes in other human/animal tissues has been detailed in the bibliography of exopeptidases by McDonald and Barrett [2]. Materials and methods
DEAE-Sephadex A50, Sephacryl S-300 and Sephacryl S-100 were obtained from Pharmacia, London, UK; aminoacyl-7-amino-4-methylcoumarin derivatives from Bachem, Bubendorf, Switzerland and Cambridge Biochemicals, Cambridge, UK; all other materials (including enzyme inhibitors and oligopeptide substrates) were obtained from Sigma, London, UK and BDH, Poole, UK, and were of analytical grade where available.
31
Enzyme extraction
Approx. 35 g of human quadriceps muscle (obtained at autopsy within 12 h of death) were homogenized using an Ultra-Turrax homogenizer (2 X 30 s at 1500 rpm). A 1: 4 tissue homogenate was prepared in the following extraction buffer: 50 mmol/l glycylglycine, 5 mmol/l EGTA, 1 mmol/l 2-mercaptoethanol, 1 mmol/l di~ot~eitol and 3 mmol/i NaN,, pH 7.5, at 6°C. The homogenate was centrifuged at 20 000 x g for 30 min at 6 o C and the soluble fraction (Sl) retained for enzyme separation. Enzyme puri~cation Step 1: separation of aminopeptidases by anion exchange
chromatography of Sl
Approximately 120 ml of Sl (NaCl cont. adjusted to 0.1 mol/l) were applied to a column (50 X 3 cm) of DEAE-Sephadex A50 anion exchange gel equilibrated with extraction buffer containing 0.1 mol/l NaCI. The column was washed with 2 bed volumes of equilibration buffer and then eluted with a linear NaCl gradient (0.1-0.5 mol/l, 1.5 1 total volume) in extraction buffer at a flow rate of 25 ml/h; 4 ml fractions were collected and assayed for enzyme activity. Step 2: gel filtration chromatography of leucyl- and pyroglutamyl aminopeptidases Fractions comprising the peaks of leucyl or pyroglutamyl aminopeptidase activity identified in Step 1 were further purified via gel filtration chromatography on Sephacryl S-300 or Sephacryl S-100 respectively. Samples were concentrated by ultrafiltration [Amicon cell, molecular mass exclusion membrane 100 kDa (leucyl aminopeptidase) or 2 kDa (pyroglutamyl aminopeptidase)] and applied to a gel column (90 x 1.5 cm) of Sephacryl S-300 or S-100 equilibrated with 50 mmol/l T&/acetate buffer pH 7.5 at 6’C containing 0.1 mol/l KCl, 1 mmol/l EDTA, 1 mmol/l mer~pt~th~ol, 1 mmol/l dit~ot~eitol and 3 mmol/l NaN, at a flow rate of 15 ml/h. Myoglobin, pepsin, creatine kinase, alcohol dehydrogenase, catalase and ferritin were used as molecular mass markers. The void volume of the column was determined using dextran blue, with fraction volumes (approximately 3 ml) determined by weight. Step 3: preparative polyacrylamide gel electrophoresis of leucyl- and pyroglutamyl aminopeptidases Fractions containing leucyl- or pyroglutamyl aminopeptidase ac-
tivity from Step 2 were pooled and concentrated to a volume of approximately 1 ml respectively (10 ml Amicon cell, 2000 kDa exclusion membr~e) and further purified by preparative polyacrylamide gel electrophoresis. Following addition of glycerol (30% v/v) and bromophenol blue (O.OOSW),samples were run at 25 mA/gel for 24 h at 6” C, using either 5% (leucyl aminopeptidase) or 12% (pyroglutamyl ~nopeptid~e~ slab gels (14 x 12 cm) and a continuous Tris/acetate buffer system (0.1 mol/l, pH 8, at 6 o C>. Gel strips 1 cm in width were removed from each slab and stained for protein following the method of Oakley et al. [6]; the gel slabs were then sectioned (3-mm slices) and the enzymes eluted by soaking the slices in assay
38
buffer for 24 h at 6” C. The protein-stained gel strips were scanned densitometer (Bio Rad model 1650, transmission mode).
using a
Assay of aminopeptidare activity Enzyme containing solution (0.1 ml) was incubated with the appropriate assay medium (total volume 0.3 ml) for 1 h at 37” C, and the reaction terminated by addition of 0.6 ml of ethanol. The fluorescence of the liberated aminoacyl-7-amino4-methylcoumarin (AMC) was measured by reference to a fluorescence standard (h,, 380 nm, A,, 440 nm). Assay blanks were run in which the enzyme was added to the medium immediately before the addition of ethanol. Assay conditions were modified for samples with high enzyme activity so that the extent of substrate utilization never exceeded 15%. Optimum assay conditions for leucyl- and pyroglutamyl aminopeptidase were as follows: leucyl aminopeptidase: 50 mmol/l glycine/NaOH buffer, pH 9.5 at 37 o C, 0.5 mmol/l MnCl, and 2 mmol/l leucylAMC; pyroglutamyl aminopeptidase: 50 mmol/l glycine/NaOH buffer pH 8.5 at 37” C, 1 mmol/l dithiothreitol and 0.125 mmol/l pyroglutamyl-AMC. The characteristics for each aminopeptidase type following purification were determined by substitution of the appropriate buffers, effecters and substrates in the above assay procedures. Protein concentrations were determined by the method of Sedmak and Grossberg [ 71. Analysis of oligopeptide degradation by aminopeptidases via high performance liquid chromatography The activity of leucyl aminopeptidase and pyroglutamyl aminopeptidase (following purification) on a series of oligopeptides was analysed by reverse phase high performance liquid chromatography, using a Waters automated system incorporating a Z-module radial compression unit and Nova-Pak C-18 cartridge [8]. Column elution was carried out with a linear gradient of acetonitrile [2-49% (v/v), 20 min at 1 ml/nun] in 11 mmol/l trifluoroacetic acid; product elution times were monitored by UV absorption at 214 nm. Degradation products were identified by comparison of elution times with those for appropriate standards. For each of the aminopeptidase types investigated, oligopeptides (10 pg) were incubated in the appropriate assay medium described above for l-5 h at 37 o C, and the reaction terminated by addition of trifluoroacetic acid (6 mmol/l final cont.). Results and discussion Purification of leucyl- and pyrogkwnyl aminopeptidases For both leucyl- and pyroglutamyl aminopeptidases, at least 90% of the enzyme activity measured in muscle homogenate was present in the soluble fraction (Sl). The separation of aminopeptidase activities in muscle soluble fraction by anion exchange chromatography is shown in Fig. 1. The data shown are derived from one muscle sample from a single individual (aged 40 yr, 12 h autopsy delay); experiments using muscle samples from other individuals differing in age (25-66 yr), sex or autopsy delay (6 h, 24 h) gave similar results. Leucyl aminopeptidase activity
39
ELUTION
VOLUME
(ml]
Fig. 1. Anion exchange chromatography of muscle soluble fraction. Aminopeptidase activities were separated by anion exchange chromatography of muscle soluble fraction, as described under ‘Materials and Methods’. The separation of leucyl aminopeptidase and pyroglutamyl aminopeptidase activities is shown in Fig. la (Leu-AMC substrate) and lb (pyroglutamyl-AMC substrate), respectively; the separation of these enzymes from alanyl- (Fig. lc; Ala-AMC substrate) and arginyl- (Fig. Id; Arg-AMC substrate) aminopeptidases previously described in skeletal muscle extract is also shown. The interpretation of the various peaks of activity is given under Results and Discussion. The [NaCl] gradient is shown (- - -). The protein elution profile (AZsOnm, 1 cm pathlength cell) is shown (-).
(equivalent to 1% of the total soluble aminopeptidase activity) eluted from the anion exchange column at a NaCl cont. of 0.1 mol/l (Fig. la), well separated from the other aminopeptidase enzyme types identified in muscle soluble fraction (Fig. lb-d). The peak of apparent leucyl aminopeptidase activity eluting from the gradient at a NaCl cont. of 0.27 mol/l (mol mass 102 kDa) resulted from the hydrolysis of leucyl-AMC by the major (alanyl-) aminopeptidase described previously [4] (Fig. lc). F’yroghttamyl aminopeptidase activity (equivalent to 2.5% of the total soluble aminopeptidase activity) eluted from the anion exchange column at a NaCl cont. of 0.22 mol/l (Fig. lb), partially overlapping the peak of arginyl aminopeptidase activity eluting at 0.17 mol/l NaCl described previously [5] (Fig.
ELUTION
C
VOLUME
(ml)
MIORATION DISTANCE (.rn)
E!
Fig. 2. Further purification of leucyl- and pyroglutamyl aminopeptidases via gel filtration chromatography and preparative ektrophoresis. The further pu~~~tioR of leucyl ~~tidase via gel filtration chromatography on Sephacryl S-300 and preparative polyacrykmide gel electrophoresiis(5% gel) is shown in Fig. 2a and c, respectively. The further purification of pyrogtutamyl aminopeptidase via gel filtration chromatography on Sephacryl SlOO and preparative polyacrylamide gel ektrophoresis (12% gel) are shown in Fig. 2b and d, respectively. Experimental details for the above procedures are given under and protein absorbance ( -) ‘Materials and Methods’. Enzyme activity (0 -0) are shown, respectively. For the preparative electrophoresis step, the direction of migration is from the cathode to the anode; the positions and intensities of the stained bands are shown.
Id). The second peak of apparent arginyl aminopeptidase activity eluting at a NaCl cont. of 0.27 mol/l in Fig. Id results from the hydrolysis of arginyl-AMC via alanyl aminopeptidase. Fractions comprising leucyl aminopeptidase or p~~ut~yl ~~op~ti~se activity peaks identified in Step 1 were pooled, concentrated by ultrafiltration and further purified by gel filtration chromatography on Sephacryl S-300 (Fig. 2a) or Sephacryl S-100 (Fig. 2b) respectively, as described under ‘Materials and Methods’. A single peak of activity was obtained for each enzyme type in this procedure; complete separation of arginyl- and pyroglutamyl aminopeptidases was obtained following this step. Fractions #~~on~g to leucyl- or p~o~u~yl aminopeptidase activities in Step 2 were concentrated and further purified via preparative
(Sl)
5.4
9.1
105
0.115
11.4
12.7
450
1176
47.0
0.087
0.025
0.011
Details of the enzyme extraction and purification procedures, determination Methods. Enzyme activity in Sl was determined in the presence of 1 mmol/l and pyroglutamyl aminopeptidase substrates.
Anion exchange chromatography Gel filtration chromatography Preparative polyacrylamide gel electrophoresis
Soluble extract
0.090
3
29
1176
5.9
19.9
28.5
31.7
65.6
6.63
0.980
0.027
( u mol/h/mg)
2429
243
36
_
of enzyme activity and protein concentration are given under Materials and puromycin, to inhibit the action of the major muscle aminopeptidase on leucyl-
4272
7.9
2.3
_
42
polyacrylamide gel electrophoresis (Fig. 2c and 2d, respectively), as described under ‘Materials and Methods’. For each enzyme type, a single protein staining band corresponding with the respective enzyme activity was obtained after re-electrophoresis of purified enzyme, following the above procedure; we would emphasize however that the latter is not an absolute criterion for enzyme homogeneity, but represents a minimum standard of enzyme purity required prior to undertaking the characterization work described in this paper. The results of a typical purification of leucyl- and pyroglutamyl aminopeptidase from skeletal muscle soluble extract are shown in Table I. Enzyme characterization
following purification
Following purification as outlined above, leucyl aminopeptidase and pyroglutamy1 aminopeptidase were found to have the following characteristics (determined as described under ‘Materials and Methods’): pH optimum of enzyme activity Optimal activity for leucyl aminopeptidase was at pH 9.5, with 50% of optimal activity at pH 8 and pH 11. Optimal activity for pyroglutamyl aminopeptidase was at pH 8.5, with 50% of optimal activity at pH 7 and pH 9.5. Effect of cations on enzyme activity Maximum activity for leucyl aminopeptidase was obtained in the presence of 5 mmol/l Mg*+ or 0.5 mmol/l Mn*‘. There was no significant enzyme activity in the absence of these cations, or on addition of the following (0.05-5 mmol/l): Ca*+, Co*‘, Sr*+, Ba*+ Cu*+, Zn*‘. The cations listed above were added in the form of their chloride salts. Maximum activity for pyroglutamyl aminopeptidase was obtained in the absence of divalent cations (1 mmol/l EDTA). Addition of the divalent cations listed above (0.05-5 mmol/l) resulted in partial or complete loss of enzyme activity.
For leucyl aminopeptidase, only Enzyme activity on aminoacyl-AMC derivatives the leucyl-AMC and methionyl-AMC (36% relative hydrolysis rate) derivatives were appreciably hydrolysed. With pyroglutamyl aminopeptidase, only the pyroglutamylAMC derivative was appreciably hydrolysed. For both aminopeptidases there was no appreciable hydrolysis of the following aminoacyl-AMC derivatives: alanyl-, arginyl-, glycyl, cY-glutamyl-, isoleucyl-, lysyl-, ornithyl-, phenylalanyl-, prolyl-, seryl-, tyrosyl- and valyl-. The effect of a series of potential inhibiEffect of inhibitors on enzyme activity tors on the activity of leucyl- or pyroglutamyl aminopeptidase is shown in Table II. For leucyl aminopeptidase, approximately 50% inhibition of activity was obtained with the following: o-phenanthroline (5 mM), p-hydroxymercuriphenyl sulphonate (10 pmol/l), bestatin (0.1 pmol/l), amastatin (1 pg/rnl) and bacitracin (1 mmol/l). For pyroglutamyl aminopeptidase, approximately 50% inhibition of activity was obtained with o-phenanthroline (4 rnmol/l) and antipain (7 pg/rnl). Neither enzyme
43 TABLE
II
Effect of inhibitors
on leucyl- and pyroglutamyl
aminopeptidase
activity
Compound
Cont.
Leucyl aminopeptidase
Pyroglutamyl aminopeptidase
phenylmethanesulphonyl fluoride lima bean trypsin inhibitor leupeptin chymostatin pepstatin EDTA
1 mmol/l 100 KS/ml 100 /%/ml 100 PLg/ml 100 M/ml 5 mmol/l 5 mmol/l 4 mmol/l
100 115 97 86 97 50 50
100 122 80 83 98 120
2 mmol/l IO pmol/l
50 50 88
ND ND
(
o-phenanthroline N-ethylmaleimide p-hydroxymercuriphenylsulphonate antipain elastinal arphamenine puromycin
B
100 M/ml 7 &ml 100 /%/ml 100 M/ml 2 mmol/l 100 /G/ml I k%/ml
bacitracin bestatin
1 mmol/l 100 j.tmol/l 0.1 gmol/l
50
70 97 81
50 70 95 95 85
50 50
122 90
50
Enzyme and inhibitor were preincubated at the concentrations for 2 min in ice. Assays were started by addition of substrate, followed. ND: not determined.
shown in assay medium minus substrate and the standard assay procedure then
showed inhibition of activity following addition of phenyl methanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 pg/ml) or pepstatin (100 pg/ml). Effect of sulphyldryl reagents on enzyme activity With leucyl aminopeptidase, maximum activity was obtained in the absence of sulphhydryl reagents; no increase in enzyme activity was observed following addition of 2-mercaptoethanol, dithiothreitol, cysteine or glutathione (0.05-5 mmol/l). For pyroglutamyl aminopeptidase, the presence of a sulphhydryl reagent was an absolute requirement for the maintenance of enzyme activity; optimum activity was obtained in the presence of 1 mmol/l dithiothreitol.
Estimation of molecular mass The mol mass of leucyl aminopeptidase (determined by gel filtration chromatography on S-300) was estimated as 280 kDa. The mol mass of pyroglutamyl aminopeptidase (determined via gel filtration chromatography on Sephacryl S-100) was estimated as 22 kDa.
Estimation of K,,, dase and leucyl-AMC
A value for K, for the reaction between leucyl aminopeptiwas estimated (via linear regression analysis) as 1.5 mmol/l
44
from a Lineweaver-Burk plot of l/v vs l/[S] over the substrate cont. range 0.2-4 mmol/l [correlation coefficient (r) = 0.991. The K, value for the reaction between pyroglutamyl aminopeptidase and pyroglutamyl-AMC was similarly estimated as 80 pmol/l (substrate cont. range 20-250 pmol/l; r = 0.99). Enzyme activity on oligopeptide substrates The following oligopeptides were hydrolysed by leucyl aminopeptidase (N-terminal amino acid and relative hydrolysis rate shown in parentheses): luliberin 7-10 (Leu-Arg-; 100%); [Leu’lenkephalin (Tyr-Gly; 43.8%); proctolin (Arg-Tyr-; 9.7%). Peptides which were not appreciably hydrolysed by leucyl aminopeptidase included neurotensin (pGlu-Leu-), angiotensin-1 (Asp-Arg-), substance-P (Arg-Pro-) and bradykinin (Arg-Pro-). For the hydrolysis of [Leu’]enkephalin by leucyl aminopeptidase, only the N-terminal Tyr residue was removed; in the absence of Mg2+ (5 mmol/l) or Mn” (0.5 mmol/l) enzyme activity on the latter peptide substrate was reduced to less than 1.0% of optimum. Thyroliberin (pGlu-His-, loo%), neurotensin (pGlu-Leu, 59%) and luliberin (pGlu-His, 50%) were hydrolysed by pyroglutamyl aminopeptidase (N-terminal amino acid and relative hydrolysis rate in parentheses). In each case, only the N-terminal pGlu-residue was removed. Oligopeptides with free N-termini (e.g. [Leu’]enkephalin) or N-acetylated N-termini (e.g. melanocyte stimulating hormone) were not appreciably hydrolysed by pyroglutamyl aminopeptidase. In summary, we have described the characteristics of leucyl aminopeptidase and pyroglutamyl aminopeptidase following purification (as judged by non-denaturing polyacrylamide gel electrophoresis) from human post-mortem skeletal muscle tissue. Although each of these aminopeptidase types has been previously characterized to a varying degree in several other tissues/species [2], as far as we are aware this is the first report describing the purification and characterization of these enzymes from muscle tissue, particularly with respect to their action in the degradation of oligopeptides. The data obtained suggest that leucyl- and pyroglutamyl aminopeptidases are metalloenzymes with active site thiol group participation, although these enzymes differ otherwise in biophysical and optimal assay characteristics; for leucyl aminopeptidase the relevance of the high pH optimum of activity, and activation by Mg*+ or Mn” ions, to the subcellular localization and mode of action of the enzyme in vivo remains to be established. The specificity of pyroglutamyl aminopeptidase in hydrolysing oligopeptides or AMC derivatives is very restricted, with only substrates with an N-terminal pyroglutamyl residue being recognized. The specificity of leucyl aminopeptidase in hydrolysing oligopeptides is less restricted than for the latter enzyme, although not as broad as that expressed by alanyl aminopeptidase purified from human muscle [4]. The characteristics of leucyl aminopeptidase and pyroglutamyl aminopeptidase purified from human skeletal muscle are very similar to those for corresponding enzymes purified from human brain [9] and human kidney [lo] tissues, using a similar experimental approach. The occurrence of such enzymes, present in similar proportions in each tissue, with correspondingly similar characteristics, in such functionally dissimilar tissues as skeletal muscle, brain and kidney suggests that these enzymes are of fundamental importance in the general cellular protein catabolic process. The work described in
45
this paper completes the systematic purification and characterization of the aminopeptidase group of enzymes previously identified in human skeletal muscle tissue [3]; further work will now be required to establish the role of this group of enzymes in the degradation of key structural proteins within the muscle cell. References 1 Pennington RJT. Proteinases of muscle. In: Barrett AJ, ed. Proteinases in mammalian cells and tissues. Amsterdam: North-Holland, 1977;515-545. 2 McDonald JK, Barrett AJ. Mammalian proteases: a glossary and bibliography. Vol. 2: Exopeptidases. London: Academic Press, 1986. 3 Lauffart B, Mantle D. Rationalization of aminopeptidase activities in human skeletal muscle soluble extract. Biochim Biophys Acta 1988;956:304l-306. 4 Mantle D, Hardy MF, Lauffart B, McDermott JR, Smith AI, Pennington KJT. Purification and characterization of the major aminopeptidase from human skeletal muscle. Biochem J 1983;211:567573. 5 Mantle D, Lauffart B, McDermott JR, Kidd AM, Pennington RJT. Purification and characterization of two Cl- activated aminopeptidases hydrolysing basic termini from human skeletal muscle. Eur J Biochem 1985;147:307-312. 6 Oakley BR, Kirsch DR, Morris NR. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 1980;105:361-363. 7 Sedmak JJ, Grossberg SE. A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G250. Anal B&hem 1977;79:544-552. 8 Smith AI, McDermott JR. High performance liquid chromatography of neuropeptides using radially compressed polythene cartridges. J Chromatogr 1984;306:99-108. 9 Mantle D, Lauffart B, Perry EK, Perry RH. Comparison of major cortical aminopeptidase activity in normal brain and brain from patients with Alzheimer’s disease. J Neurol Sci 1989;89:227-234. 10 Mantle D, Lauffart B, McDermott JR, Gibson AM. Characterization of aminopeptidases in human kidney soluble fraction. Clin Chim Acta 1990;187:105-114.
