Isolation and partial characterization of rat elastolytic enzymes from various cells and tissues

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 250, No. 1, October, pp. 63-69, 1986

Isolation and Partial Characterization of Rat Elastolytic Enzymes from Various Cells and Tissues’ CONCETTA GARDI

AND

GIUSEPPE LUNGARELLA’

Institute of General Pathology, University of Siena, Siena 53100,Italy Received December 2, 1985, and in revised form May 6,1986

Different elastolytic enzymes were isolated from rat aorta and platelets, as well as from granulocyte and pancreatic extracts. The active fractions were purified to electrophoretic apparent homogeneity by precipitation with ammonium sulfate, sequential batch fractionation on DEAE-Sephadex A-50, and finally by isoelectric focusing (IF) on Sephadex G-75 Superfine. The molecular weight and the isoelectric point of the isolated enzymes were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by analytical IF, respectively. All the enzymes have elastolytic activity as well as activity toward Sue-(Ala)a-NA. The inhibition profile of the different isolated enzymes toward various inhibitors indicates that aortic, pancreatic, and granulocyte enzymes all belong to the group of serine proteinases, unlike the platelet elastase which is a metalloprOtekw%

0 1986AcademicPress,Inc.

During the past several years much research has been directed toward identification and isolation of elastolytic enzymes from various cells and tissues. Such enzymes are thought to play important roles in a diverse range of physiological processescharacterized by limited proteolysis, or in several pathological conditions characterized by uncontrolled destruction of structural proteins (1). In particular elastic tissue destruction, as observed in emphysema, atherosclerosis, and other pathological processes, is usually attributed to an imbalance between elastase(s) and its naturally occurring inhibitors (2, 3). This view is supported by the fact that in several experimental animal models the release of elastolytic enzymes in extracellular fluids in concentrations sufficient to overwhelm available inhibi-

tar(s) results in a variety of tissue lesions (4, 5). However, these “in vim” models (which involve a plurality of cell types, proteases, and substrates) are still very complex, and make interpretations of interactions between individual proteasecontaining cells and protein substrates very difficult. Therefore the identification and the characterization of enzymes with elastolytic activity in cells and tissues of various experimental animals should be of great interest to laboratories involved in studies on the pathogenetic role of these proteases. In a previous paper (6), a method was described for isolation of rat pancreatic and leukocyte elastases, and some physical properties of these enzymes were identified. The presence of an elastase-like activity in rat aorta smooth muscle cells has been reported (7), and this enzyme was partially purified and characterized (8). However, presumably due to the difficulty in obtaining sufficient quantities of material, detailed studies have not been done. In addition, although the purification of elas-

1 This work was supported by a grant from Consiglio Nazionale delle Ricerche, Rome, Italy (Progetto Finalizzato Oncologia). 2 To whom correspondence should be addressed. 63

0003-9861/86 $3.00 Copyright 0 1986by AcademicPress,Inc. All rights of reproductionin any form reserved.

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tase-like enzyme from human platelets (9) has been reported, little is known about its rat counterpart. In this paper we described the purification to homogeneity of elastolytic enzymes from rat platelets and aortas. The properties of these enzymes were compared with those of pancreas and granulocyte enzymes, MATERIALS

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METHODS

Materials. The following materials were obtained from the indicated sources: DEAE-Sephadex A-50 and Sephadex G-75 Superfine, from Pharmacia Fine Chemicals, Sweden; SAPNAT elastin-Congo red, elastatinal, o-phenanthroline, soybean trypsin inihibitor, chicken ovoinhibitor, PMSF, pepstatin, and leupeptin from Sigma; carrier ampholytes from LKB, Sweden; molecular weight standards from Bio-Rad. Other reagents were of the highest quality available and were used without further purification. Aniwtak Male Sprague-Dawley rats (250-350 g) maintained on a pellet diet (Nossan, Correzzana, Milan, Italy) were used. Purijicatim of rat ehtolytic enzymes. All the following operations were carried out at 4°C unless otherwise stated. Step 1. The pancreatic tissue (from 100 animals) cleaned from the extraneous tissues was frozen immediately in dry ice, stored at -2O”C, and thawed just before use. Pancreatic tissue (45 g) was homogenized in a 1:5 (w/v) volume of 0.2 M NaHCOa-NaOH buffer, pH 8.4, with an Ultraturrax tissue homogenizer. The suspension was filtered through glass wool to remove pieces of fat tissue, stirred for 2 h at room temperature (to activate proelastase), and dialyzed overnight against distilled water. Then the suspension was stirred with an equal amount (200 ml) of 0.2 M sodium acetate buffer, pH 4.5, for 3 h and centrifuged for 30 min at 5OOOg.The supernatant, amounting to 300 ml, designated “pancreas acetone extract,” was stored at -20°C and thawed just before use. The granulocyte lysosomal fraction was obtained from 1.2 liters of rat blood according to the procedure described for rabbit granular (lysosomal) fraction (10). The granulocyte lysosomal fraction was homogenized in 0.1 M sodium acetate buffer, pH 4.5, with a glass/glass Potter-type homogenizer. Homogenization was performed in several batches using a minimum volume

a Abbreviations used: SDS-PAGE, sodium sulfate-polyacrylamide gel electrophoresis; polyacrylamide gel isoelectric focusing; IF, tric focusing; PMSF, phenylmethylsulfonyl SAPNA, Sue-(Alah-NA.

dodecyl PAGIF, isoelecfluoride;

LUNGARELLA of buffer; each aliquot of granule preparation was subjected to 50 strokes with the glass homogenizer. Homogenates were centrifuged at 20,OOOgfor 15 min, and the supernatants poured off and stored at -20°C. The preparation of rat platelet lysate was carried out as follows: 1.2 liters of blood, drawn from 100 animals, was mixed with an ACD solution (citric acid, 7 mM; sodium citrate, 0.1 M; dextrose 0.14 M) in a ratio of nine parts of blood to one part of anticoagulant. The platelet-rich plasma was separated by centrifugation for 15 min at 100s at 20°C reacidified with a 1:20 volume 0.1 M EDTA, and further centrifuged at 30009 for 15 min to precipitate platelets. The precipitate was washed twice with modified Tyrode’s buffer (11) (containing 2 mM EDTA and 0.35% bovine serum albumin) by centrifugation, yielding a platelet preparation of over 99% purity, as judged by phase-contrast or bright-field microscopy using a differential Wright stain. The washed platelets were lysed by sonication with a 100-W MSE ultrasonic disintegrator, equipped with “microtip” at 12 pm wave amplitude (four sonications, 15 s each) in 0.1 M sodium acetate buffer, pH 4.5, and extracted twice with the same buffer for 24 h. The preparation was centrifuged at 30009 for 30 min, and the supernatant designated “platelet acetone extract” was used for the enzyme separation. For the preparation of rat aortic crude extract, the adventitia of aorta was carefully stripped off, and the two layers (intima and media) (18 g from 100 animals) were cut into small pieces, homogeneized (with an Ultraturrax tissue homogenizer) in a 1:5 (w/v) volume of saline, and washed with the same solution, until the supernatant was colorless. After centrifugation at 3000g for 15 min, the residue was extracted with a 1:5 (w/ v) volume 0.5 M sodium acetate, pH 4.0,0.2 M EDTA, 0.2% (w/v) sodium azide, during 24 h under mechanical stirring. This extraction was repeated twice, and the pooled supernatants, designated “aortic acetone extract,” were dialyzed, stored at -2O”C, and thawed just before use. Further purification of the enzyme activities from the above-mentioned extracts of cells and tissues was achieved by two additional steps consisting of (a) ammonium sulfate precipitation, followed by a batchwise fractionation on DEAE-Sephadex A-50, and (b) preparative IF in Sephadex G-75 Superfine. Step2. Fractional precipitation of the various preparations with ammonium sulfate was accomplished by adding an adequate amount of ammonium sulfate for a final concentration of 45% of saturation (for pancreatic extract) and of 60% of saturation (for aorta, granulocyte, and platelet preparations). The resulting suspensions were stirred 1 h at room temperature and left overnight at +4’C. The sediments obtained by centrifugation at 40,OOOgfor 15 min were then dissolved in 0.05 M Na&03-HCI buffer, pH 8.8, and extensively dialyzed against water to remove salt. The samples were centrifuged at 4O,OOOg,the supernatants

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were discarded, and the resulting precipitates were recovered and washed twice with distilled water. The dialyzed ammonium sulfate fractions were further processed by a batchwise fractionation procedure on DEAE-Sephadex A-50, at pH 8.8, essentially similar to that of Smillie and Hartley (12). The filtrates obtained after DEAE-Sephadex fractionation were extensively dialyzed against several changes of 1 mM acetic acid and freeze-dried. The salt-free, freeze-dried DEAE-Sephadex filtrates were dissolved in distilled water (to give a final protein concentration of about 50 mg/ml) and then subjected to preparative IF in a bed of Sephadex G-75 Superfine to further purify the enzyme activities. Step 3. IF was performed in (110 X 230 X 3.5-mm) layer gels, with a pH range 7-10, containing 4% Sephadex G-75 Superfine and 2% carrier ampholytes (pH 7-9 and 9-11 in equal amount) in an initial slurry volume of 100 ml. The corresponding bed volume was about 70 ml. The procedure used for preparing granulated gels was similar to that described in LKB Application Note 198. The samples (3 ml for each gel) were applied 1.5 cm from the anode; 1 M NaOH and 1 M H3PO1, respectively, were used as cathode and anode electrolytes. IF was carried out at 16 W constant power for 6 h using a LKB 2117 Multiphor II, with a LKB 2197 power supply, maintained at 4°C by a circulating water bath (Ministat, Huber, Germany). After completion of the electrophoretic run, the proteins with enzyme activity toward SAPNA were localized in the focused gels by the aid of a zymogram method as described previously (6). The gel within areas containing enzyme activity was then removed, transferred into centrifuge tubes, suspended 1:1.5 (v/v) in distilled water, and centrifuged at 35,000g for 20 min. The enzymes contained in the supernatants were recovered and separated from ampholytes by ammonium sulfate precipitation. The homogeneity of the purified material was checked by SDS-PAGE and analytical IF. Pooled fractions obtained during various steps of purification were extensively dialyzed and assayed for enzyme activity against SAPNA (13) and for protein concentration (14). Protein determination. The protein concentration was estimated by the method of Lowry et el (14) using bovine serum albumin as standard. Enzyme assays. Elastolytic activity of purified enzymes was determined by the method of Shotton (15) with Congo red-elastin as substrate. Enzyme activity of the purified enzymes and of the pooled fractions obtained during the various steps of purification was assayed with the elastase-specific chromogen substrate SAPNA according to Bieth et el (13). A unit of enzyme activity was defined as the hydrolysis of 1 pM substrate produced per minute at pH 8.0 at 25°C. Tryptic and chymotryptic activities were assayed according to Erlanger et el (16) and Del Mar et el (17), respectively.

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Kinetic studies. Hydrolysis of SAPNA at 410 nm was employed for K,,, studies. All assays were performed in 0.2 M Tris-HCl, pH 8.0. Substrate was used at a minimal range of concentration of 0.1-3 times K,. Purified enzymes were employed at a final concentration of 0.2,0.4, or 0.6 pg/ml. The data were plotted according to the method of Lineweaver and Burk (18) and the parameters K,,, and V,, were estimated by an interactive least-squares fit to the MichaelisMenten equation. Inhibitor studies. Seven proteinase inhibitors were used to characterize the enzyme activities. These included elastatinal, o-phenanthroline, soybean trypsin inhibitor, chicken ovoinhibitor, PMSF, leupeptin, and pepstatin. The enzymes were preincubated with each inhibitor in 0.2 Tris-HCl buffer, pH 8.0, at 25°C for 30 min before assaying. The residue enzyme activities were measured at 410 nm toward the synthetic substrate SAPNA (13). Acqhmide elextrophuresis. SDS-PAGE was carried out according to Laemmli (19). For the molecular weight determination of purified enzymes, bovine serum albumin (ilf, 66,500), ovalbumin (&& 45,000), carbonic anhydrase (Af, 30,000), soybean trypsin inhibitor (il4,21,500), and lysozyme (iV& 14,400) were used as marker proteins. IF was performed in an LKB flatplate apparatus (LKB 2117) using 5% polyacrylamide gels, pH 7.9-10.0, containing 2% carrier ampholytes. RESULTS

Different rat proteinases with enzyme activity toward SAPNA were purified to homogeneity from various cell and tissue preparations. The purification scheme for the isolation of rat enzymes, the protein recoveries, and the enzyme activities on SAPNA at each step of purification are given in Table I. As can be seen, the initial two steps of purification provided reasonably pure enzyme activities in good yields from all the different preparations. In addition, the pancreatic filtrate obtained after DEAE-Sephadex A-50 fractionation showed no detectable enzyme activities when assayed for tryptic and chymotryptic activities. Furthermore this fraction showed at SDS-PAGE analysis the disappearance, in the molecular weight region ranging from 21.5K to 31K (Fig. l), of several protein bands usually occurring in ammonium sulfate fraction. A further substantial purification of the enzyme activities (as judged by the marked increase in the specific activity) was observed for all the different enzyme prepa-

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TABLE

I

SUMMARY OF PURIFICATION OF RAT ELASTASES FROM VARIOUS CELLS AND TISSUES

Source

Step”

Total protein (mg)

Total activity (units)

Specific activity (units/mg protein)

Yield (W)

Pancreas

I IIa IIb III

1113.6 184.1 44.5 21

557 534 436 295

0.5 2.9 9.8 14

100 95.9 78.3 52.9

Aorta

I IIa IIb III

2966.1 111.5 25.5 4.5

359 301 171 62

0.1 2.7 6.7 13.8

100 83.8 47.6 17.2

Granulocyte

I IIa IIb III

1420.3 290.1 34.8 3.1

298 253 122 46

0.2 0.9 3.5 14.9

100 84.8 40.9 15.4

Platelet

I IIa IIb III

888.3 63 28.3 1.8

45 41 31 19

0.05 0.65 1.1 10.5

100 91.1 68.8 42.2

Purification factor 1 5.8 19.6 28 1 27 67 138 1 4.5 17.5 74.5 1 13 22 210

a I, II, and III represent the purification steps described under Materials and Methods. IIa: ammonium sulfate precipitation; IIb: DEAE-Sephadex A-50 fractionation. The protein content and the enzyme activity toward SAPNA were determined as described under Materials and Methods.

rations after preparative IF. The purification procedure detailed above yielded enzyme preparations with approximately 2%fold (pancreas), 13%fold (aorta), 74-fold (granulocyte), and 210-fold (platelet) purification compared with the initial extracts. Samples of the purified enzymes were subjected to analytical PAGIF and to SDSPAGE according to a procedure previously described in detail (6). These proteins were judged pure by the presence of only single bands with enzyme activity on SDS-acrylamide gels (Figs. 1 and 2) after IF. Molecular weights and isoelectric points of the purified enzymes are reported in Table II. The K, for SAPNA as well as the proteolytic activity on elastin-Congo red by the various isolated enzymes are also given in Table II. The K, values for the hydrolysis of SAPNA (the most commonly used substrate for porcine pancreatic elastase) are quite similar to pancreatic, aortic, granulocyte, and platelet enzymes, even if the inherent efficiency (as judged from the

values of the specific activities of the different rat enzymes; Table I) is somewhat lower for the platelet enzyme. In addition, as can be seen in Table II, aortic, pancreatic, granulocyte, and platelet proteases all hydrolyze insoluble elastin but to a very different extent. The results obtained with the variety of inhibitors tested (Table III) provide evidence that platelet elastase is a metalloproteinase, whereas aortic enzyme is a serine proteinase. The activity of the aortic enzyme toward SAPNA was, in fact, completely inhibited by PMSF, a potent inhibitor of serine proteinases, whereas it was unaffected by o-phenanthroline, a potent inhibitor of metalloproteinases. On the contrary, platelet enzyme was completely inhibited by o-phenanthroline, but insensitive to PMSF. In addition, the pancreatic, granulocytic, and aortic elastases show a different inhibition profile toward some inhibitors (like soybean trypsin inhibitor or chicken ovoinhibitor) even if they all belong to the serine proteinase group.

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ELASTASES

-i

a

b

b

a

c

FIG. 1. SDS-PAGE of various rat pancreatic preparations in an 8-15% gradient gel of 0.75mm thickness. (a) Purified rat pancreatic elastase; (b, c) samples of pancreatic extracts after DEAE-Sephadex A-50 fractionation and ammonium sulfate precipitation, respectively; (d) reference protein mixture comprising ovalbumin (A& 45,000), carbonic anhydrase (M, 31,000), bovine trypsinogen (M, 24,000), soybean trypsin inhibitor (iWr 21,5000), and lysozyme (iW, 14,400). After electrophoresis, the gel was fixed and stained with Coomassie brilliant blue. DISCUSSION

Several rat elastolytic enzymes from various cell and tissue extracts were puTABLE

c

d. e,

f

FIG. 2. SDS-PAGE of purified enzymes in an 8-15% gradient gel of 0.75-mm thickness. (a) Rat aortic elastase; (b) rat pancreatic elastase; (c, d), enzyme activities from rat leukocyte extract, focused at pH 8.2 and 8.8, respectively; (e) rat platelet elastase; (f) low molecular weight SDS standard protein mixture with (top to bottom) carbonic anhydrase (Afr 31,000) and soybean trypsin inhibitor (Mr 21,500). The slab gel was stained with Coomassie brilliant blue.

rified to apparent electrophoretic homogeneity by preparative isoelectric focusing on Sephadex G-75 Superfine after a second purification step consisting of ammonium sulfate precipitation and sequential batch fractionation on DEAE-Sephadex A-50. II

COMPARISON OF SOME PROPERTIES OF PURIFIED RAT ELASTASES FROM PANCREAS, GRANULOCYTE, AORTA, AND PLATELET Pancreatic Molecular weight Isoelectric point K, (mM) for hydrolysis of SAPNAd Proteolytic activity on elastin-Congo Redf

23,600 8.75

Aortic

Granulocyte 26,000” 8.8”

26,000 b 8.2’

24,000” 8.2”

Platelet

22,300 8.7

25,500 8.45

0.93 + 0.03

1.16’ & 0.02

1.86 f 0.04

2.1 ?I 0.04

100

17”

21

4

amcDifferent forms of granulocyte elastase (6). d Expressed as mean + SD of triplicate determinations. e Values obtained for pooled forms. jThe activity of the same amount of protein of the different enzymes was determined under the same experimental conditions. The enzyme activities were compared with that of pancreatic enzyme and expressed as percentage of this value. Values represent the means of triplicate analyses.

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TABLE

III

EFFECTS OF INHIBITORS ON DIFFERENT RAT ELASTASES Enzyme activity

Inhibitor PMSF” o-Phenanthroline Elastatinal Soybean trypsin inhibitor Chicken ovoinhibitor Pepstatind Leupeptin

Concentration

Pancreatic elastase

Granulocyte elastase*

(% of control

value)”

Aortic elastase

Platelet elastase

1.0 mM 1.0 mM 100 pgg/ml

0 100 5

0 100 17

0 95 0

100 0 7

100 100 10 10

90 0 100 100

23 21 100 100

56 0 100 97

0 2 100 100

pg/ml rg/ml pg/ml pg/ml

Note. The enzymes were preincubated with each inhibitor in 0.2 M Tris-HCl (pH 8.0) at 25°C for 30 min before assaying. The activity was determined on SAPNA and was expressed as a percentage of the control activity in the presence of each solvent. “Values obtained from three different measurements. *Values obtained for pooled forms of granulocyte elastases. ’ Isopropanol (4% ,v/v) present in preincubation mixture. d Dimethyl sulfoxide (4%,v/v) present preincubation mixture.

The second purification step turned out to be necessary to avoid the disturbances detected, in preliminary experiments, when the initial extracts from the different sources were subjected to preparative IF in a narrow pH range. These disturbances (revealed as protein precipitation, gradient drift, and wavy bands), caused by protein overloading and by the presence of anionic proteins, disappear after the second purification step. In addition the consistent increase in the specific activity observed for all the different preparations after the second step of purification enabled us to apply, on IF gels, sample laods with high activity and thereby to recover enzyme activities in a good yield. Even if this procedure (because of the consistent losses of some enzyme activities) appears much less convenient than that described by others for purifying elastase activities (20-22), actually it provided very pure enzyme preparations readily available for further characterization. Although several investigators (see Ref. (9) for a review) have raised some doubts about the existence of true elastase activity in platelet and aorta, our own results clearly indicate that rat platelet and aorta enzymes are true elastases. In addition, the

enzyme activity isolated from aorta and platelet extracts is undoubtedly due to elastases different from those isolated from pancreatic and granulocyte extracts. This fact is clearly supported by the different physical and biochemical properties of the various isolated enzyme preparations. Recently, Hornebeck and Legrand (9) purified and partially characterized elastolytic enzymes from pig and human aortas. Aside from minor differences in substrate specificity toward SAPNA, rat aortic elastase can be said to be analogous to human and pig counterparts with respect to the enzymatic activity on insoluble elastin, molecular weight, and inhibition profile. Unexpectedly, a marked difference was found between the elastin-degrading enzyme isolated from rat platelets and that isolated from human platelets (9). The latter enzyme, in fact, was found inactive toward synthetic substrate SAPNA, and furthermore was characterized on the basis of its inhibition profile as a serine proteinase. Further characterization of the catalytic mechanisms, substrates, inhibitors, and interrelationships among different enzyme forms will be necessary to help define the potential biological functions of aortic and

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platelet elastases. Nevertheless, the availability of pure elastases from platelet and aorta is expected to facilitate studies on the possible role played by elastolytic enzymes in different pathological processes. The question of the possible involvement of different elastases (pancreatic, aortic, or platelet) in atherosclerosis has yet to be clearly answered. REFERENCES 1. WERB, Z., BANDA, M. J., MCKERROW, J. H., AND SANDHAUS, R. A. (1982) J. Invest. Dermatol. 79, 154s-159s. 2. STOCKLEY, R. A. (1983) Clin S& 64,119-126. 3. SCHNEBLI, H. P. (1985) in Handbook of Inflammation, Vol. 5, The Pharmacology of Inflammation (Bonta, I. L., Bray, M. A., and Parnham, M. J., eds.), pp. 321-333, Elsevier, Amsterdam. 4. LUNGARELLA, G., GARDI, C., DE SANTI, M. M., AND LUZI, P. (1985) Exp. MoL Puthol. 42,44-59. 5. ABRAMS, W., COHEN, A. B., DAMIANO, V., ELIRAZ, A., KIMBEL, P., MERANZE, D. R., AND WEINBAUM, G. (1981) J. Clin Invest. 68,1132-1139. 6. GARDI, C., AND LUNGARELLA, G. (1984) And B&hem. 140,472-477. 7. BOURDILLON, M. C., BRECHEMIER, D., BLAES, N., DERQUE~E, J. C., HORNEBECK, W., AND ROBERT, L. (1980) CeU BioL Id. Rep. 4,313-320. 8. HORNEBECK, W., BRECHEMIER, D., BOURDILLON, M. C., AND ROBERT, L. (1981) Connect. Tks. Res. 8,245-249.

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9. HORNEBECK, W., AND LEGRAND, Y. (1980) in Frontiers of Matrix Biology (Robert, L., ed.), Vol. 8, pp. 199-215, Karger, Bagel. 10. FONZI, L., AND LUNGARELLA, G. (1979) Exp. Mol. PuthoL 31,486-491. 11. TYRODE, M. V. (1910) Arch Int. Pharmacodyn. Ther. 20,205-212. 12. SMILLIE, L. B., AND HARTLEY, B. S. (1966) Biochem. J. 101,232-241. 13. BIETH, J. G., SPIESS,B., AND WERMUTH, C. G. (1974) Biochem. Med. 11,350-357. 14. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol Chem. 193, 265-275. 15. SHOWON, D. M., in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 19, pp. 113-140, Academic Press, New York. 16. ERLANGER, B. F., KOKOWSKY, N., AND COHEN, W. (1961) Arch B&hem. g&271-276. 17. DEL MAR, E. G., LARGMAN, C., BRODICK, J. W., AND GEOKAS, M. C. (1979) Anal. Biochem. 99, 316-325. 18. LINEWEAVER, H., AND BURK, D. (1934) J. Amer. Chem. Sot. 56,658-665. 19. LAEMMLI, U. K. (1970) Nature @on&m) 227,680685. 20. SCHMIDT, W., AND HAVEMANN, K. (1974) HoppeSeyler ‘s 2. Physiol Chem 355,1077-1082. 21. BAUGH, R. J., AND TRAVIS, J. (1976) Biochemistry 15,836-841. 22. ARDELT, W., TOMCZAK, Z., KSIEZNY, S., AND DUDEKWOJCIECHOWSKA, G. (1976) Biochim. Biophys. Acta 445, 683-693.