Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation

Biochimica et Biophysica Acta. 1094( 1991) 281-287

28 l

© 1991 ElsevierScience PublishersB.V. All rights reserved f1167-4889/91/$03.50 ADONIS 016748899100244C

Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation H i r o k o T a n a b e t, N o r i o K u m a g a i ~, T o s h i f u m i T s u k a h a r a 2, S h o i c h i I s h i u r a 2, Eiki K o m i n a m i 3, H i r o m i c h i N i s h i n a t a n d H i d e o S u g i t a 2 t Department of Plastic and Reconstrttcti~'e Surgery. St. Marianna Unit'e~vity School of Medicine. Kawasaki (Japan). 2 National Institute of Neuro.science. National C~'nterof Nt,arolo,~ and P~)'chiat,T. Tokyo (Japan) and "~Department of Biochemistry. JIintendo Unicerxity School of Medic#It,. Tokyo (Japan)

(Received 18 April 19911 (Revised manuscriptreceived 21 June ITS91)

Key words: Lysosomalproteinase; Cathepsin; Differentiation; (Rat keratinocyte) The cathepsins B, H and L, lysosomal cysteine proteinases, play a major role in intracellular protein degradation. These proteinase activities and expressions were examined in a Ca z+ regulated epidermal culture system which consists of two morphological cell types: undifferentiated cells grown in low Ca z+ (0.1 mM concentration) and differentiated cells grown in high Ca 2+ (1.8 mM concentration), respectively. Cathepsin B and L activities of the differentiated cells showed a several-fold increase compared to that of the undifferentiated cells. In addition, by using CM-cellulose column chromatography, cathepsin B and L were separated and the level of cathepsin L activity increased significantly. Cathepsin B, L and H were also detected by using an immunoblotting procedure in which their bands were expressed after differentiation was induced by the increasing calcium concentration. Cathepsin L activity and immunostaining intensity reached a maximum at l or 2 days of differentiation. In contrast, cystatin a (an endogenous inhibitor of cysteine-dependent cathepsins) appeared in tile final stage of differentiation. These results indicate that the expression of epidermal cathepsins and their endogenous inhibitor are involved in part of the prowam of cell differentiation and the terminal differentiation process in cultured rat keratinocytes.

Introduction During differentiation, epidermal keratinocytes form cornified cells that make up the anuclear stratum corneum. In this process, the population of intracellular proteins such as involucrin, histidine-rich proteins (profilaggrin and filaggrin), which are specifically related with cornifieation of epidermal keratinocytes, dramatically changes [I-6]. Recently, it has been suggested that the synthesis and breakdown of these proAbbreviations: FCS.fetal calf serum; PBS, phospilate-bufferedsaline; Z, benz-yloxyearbonyl;MCA, 4-methylcoumar/l-7-amide: 2-ME, 2mercaptoethanol; STI, soybean trypsin inhibitor: DFP, diisopropylfluorophosphate; E-64-C, L-trans-epox'isuccinyl-leucylamide (3methyl)butane; N-CBZ-Phe-AIa-CH2N2,carbobenzoxyphenylalanyldiazomethylketone; SDS, sodium dodecyl sulfate; PAGE. polyaerylamide gelelectrophoresis. Correspondence: H. Tanabe. Department of Plastic and Reconstructive Surgery. St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki 213, Japan.

teins may occur through the increased proteolytic activity of epidermal proteinases. For instance, cathepsin D and the epidermal serine proteinase cleave rat profilaggrin to monomerie filaggrin repeat-units [7-8]. Harvima et al. reported that cathepsin B, D, H and L were separated and identified in newborn rat epidermis [9]. In addition, immunohistochemical studies identified these e n ~ m e s and also their inhibitors in epidernal tissue [10-12]. It has been shown that growth, proliferation and differentiation of cultured keratinocytes are induced by changing Ca -'+ levels in the culture medium [13]. Therefore, it is thought that the Ca2+-regulated epidermal culture system is a good model for studying cell differentiation, using undifferentiated and differentiated cells, respectively. In this report, using a Ca2+-induced differentiation system of cultured rat keratinocytes, we present data in support of the proposition that lysosomal cysteine proteinases, cathepsin B, H and L, may partly regulate protein breakdown and intracellular protein recta-

282 bolism during the course of cell differentiation and terminal differentiation. We also demonstrate that cystatin a, an epithelial cell specific endogenous inhibitor of cysteine proteinases [12,14,15], was detected in the final stage of differentiation in the cultured rat keratlnocyte. Materials and Methods

Materials Benzyloxycarbonyl (Z)-Phe-Arg-methylcoumarylamide (MCA) and Z-Arg-Arg-methylcoumarylamide were purchased from the Peptide Institute (Osaka, Japan), and E-64-c from Taisho Pharmaceutical. Soybean trypsin inhibitor (STI), diisopropylfluorophosphate (DFP), aprotinin and carbobenzoxyphenylalanyldiazomethylketone (N-CBZ-Phe-Ala-CH2N z) were obtaided from Sigma. DEAE-eellulose (DE-52) and CM-cellulose (CM-52) were obtaided from Whatman. Antisera against rat liver cathepsin B, H, L and cystatin a were prepared as previously described [1618].

Preparation of cell extracts The keratinocytes harvested from 100 mm dishes were rinsed three times with 5 ml of phosphate-buffered saline (PBS) and lysed in 0.5 ml PBS with a sonicator.

Assaying proteinase activities Proteinase activities were determined by measuring the fluorescence of aminomethylcoumarin (NH2-Mec) liberated from peptides [17] with Z-Phe-Arg-MCA (for cathepsin L) and Z-Arg-Arg-MCA (for cathepsin B) as substrates, as described by Barrett and Kirschke [18]. The reaction mixture (100 ~l) was comprised of 0.1 M sodium acetate (pH 5.5 for cathepsin L, pH 6.0 for cathepsin B), containing 1 mM 2-mercaptoethanol and 1 mM EDTA. After incubation at 37 °C for 30 min, the reaction was terminated by the addition of 100/1,1 of 10% sodium dodecyl sulfate (SDS) and 1.3 ml of 0.2 M Tris-HCl (pH 9.0).

Polyacrylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis (PAGE) was performed by the method of Laemmli in 12.5% and 15% gel [20].

Cell culture Newborn rat keratinocytes were cultured with a feeder of mitomycin C treated 3T3 cells, as described by Rheinwald and Green [19]. Newborn rat skin was cut into small pieces. The skin specimens were incubated in phosphate-buffered saline (Ca 2÷ and Mg 2+ free, pH 7.4) containing 0.2% trypsin (Difco Laboratories) at 4 ° C for 15 h. After incubation, the effect of trypsin was blocked by adding fetal calf serum (FCS) (Micro Biological Inc.). The specimens were then gently stirred in a solution containing Dulbecco's modified Eagle's medium (Gibco laboratories) plus 20% FCS, and were filtered to remove dermal debris through two layers of sheets of 50 tzM pore sized nylon gauze. The cells were centrifuged for 7 min at 120 × g. The pellet were suspended in medium, which consisted of DMEM plus 10% FCS supplemented with penicillin O (100 U / m l ; Sigma), kanamycin (0.1 mg/ml; Sigma), fungizone (0.25/zg/ml; Sigma), hydroeortisone (0.5/zg/ml; Sigma), choleratoxin (10 ng/ml; Sigma) and mouse epidermal growth factor (10 ng/ml; Sigma). The keratinocytes were incubated at 4. 104/cm 2 cell density under a 5% CO 2 atmosphere at 37°C. Primary cultures were expanded approximately subconfluent, and then trypsinized into suspension and stored frozen at - 1 9 0 °C. For the present study, first passage cells, which were cultured without 3T3 cells and any supplement except antibiotics, were used. The level of calcium concentration of the medium was adjusted to 0.1 mM or 1.8 mM by adding sterile CaCI 2. Accordingly, the culture media were first prepared by omitting calcium salts from the DMEM and adding the 10% FCS pretreated with Chelex-100 resin (Bio-Rad).

Immunoblotting procedures E!ectrophoretic transfer of proteins from the polyacrylamide gel to nitrocellulose membranes was performed by the method of Towbin et al. [21]. The proteins were developed with rabbit anti-cathepsin B, H, L and cystatin ot serum followed by the avidin-biotin-peroxidase method with 4-ehloro-l-naphthol as a substrate [22].

Protein determination The protein concentration was determined by the method of Lowry et al. with bovine serum albumin as a standard [23]. Results

Proteinase activities after induction of differentiation Rat cultured keratinocytes showed clear morphological changes, when the cells were shifted to a high Ca 2+ medium (1.8 mM) from a low Ca z+ medium (0.1 mM). Therefore, we examined the proteinase activities of undifferentiated keratinocytes grown in 0.1 mM Ca 2+ and those of differentiated keratinocytes after induction of differentiation by a change in the calcium concentration for 24 h (Fig. 1). To determine the quantitative amounts of lysosomal enzymes, each keratinocyte extract was applied to a DEAE-cellulose column and Z-Phe-Arg-MCA hydrolytic activity, tentatively regarded as cathepsin B and L, was measured. In the differentiated keratinocytes, the Z-Phe-Arg-MCA hydrolytic activity was separated into two peaks (A and B). The highest peak was shown in differentiated kera-

283

Fig. 1. Phase contrast photograph of cultured rat keratiaocytes before and after induction of differentietion. Rat kcratinocytes were cultured 5 days in 0.1 mM Ca medium until growing in confluent, following the calcium concentration in the medium raised to 1.8 raM. After 24 h, cultured keratinocytcs showed clear morphological changes. (a) shows the form of undifferentiated keratinocytes in 0.1 mM Ca medium and (b) shows the form of differentiated kecatiocytes in 1.8 mM Ca medium.

Effect of z'arious inhibite,rs on two activities eluted from DEAE-celhdose cohmm chromatography

t i n o c y t e s in w h i c h a n a p p r o x . 6-fold i n c r e a s e n c c u r r e d , as compared with that of undifferentiated keratino~ t e s ( F i g . 2). S i n c e t h e f o r m e r , o f c o u r s e , c o n t a i n s both major differentiated and minor undifferentiated cells, t h e 6+fold i n c r e a s e is a m i n i m a l e s t i m a t i o n .

I n o r d e r to c o n f i r m w h e t h e r t h e s e t w o p e a k s ( A a n d B) h a v e s i m i l a r p r o p e r t i e s o r not, w e e x a m i n e d t h e e f f e c t s o f v a r i o u s specific i n h i b i t o r s o f t h e p r o t e i n a s e

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Fig. 2. Elution profile of the Z-Phe-Arg-MCA hydrolytic activity on DE 52 column. DEAE-cellulose column chromatography of cultured rat keratin0t3'tes of undifferentiated and differentiated cell types, as shown in Fig. I. Cell extract was loaded onto a DE 52 column ('7.5 ml) equilibrated with 5 mM phosphate buffer (buffer A) and eluted at a flow rate of 60 ml/h with a linear 60 ml-60 ml gradient of NaCI (0 to 0.5 M in buffer A fraction l to 30), and 2 ml frac+:ion was conecled. The flowthroagh which contained unbound materials s~owed no activity. Z-Phe.Arg-MCA hydrolytic activity was determined as described in Materials and Methods. (Left) The elution profile of Z-Phe-Arg-MCA hydrolytic activity in undifferentiated cell extracts. (Right) The elution profile of Z-Phe-Arg-MCA hydrolytic activity in differentiated cell extract. Z-Phe-Arg+MCA hydrolytic activity in differentiated cells was separated into two peaks CA and B). Open circles indicate enzyme activity and broken lines indicate protein absorbance.

284 TABLE l

Effect of carious inhibitors on two acth'ities elnted from DEAE-celhdose cohmm chromatography Z-Phe-Arg-MCA hydrolytic activity was measured in the presence of indicated inhibitors. Peak A and B were further purified by HPLC gel filtration (TSK G 3000 SW). Residual activities of t~th Feaks (A and B) markedly decreased by the addition of a specific z>steine proteinase inhibitor (E-64-c), 2-ME. 2-mercaptoethanol; STI. soybean trypsin inhibitor; DFP, diisopropyifluorophosohate; E-64-c. Ltrans-epoxysuccinylleucylamide(3-methyl)butane; N-CBZ-Phe-AIaCH,N,, carbebenzoxyphenyllalanyldiazomet hylketone.

20

v~"

20, .20

• )

10' Concentration

Reagent

Residual activity (%) peak A

peak B

100 mM

100

0.5 mg/ml 1.0 mg/ml 0.5 mM 10 ~g/ml

198 70.2 107.9 0.4

100 12.3 192 86.8 122.4 0.3

1.1 0.9 1.3 23.2 74.2

2.5 0.7 0.7 22.7 78.1

2-ME + -

4

STI Aprotinin DFP E-64-e N-CBZ-Phe-Ala-CH 2N2

tO- 10 -3 tO. tO -4 I0.10 -5 10.10 -6 I0' lO -7

M M M M M

'10

O' 30

40

0

5o

Fraction Number

3

2

20

Fig. 3. Separation of cathepsin B and L with CM 52 column chromatography. The sample was applied to a l0 ml CM-cellulose column and eluted by a stepwise NaCI gradient (0 to 0.4 m, fraction 10 to 40). Cathepsin B and L activities were assayed by using as substrates, Z-Phe-Arg-MCA (cathepsin L) and Z-Arg-Arg-MCA (cathepsin B). The open circles show eathepsin B and closed circles show cathepsin L.

( T a b l e l). T h e activity is e n h a n c e d by 2 - m e r c a p t o e t h a n o l a n d strongly i n h i b i t e d by E-64-c, b u t n o t by s o y b e a n trypsin inhibitor ( S T I ) o r d i i s o p r o p y l f l u o r o p h o s p h a t e ( D P E ) . In a d d i t i o n , n o s i g n i f i c a n t d i f f e r e n c e w a s o b s e r v e d w i t h a d o s e - d e p e n d e n t i n c r e a s e in carbobenzoxyphenylalanyldiazomethylketone (N-CBZP h e - A I a - C H 2 N 2 ) . So w e c o n c l u d e t h a t cysteine p r o t e i n a s e s a r e p r e s e n t in d i f f e r e n t i a t e d k e r a t i n o c y t e s .

1

•

10

Separation o f cathepsin B and L by CM-52 column chromatGgraphy T h e f l u o r o g e n i c s u b s t r a t e Z - P h e - A r g - M C A is o f t e n u s e d f o r t h e a s s a y o f c a t h e p s i n L, b u t this s u b s t r a t e is also c l e a v e d by c a t h e p s i n B [18], H o w e v e r , c a t h e p s i n B is usually e l u t e d w i t h 150 m M N a C l a n d c a t h e p s i n L w i t h 300 r a M N a C l o n C , ~ - 5 2 c o h t m n c h r o m a t o s r a p h y

4

5

6

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43~ 30~-

,

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20 14 kDa Fig. 4. Comparison between differentiated and undifferentiated rat keratinocytes on immunobloUing analysis. Cell were lysed in phosphatebuffered saline (PBS) and centrifuged. Resulting supernatants (15 p.g protein) were subjected to 12.5% SDS-PAGE followed by intmunoblotting as described io Materials and Methods. The electrophoretic pattern of proteins was visualized with amido black. Three proteinases, eathepsin B, L and H were detected using the respective antisera. Lane 1, 3, 5, undifferentiated keratinocytes (0.1 mM Ca). Lane 2, 4, 6, differentiated keratinocytes (1.8 mM Ca). M r standards (phosphorylase B, albumin, ovalbumin, carbonic anbydrase, trypsin inhibitor, ~t-lactalbumin) are shown at the left. The bands of 39 kDa are observed in lane 2 (cathepsin B). The band of 28 kDa is observed in lane 4 (cathepsin H). The bands of 39 kDa and 23 kDa are observed in lane 6 (cathepsin L).

285 [17]. Therefore, the differentiated keratinocyte extracts were applied to a CM-52 column, and hydrolytic activity for Z - P h e - A r g - M C A and Z - A r g - A r g - M C A was m e a s u r e d in e a c h fraction (Fig. 3). A l t h o u g h cathepsin B a n d L were not completely s e p e r a t e d by this column, the activity of cathepsin I. was higher than that of cathepsin B.

1

2

3

4

5

6

7

l)ays

M

3O 14 kl)e t000. •

--~ w

800,

=m

600~

I=

1.a mM Ca

M~.m

~y-

94 67 43

Fig. 6. Expression of endogenous inhibitor (cystatin a ) for cathepsin L during differentiation. After growing in 0.l mM Ca medium, the rat keratinoc~tes were cultured for 7 days in 1.8 mM Ca medium. 8 ~g from each sample were subjected to SDS-PAGE and visualized by anti-cystatin antibody. The specific band of 13 kDa is observed in 5 to 7 days.

Comparison between differentiated and undifferentieted keratinocytes on immunoblotting analysis

400

q¢

M

41 1 3

C 1 2 34

5 6 7 9

Days

" " . . . . .

439

--

• 23

l m m u n o b l o t t i n g results o f cultured rat keratinocyte extracts are shown in Fig. 4. Cathepsin B, H a n d L, which are the major eysteine proteinases, were detected using the respective antisera. In differentiated keratinocytes, cathepsin B was detected as a 39 k D a p r e c u r s o r polypeptide a n d a 26 k D a m a t u r e enzyme, while cathepsin L was detected as a 39 kDa p r e c u r s o r a n d a 23 k D a m a t u r e enzyme. Cathepsin H was detected as a 28 k D a m a t u r e form of the enzyme, but not as a p r e c u r s o r polypeptide~. Cathepsin L strikingly increased after differentiation was induced by raising the calcium concentration. In contrast, no significant expression of these cathepsin B, H, or L, was observed in undifferentiated keratinoeytes.

30 20 14 kDa Fig. 5. Time-dependent change in Z-Phe-Arg-MCA hydrolytic activit,/ and cathepsin L immunostaining intensity after induction of differentiation. The upper panel shows the time-course of Z-PheArg-MCA hydrolytic activity during the differentiation of cultured rat keratinocytes. Alter 1, 2, 3, 4 and 5 days of culture, undifferentiated and differentiated keratinoodtes were collected and lysed in PBS. Proteinase activities and protein concentration were determined as described in Materials and Methods. The lower panel shows the immunostaining of cathepsin L. After l, 2, 3, 4, 5, 6, 7 and 9 days of culture, 8 /~g from each sample were subjected to SDSPAGE and visualized by anti-cathepsin L antibody. The specific bands of 39 kDa and 23 kDa are observed in each stage. C, control, undifferentiated keratinocytes.

The time-dependent change in ZoPhe.Arg-MCA hydrolytic activity and cathepsin L immunostaining intensity after induction of differentiation Fig. 5 sl~ows the time-course of Z - P h e - A r g - M C A hydrolytic activity a n d cathepsin L immunostaining intensity d u r i n g induction of differentiation. The enzyme level r e a c h e d a maximum after 1 o r 2 days, a n d g r a d u ally d e c r e a s e d with progression of differentiation, with a c o n c o m i t a n t decrease in the processing into the m a t u r e enzyme. T h e m a t u r e enzyme (23 k D a ) could be found at day l. This implies the activation of o t h e r cathepsins which digest pro-eathepsin L into the mature form.

Expr,cssion of endogenous inhibitor for cathepsin L during differentiation Cystatin a , a n inhibitor of cysteine proteinases, was detected by immunoblotting only after 5 to 7 days of

286 differentiation (Fig. 6). The appearance seemed to be delayed compared with that of cathepsin L. Discussion

In other tissues except skin, there have been marly reports concerning lysosomal proteinase activities and their expression during cell differentiation. For example, cathepsin B, H, L and cysteine proteinase inhibitor increase during muscle differentiation (myogenesis and myotube formation) in vitro [24]. Recently, Colella et al. have reported an increase of expression of cathepsin B and D genes during myogenesis [25]. Cathepsins B and D have been induced by the addition of heroin in K562 erythroleukemia cells [26]. These reports suggest that cathepsins are significantly involved in the induction of cell differentiation as lysosomal endopeptidases. In the case of cultured keratinocytes, a change in lysosomal proteinases duri::~ dif~=rentiation has not been reported, although cathepsin B, D, H and L have been previously separated and identified in extracts of rat epidermis tissue origin [9]. In this paper, we have demonstrated that the immunologically detectable protei11 bands of cathepsin B, H and L significantly increased in response to Ca2+-in duced differentiation in cultured rat keratinocytes. We could not clearly separate the proteinase activities of cathepsin B and L in differentiated keratinocytes, because Z-Phe-Arg-MCA as a substrate is unaffected by cathepsin H but is sensitive to cathepsin B as well as L [18]. However, the activity of cathepsin L, separated by CM-column chromatography, was seen to be greater than that of cathepsin B. Therefore, in our present study, we attached importance to the presence and movement of cathepsin L during differentiation. In the time-dependent changes, the activity as well as the immunostaining intensity of catbepsin L were remarkable increased after 1 or 2 days and gradually decreased with progression of differentiation. Furthermore, the processing into the mature enzyme (23 kDa) of eathepsin L also decreased. The inactive proenzyme (39 kDa) appeared in the first stage of the differentiation and gradually decreased. The total amount of cathepsin L precursor plus mature enzyme underwent similar transitions. These results suggest that cathepsin L plays a major role in the initial stage of differentiation. In contrast, cystatin a, an endogenous inhibitor of cysteine proteinases, showed increase at the final step of differentiation. Although it seems natural that both changes would be parallel during differentiation, their appearance was completely different. During differentiation, in general, the content of lysosomal cysteine proteinases increased, as did their specific endogenous inhibitors. Cystatin a was found at a higher concentra-

tion in epithelial cells than in non-epithelial cells [12,14,15]. Our results first indicated an epidermal specific mode of increase in eathepsins and their inhibitor protein. This evidence may be partly related to the programs in terminal differentiation in cultured keratinocytes. On the other hand, polymorphonuclear leucocytes also contained cystatin in remarkably higher amounts [27]. In addition, Korant has pointed out that cystatin alters viral protein cleavages in infected cells [28]. The drastic increase of cystatin a in the terminal differentiated cells may be one of the protection mechanisms against the invading microorganisms from the environment [12,14,15]. In epidermal keratinocytes, differentiation is a multi-step process leading to the formation of cornification and finally reaching anuclearization. For this morphological event, it is enough to postulate that the activation of these lysosome enzymes may occur, because the lysosomal proteolytic enzyme activity is very stable and possesses the potential of inducing dramatical changes. Whether the eathepsin mediated proteolysis is directly involved in the control of the differentiation of cultured keratinocytes or not remains to be demonstrated by further experiments. References

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