The appearance of free hydroxyproline as the major product of degradation of newly synthesized collagen in cell culture

Biochimica et Biophysica A cta, 719 (1982) 480-487

480

Elsevier Biomedical Press BBA 21304

THE APPEARANCE OF FREE HYDROXYPROLINE AS THE MAJOR PRODUCT OF DEGRADATION OF NEWLY SYNTHESIZED COLLAGEN IN CELL CULTURE MICHAEL IMBERMAN, FRANK OPPENHEIM and CARL FRANZBLAU

Department of Biochemistry, Periodontics and Oral Biology, Boston University Medical Center, Boston, MA 02118 (U.S.A.)

(Received April 27th, 1982)

Key words: Collagendegradation, Hydroxyproline, Cellculture

Embryonic lung fibroblasts and rabbit vascular smooth muscle cells have the ability to degrade newly synthesized collagen. Analysis of 24-h pulse media from cultures given [14C]proline demonstrates that greater than 90% of the degraded collagen is represented by free hydroxyproline rather than the peptide-bound imino acid. The addition of cycloheximide or a-a-dipyridyl to the culture medium during the pulse period severely diminished the formation of the free hydroxyproline demonstrating its enzymatic and protein (collagen) origin. It is proposed that assessment of free hydroxyproline formation may allow us to distinguish between intracellular and extracellular collagen degradation.

Introduction The degradation of newly synthesized collagen in cell cultures has been demonstrated to be an intracellular event with the lysosome probably playing a major role [1,2]. However several questions exist relating to the products of this degradation and its relationship to the in vivo situation. If the cell produces hydroxyproline containing peptides [3] one would expect to see such peptides in urine since they have been reported to be resistant to further degration [4]. Approx. 95% of the hydroxyproline in urine is peptide-bound [5] and its level is usually taken as a mearure of collagen turnover [6]. It is known that mammals have enzyme systems capable of metabolizing free hydroxyproline [7] which is present in higher quantities in serum than in urine [8]. Thus if free hydroxyproline is a product of collagen degradation, it may be further modified before entering the urine and thus not detected. Although proteases have been identified which could degrade extracellular collagen to free amino acids, the exact sequence of these proteolytic reactions is not un0304-4165/82/0000-0000/$02.75 © 1982 Elsevier BiomedicalPress

derstood [6]. It has also been suggested that the existence of free hydroxyproline or hydroxyproline-containing peptides could be the result of a nonenzymatic hydroxylation of proline and therefore not necessarily represent collagen degradation [9]. Results presented in this communication demonstrates that the major product of intracellular degradation of newly synthesized collagen is indeed free hydroxyproline which is of enzymatic and protein (collagen) origin.

Materials and Methods (,4) Cell culture (1) Human embryonic lung fibroblasts. IMR-90 fibroblasts were used at PDL 20. The cells in culture were maintained in 75-cm2 plastic tissue culture flasks with 40 ml Eagle's minimal essential medium containing Earle's balanced salts supplemented with glutamine (2 mM), 10% fetal bovine serum, penicillin (100 units per ml) and streptomycin (100 /~g per ml) [10]. The flasks were incubated at 37°C in a humidified atmosphere of 5% CO 2 and 95% air and subcultured by trypsini-

481

zation. All subsequent seedings involved the addition of 0.75 x 106 cells to a flask containing 40 ml of complete medium. (2) Vascular smooth muscle cells. Smooth muscle cell cultures were prepared as outgrowths from 1 mm 3 explants of the inner media of the thoracic aorta arch of weanling New Zealand white rabbits [11]. Primary cells were detached from explants after 2 weeks in culture with 0.05% trypsin/0.02% EDTA solution and seeded into 75 cm 2 plastic tissue culture flasks at a density of 1.5 x 106 cells. After 1 week, at which time they were confluent, the cells were subcultivated a second time. These second passage cells, also plated at 1.5 x 106 cells per flask, were maintained in the same flasks for approx. 1-2 weeks. They were fed twice weekly until used with 20 ml of growth medium (Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and per ml, 100 units of penicillin and 100 #g streptomycin).

(B) Radiolabeling and measurement of degradation of newly synthesized intracellular collagen IMR-90 fibroblasts (PDL 20) and rabbit aortic smooth muscle cells (2nd passage) were plated at 0.75 x 106 and 1.5 x 106 cells, respectively in 75cm 2 flasks and maintained at 37°C (5% Co 2 in a humidified atmosphere). All experiments were performed on cells which had reached confluence, usually 7-8 days after seeding. Cells were prefed 24 h prior to labeling with media containing 50 /~g/ml sodium ascorbate. To evaluate collagen degradation, media were replaced 1 h before pulsing with radioactive precursors in 10 ml of minimal essential medium (containing 10% dialysed fetal bovine serum, nonessential amino acids without proline, ascorbate, sodium pyruvate, penicillinstreptomycin). Cells were then pulsed with the above media containing 2 / t C i / m l [laC]proline (NEC-285E 286.0 mCi/mmol). After 24 h the media was separated from the cells and combined with a saline wash. After removing the cell layer from the flask with saline, the separated media and cell layer were placed in a 100°C water bath for 15 min to solubilize native collagen and destroy any protease activity. Following homogenization of the cell layer in a glass homogenizer, non-degraded collagen ( M r > 25000) was sep-

arated from collagen degradation products ( M r < 25 000) by placement on Amicon centriflow membrane cones (CF 25). After soaking of filters for 1 h in deionized H20, the filters were prespun with 2 ml H 2 0 to remove any detergent. Following placement of a maximum of 3 ml onto the filter, the sample was centrifuged at 1000 x g for 60 min. After a wash of 1 ml H20, the filtrate was analyzed. Samples of the media and cell layer were taken before and after separation and hydrolyzed in 6 N HC1 at 110°C for 20 h. Hydrolysates were placed on a Beckman amino acid analyzer equipped with a split stream arrangement to determine the presence of [14C]hydroxyproline. The level of contaminating [14C]hydroxyproline contained within the [laC]proline used in the above experiments was monitored.

(c) Demonstration of the enzymatic nature of hydroxylation of proline To evaluate the origin of the hydroxyproline, either a,a-dipyridyl (0.2 mM) or cycloheximide (20 /~g/ml) was added to the culture medium during the 1 h prefeed and the pulse period. The analysis of total hydroxyproline and degradation of newly synthesized collagen are described above.

(1)) Separation of small peptides and free amino acids Separation of hydroxyproline containing peptides from free hydroxyproline was accomplished on a Bio-Gel P2 column. Pulsed media, after removal of the large radioactive proteins with the aid of centriflo cones, was lyophilized, dissolved in water and placed on a Bio-Gel P2 column (1.6 x 86 cm, 400 mesh). The column was equilibrated and eluted with 0.05 M ammonium bicarbonate, pH 8.0, and 0.5% chloroform. Fractions of 3.4 ml (20 min) were collected, hydrolyzed in 6 N HCI and analyzed for the presence of [lac]hydroxyproline. To standardize the column 5 mg each of proline ( M r 115), hydroxyproline ( M r 131), Gly-pro ( M r 172), Gly-Pro-Hypro ( M r 325) and albumin ( M r 60000) were applied separately and in combination. Pulse media treated as in previous experiments without the presence of cells was used as a control. The effect of boiling on the distribution of the hydroxyproline was also analyzed.

482

(E) Demonstration of existence of free hydroxyproline (1) Amino acid analysis before and after hydrolysis. The presence of [14C]hydroxyproline and

grams were divided into 1 cm sections, vortexed in 1 ml of 30% ethanol and analyzed for the presence of 14C radioactivity using a scintillation counter.

[t4C]proline in the medium and cell layer was determined by first hydrolyzing each sample in 6 N HC1 at 110°C for 20 h. These hydrolysates were dried under nitrogen, reconstituted with 0.01 N HC1, and placed on a Beckman amino-acid anlayzer with a split-stream arrangement. To demonstrate the existence of free hydroxyproline, samples of pulsed media (after the removal of large proteins with centriflo cones) were either adjusted to pH 2.0 and placed directly on the analyzer or hydrolyzed as above. A comparison of the amount of 14C-eluting under the hydroxyproline peak (as determined by standards) were made. An increase in [14C]hydroxyproline after hydrolysis would suggest the existence of a significant level of hydroxyproline containing peptides. Little or no increase would demonstrate the presence of primarily free hydroxyproline. An additional control was the use of the peptide Gly-Hypro in the media. Any breakdown of the peptide as detected by an amino acid analyzer might suggested additional degradation of small collagen derived peptides after secretion by the cells. (2) Mixed paper chromatography. The existence of free hydroxyproline can be demonstrated by comparing the diffusion of the sample in various solvents in comparison with standards [12]. Pulsed media filtrates (before and after hydrolysis) were placed on an amino acid analyzer with split stream to separate the 'hydroxyproline' peak. The individual hydroxyproline fractions were desalted on a Dowex-50 column, lyophilized, and mixed with unlabeled hydroxyproline. Following this the samples were chromatographed on Whatman No. 1 paper at room temperature in two different ascending solvent systems; n-butanol-acetic acidH20 (12 : 3 : 5 v/v); and Phenol-n-butanol-methyl ethyl ketone-propionic acid-acetic acid-H2 ° (20 : 20 : 50 : 10 : 10 : 20 v/v). After 6 h, the chromatograms were removed from the chamber, dried in a 100°C oven for 3 min and sprayed with 0.2% ninhydrin in ethanol to visualize the localization of the unlabeled hydroxyproline. After visualization with ninhydrin, several lanes on the chromato-

Results

(A) Degradation of newly synthesized collagen The methods developed for the separation of large and small hydroxyproline containing peptides were both efficient and reproducible. The results within an experiment generally had standard deviations of less than 2% (Table I). The deviation was somewhat higher for comparison of repeated experiments. As mentioned earlier, the level of contamination of the [14C]proline used in these studies was constantly monitored. Commercially produced [14C]proline preserved in 2% ethanol (New England Nuclear) was the only product tested that consistantly had background levels of less than 0.1%. Approx. 5-15% of the hydroxyproline of M r less than 25 000 was found in the cell layer with the remaining 85-95% in the media. The omission of boiling from the experimental protocol had little effect on these results. Background levels of hydroxyproline were measured by incubation of [14C]proline in pulse media in the absence of cells and treated as explained above. Increases over contamination levels during the experiment were insignificant in the analysis of the data. Percent degradation of collagen was determined as follows: % Degradation cpm [14C]hydroxyproline ( M r < 25 000) × 100 total cpm [ 14C]hydroxyproline

(B) Effect of a,a-dipyridyl and cycloheximide on levels of hydroxyproline To demonstrate that the free hydroxyproline originates from previously synthesized collagen, rather than from free proline, we treated the cell cultures with either a,a-dipyridyl, an inhibitor of prolyl hydroxylase [13] or cycloheximide, a general inhibitor of protein synthesis. If a significant amount of the hydroxyproline had a nonenzymatic or non-protein origin, the use of the above inhibitors would only effect its level in collagen and have little effect on the total amount of filterable

483 TABLE I D E G R A D A T I O N OF N E W L Y SYNTHESIZED C O L L A G E N [ ~4C]Hydroxyproline determination after 24-h pulse period. Filterable hydroxyproline separated as fraction with a molecular weight of less than 25000. Data shown are averages of two flasks and demonstrates distribution of [14C]hydroxyproline in media and cell layer filterable hydroxyproline after hydrolysis. Values represent c m p / f l a s k x 10-3. % Degradation - total hydroxyproline (filterable + n o n - filterable) × 100. Cell type

Rabbit smooth muscle cell

A. B. C.

H u m a n embryonic lung fibroblasts

A. B. C.

Media Cell Layer Total Media Cell Layer Total

Total hydroxyproline

Filtrate hydroxyproline

1760

229

547 2 307

18 247

636

108

137 773

18 126

hydroxyproline. If, on the other hand, the hydroxyproline is primarily from collagen and of protein origin, the above inhibitors would effect both filterable and non-filterable hydroxyproline. When either of the above inhibitors were added to the culture system (Table II), it inhibited the formation of the major bulk of the hydroxyproline in both molecular weight fractions. Noticeably, as one inhibited the hydroxylation of proline, there was an increase in the percent degradation of the collagen produced.

%Degradation

10.8+0.4%

16.3 _+ 1.6%

While hydroxyproline synthesis was not completely inhibited in this experiment, the cycloheximide inhibited approximately 98% of its formation. The et,a-dipyridyl inhibited 91 and 75% of the hydroxyproline formation in fibroblasts and smooth muscle cells, respectively.

(C) Separation of small peptides and free amino acids To determine the size distribution of hydroxyproline containing peptides, a BioGel P2 column

T A B L E II INHIBITION OF HYDROXYPROLINE FORMATION [14 C]Hydroxyproline determination after 24-h pulse period. Data shown are cpm [t4C]hydroxyproline per flask after separation on filter cones and hydrolysis, and represent the average of two flasks. All conditions are the same except for the addition of either a, a-dipyridyl or cycloheximide beginning 1 h before the pulse period. Retentate ( M r > 25 000)

Filtrate ( M r < 25 000)

%Degradation

Smooth muscle cells A. Control B. a, a-Dipyridyl (0.2 mM) C. Cycloheximide (20 u g / m l )

2061000 476 000 24000

247000 95 000 16000

10.8 16.8 39.3

Fibroblasts A. Control B. a,a-dipyridyl (0.2 mM) C. Cycloheximide (20 btg / m l )

646000 46000 8 000

126 000 22000 4 000

16.3 32.6 44.9

484

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TABLE III PERCENT FREE [~4C]HYDROXYPROLINE IN MEDIA FILTRATE

AI

24-h pulse media after separation on centriflow cones was analyzed on BioGel P-2 column for molecular weight distribution of [ 14C]hydroxyproline. Filtrate = Mr ~<25000.

oc

(1) Smooth muscle cells a Dialyzed serum b Dialyzed serum (without boiling) c No serum (2) Fibroblasts a Dialyzed serum

4o

0

89.2% 96.4% 100.0% 98.5%

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16

26

36

40

F r a c t i o n No.

Fibroblasts

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40

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Fig. 1. Elution profile of media filtrate on Bio-Gel P-2 column. After equilibration with 0.05 M ammonium bicarbonate and 0.5% chloroform, pH 8.0, 20 min (3.4 ml) fractions were collected, hydrolyzed, and analyzed for the presence of [14Clhydroxyproline ( O - ©) and []4C]proline (zx A). Results are presented as percent of total [14C]hydroxyproline or []4C]proline present in the media filtrate after a 24-h pulse period. Position of elution of standards are labeled: albumin (ALB), Gly-Pro-Hypro (GPH) and Gly-Pro (GP). Proline and hydroxyproline standards eluted in fraction No. 33-36 with minimal separation.

was used to s e p a r a t e small p e p t i d e s from free a m i n o acids (Fig. 1). E a c h fraction f r o m the colu m n was h y d r o l y z e d before analysis for h y d r o x y proline. Over 90% of the l a b e l e d h y d r o x y p r o l i n e in the m e d i a filtrates from fibroblasts or s m o o t h muscle cells c o - m i g r a t e d on the P-2 c o l u m n with the h y d r o x y p r o l i n e s t a n d a r d s (Table I I I ) suggesting that it was free h y d r o x y p r o l i n e . A h y d r o x y p r o line s t a n d a r d was s e p a r a t e d f r o m a small p e p t i d e s t a n d a r d ( G l y - P r o ) b y 40 m i n or 6.8 ml. The a b s e n c e of dialyzed serum o r the omission of boiling from the e x p e r i m e n t a l p r o t o c o l h a d little effect on these results. As can b e seen in Fig. 1, there was m i n i m a l s e p a r a t i o n of h y d r o x y p r o l i n e f r o m proline. Small a m o u n t s of h y d r o x y p r o l i n e were d e t e c t e d in other fractions from the column, b u t no d i s c e r n a b l e p e a k was o b t a i n e d . T h e o b s e r v a t i o n that no hyd r o x y p r o l i n e was d e t e c t e d in the void volume certainly d e m o n s t r a t e s no significant quantities of h y d r o x y p r o l i n e c o n t a i n i n g p e p t i d e s in the molecular weight range of 1500-25000. This was conf i r m e d in e x p e r i m e n t s with dialysis t u b i n g with a m o l e c u l a r weight cut-off of 1000. In these experiments, all of the h y d r o x y p r o l i n e was f o u n d to be dialyzable. As above, if the gel filtration experim e n t is r e p e a t e d with m e d i a i n c u b a t e d in the presence of [14C]proline in the absence of cells, no h y d r o x y p r o l i n e f o r m a t i o n is detected.

(D) The existence of free hydroxyproline (1) Amino acid analysis before and after dialysis. T h e d e g r a d a t i o n of the newly synthesized collagen to free a m i n o acids was further d e m o n s t r a t e d b y e x a m i n a t i o n of the l a c elution profile from an

485 •

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,

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,

Hydroxy~ollne

10000

r,

,

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•

, A .~

lOO0OC

Proline

1000

o

o:_,:,

0

,oo

lO 4

8

12

16

20

F r a c t i o n No.

Fig. 2. Comparison of 14C-elution profiles of smooth muscle cell media filtrate from amino acid analyzer. (+ +) before sample hydrolysis; (O O) after sample hydrolysis. Arrows indicate elution time of standards. Data represents total cpm per flask.

80

hydroxyproline

; "

prollne

l i

>,

gly-

SO

ro

amino acid analyzer (Fig. 2). Hydrolysis of the media filtrate in 6 N HC1 before analysis did not result in a significant increase in [lac]hydroxyproline recovered over samples analyzed without prior hydrolysis. In fact, the t4C-elution profiles of the before and after hydrolysis samples were almost identical. When the dipeptide Gly-Hypro was added to the culture system and treated as explained above, it underwent no further degradation. Such results suggest that complete degradation of collagen took place within the cell before secretion. (2) Mixed paper chromatography. The migration of a hydroxyproline standard was compared with hydroxyproline containing fractions of media filtrate separated by an amino acid analyzer before and after hydrolysis (Fig. 3). In both buffer systems the 'hydroxyproline' peak from the unhydrolyzed media filtrate co-migrated with 'hydroxyproline' peak from the hydrolyzed media filtrate. Both of the radioactive peaks co-migrated with the unlabeled hydroxyproline standard as determined by ninhydrin. These experiments definitively identify the ~4C-labeled species isolated from the amino acid analyzer as free hydroxyproline. Discussion

1

2

3

4

5

Distance

~-t°°I lao B

6

8

9

10

In Cm

i BufieBr

hydroxyproline

.

7

gly-pro

proline

< >, g

60

1

2

3

4 Distance

5

6 In

7

8

9

10

Cm

Fig. 3. Evaluation of mixed paper chromatography of [14C]hydroxyproline fractions from amino acid analyzer. Open bars represent unhydrolyzed sample; closed bars, represent hydrolyzed sample. Samples of pulsed media filtrate (before and after hydrolysis) were placed on an amino acid analyzer to separate the 'hydroxyproline' peak as on Fig. 2. Paper chromatography of hydroxyproline fraction was carried out in two different buffer systems (A = n-butanol-acetic acid-H20 12 : 3 : 5 v/v;

Human embryonic lung fibroblasts (IMR-90) and rabbit aortic smooth muscle cells have the ability to degrade newly synthesized collagen intracellularly before it is secreted by the cell. This is detected by measuring the appearance of free or peptide-bound hydroxyproline of molecular weight less than 25 000. The fact that hydroxyproline is formed as a post-translational modification of peptide-bound proline, and is not recycled into protein makes it a unique marker for collagen synthesis. Degradation of other hydroxyproline containing peptides such as collagen propeptides [14] and elastin [15] or Clq component of complement [16] could contribute to the levels of free hydroxyproline qualitatively but the levels are far too low to justify the results seen in cell culture. B = phenol-n-butanol-methylethyl ketone-propionic acid-acetic acid-HzO (20:20:50: 10:10:20 v/v). The migration of the 'hydroxyproline' peak was compared with unlabeled hydroxyprofine standards as determined by ninhydrin. Arrows denote migration of standards.

486

The use of dialyzed serum containing a2-macroglobulin and the boiling of the samples to denature all proteases negates the possibility of extracellular degradation of the secreted collagen by enzymatic means. This has been demonstrated by other laboratories through the use of radiolabeled procollagen [1]. The latter is not degraded when present in the pulse media during the experiment. Also, short term pulses have demonstrated degraded collagen to be present in the cell before enough time has elapsed for the secretion of the procollagen [ 1]. The use of centriflo filter cones has provided an efficient and reproducible method for the separation of collagen plus procollagen from its degradation products. This combined with a purified [~4C]proline preparation containing minimal contamination of hydroxyproline provides for a fast and accurate method for the measurement of intracellular collagen degradation [2]. The appearance of filterable hydroxyproline in these culture systems is almost completely inhibited by the addition of et,et-dipyridyl or cycloheximide. This has been demonstrated for human skin fibroblasts by Steinmann et al. [17]. This strongly suggests a collagen origin for the hydroxyproline and rules out the non-enzymatic hydroxylation of proline as its primary source. The explanation for incomplete inhibition of filterable hydroxyproline with the use of a, et-dipyridyl in the smooth muscle cells is not entirely clear but could be due to an inability of a,a-dipyridyl to diffuse into the cell or to the possible contribution of degraded elastin hydroxyproline to these measurements. As one inhibits the hydroxylation of proline with et, a-dipyridyl, the degradation of newly synthesized collagen increases in both culture systems. This is not surprising, considering the unstable nature of the underhydroxylated procollagen molecule and its slower rate of secretion. To identify the nature of the filterable hydroxyproline, the radiolabeled components of the media filtrate were separated by molecular weight. Consistently, over 90% of the radiolabeled hydroxyproline eluted with free amino acid standards, clearly distinct from the elution of the small peptide Gly-Pro. No other definite [~4C]radioactive peaks could be detected in the analysis. 14C-Elution pro-

files of the media filtrate either before or after hydrolysis on an amino acid anlyzer were almost identical again demonstrating the lack of any significant amounts of pe~tide-bound [J4C]hydroxyproline in the media filtrate. The definitive identification of the radiolabeled species eluting under the hydroxyproline peak on the amino acid analyzer was accomplished by comparing the mobility of its radiolabeled contents with a standard on mixed paper chromatography. Thus the data clearly establish free hydroxyproline as the major product of the degradation of newly synthesized collagen in fibroblast and smooth muscle cell cultures. If one considers the lysosome with its large supply of hydrolytic enzymes as the locus for this process, complete protein degradation to free amino acids is not unexpected. Although much has been written about the existence of free hydroxyproline in biological fluids and tissues [5-7], its origin has always been questioned. It has been hypothesized that extracellular enzymes could digest collagen to free amino acids, but little evidence could be found to support this. The identification of a cell system whose major product of collagen degradation is free hydroxyproline suggests a possible source for the existence of this free amino acid. We propose that the free hydroxyproline seen in the culture medium originates within the lysosomes of cells that are degrading newly synthesized collagen. We believe that in vivo the origin of free hydroxyproline is also lysosomal and it is this hydroxyproline which is degraded in the 4-hydroxyproline oxidase pathway [7]. Further studies will determine if the intracellular degradation of newly synthesized collagen is the sole source of the free hydroxyproline in biological fluids and therefore possibly demonstrate its usefulness as an in vivo measurement.

Acknowledgements We thank George Crombie for his technical assistance, Rosemarie Moscaritolo and Valerie Verbitski for the smooth muscle cells, and Barbara Faris and Wayne Gonnerman for their suggestions and many discussions. This work was supported by grants (to M.I.) NRSA 5F32DE05238 from the

487 National Institute of Dental Research and H L 1 9 7 1 7 a n d H L 1 3 2 6 2 f r o m the N a t i o n a l H e a r t , L u n g , a n d B l o o d I n s t i t u t e , N a t i o n a l I n s t i t u t e of Health, U.S.A.

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8 Dubousky, J. and Meyer, R.O. (1975) Clin. Chim. Acta 62, 277-286 9 Trelstad, R.L., Lawley, K.R. and Holmes, L.B. (1981) Nature 289, 310-312 10 Fails, B., Snider, R., Levine, A., Moscaritolo, R., Salcedo, L. and Franzblau, C. (1978) In Vitro 14, 1022-1027 11 Fails, B., Salcedo, L.L., Cook, V., Johnson, L., Foster, J.A. and Franzblau, C. (1976) Biochim. Biophys. Acta 418, 93-103 12 Sherman, J. and Zweig, G. (1971) in Paper Chromatography and Electrophoresis (Zweig, G. and Whitaker, J.R., eds.), Vol. II, Academic Press, New York 13 Hutton, J.J., Jr., Tappel, A.L. and Udenfriend, S.A. (1966) Anal. Biochem. 16, 384-394 14 Bradley, K., McConnell-Breul, S. and Crystal, R.G. (1974) J. Biol. Chem. 249, 2674-2683 15 Bentley, J.P. and Hanson, A.N. (1969) Biochim. Biophys. Acta 175, 339-344 16 Porter, R.R. and Reid, K.B.M. (1978) Nature 275, 699-704 17 Steinmann, B., Rao, V. and Gitzelmann, R. (1981) FEBS Lett. 133, 142-144