Specificities of cell permeant peptidyl inhibitors for the proteinase activities of mu-calpain and the 20 S proteasome.

Cell-permeant peptidyl aldehydes and diazomethylketones are frequently utilized as inhibitors of regulatory intracellular proteases. In the present study the specificities of several peptidyl inhibitors for purified human mu-calpain and 20 S proteasome were investigated. Acetyl-LLnL aldehyde, acetyl-LLM aldehyde, carbobenzyloxy-LLnV aldehyde (ZLLnVal), and carbobenzyloxy-LLY-diazomethyl ketone produced half-maximum inhibition of the caseinolytic activity of mu-calpain at concentrations of 1-5 x 10(-7) M. In contrast, only ZLLnVal was a reasonably potent inhibitor of the caseinolytic activity of 20 S proteasome, producing 50% inhibition at 10(-5) M. The other inhibitors were at least 10-fold less potent, producing substantial inhibition only at near saturating concentrations in the assay buffer. Further studies with ZLLnVal demonstrated that its inhibition of the proteasome was independent of casein concentration over a 25-fold range. Proteolysis of calpastatin or lysozyme by the proteasome was half-maximally inhibited by 4 and 22 microM ZLLnVal, respectively. Thus, while other studies have shown that ZLLnVal is a potent inhibitor of the hydrophobic peptidase activity of the proteasome, it appears to be a much weaker inhibitor of its proteinase activity. The ability of the cell permeant peptidyl inhibitors to inhibit growth of the yeast Saccharomyces cerevisiae was studied because this organism expresses proteasome but not calpains. Concentrations of ZLLnVal as high as 200 microM had no detectable effect on growth rates of overnight cultures. However, yeast cell lysates prepared from these cultures contained 2 microM ZLLnVal, an amount which should have been sufficient to fully inhibit hydrophobic peptidase activity of yeast proteasome. Degradation of ubiquitinylated proteins in yeast extracts by endogenous proteasome was likewise sensitive only to high concentrations of ZLLnVal. The higher sensitivity of the proteinase activity of calpains to inhibition by the cell permeant inhibitors suggests that calpain-like activities may be targets of these inhibitors in animal cells.

Cardiac high molecular weight calmodulin binding protein contains calpastatin activity.

A high molecular weight calmodulin binding protein (HMWCaMBP) was previously identified and purified from bovine heart cytosolic fraction [Sharma, R.K. (1990) J. Biol. Chem. 265, 1152-1157]. In this study, we report the biological function of this protein. HMWCaMBP was subjected to peptide mapping and three peptides were sequenced. Two of the three peptide sequences were shown to be highly homologous to the calpain inhibitor, calpastatin. However, the third peptide did not show homology to any known proteins. The Western blot analysis of HMWCaMBP and purified calpastatin from bovine cardiac muscle showed immunoreactivity with polyclonal antibody raised against HMWCaMBP. Furthermore, HMWCaMBP inhibited calpain II and calpain I activities in a dose dependent fashion. Our data based on sequence homology, amino acid analysis, antibody reactivity and calpain inhibition suggests that HMWCaMBP is homologous to calpastatin and may be a CaM-binding form of calpastatin.

Evidence for participation of a calpain-like cysteine protease in cell cycle progression through late G1 phase.

Recent studies have demonstrated that cell-permeant protease inhibitors arrest human fibroblasts in late G1. The target for the inhibitors has been claimed to be either the proteasome, or a calpain-like cysteine protease activity. In the present investigation, the progression of serum-stimulated WI-38 fibroblasts into S-phase was partially inhibited by the cell-permeant general inhibitor of cysteine proteases, E64d, but not by its non-permeant anolog, E64c. Exposure of fibroblasts in late G1 to the proteasome inhibitor, lactacystin, produced only a modest inhibition of progression into S-phase, and did not influence the extensive inhibition produced by the calpain-selective inhibitor, ZLLY-DMK. ZLLnV-CHO and ZLLL-CHO, which are reportedly selective for the proteasome, were less potent than ZLLY-DMK as inhibitors of S-phase progression. These results argue for the involvement of a calpain-like protease acting in late G1 to allow transit into S-phase.

Inhibition of the growth of WI-38 fibroblasts by benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone: evidence that cleavage of p53 by a calpain-like protease is necessary for G1 to S-phase transition.

The effect of a calpain-selective cell permeant inhibitor, benzyloxycarbonyl Leu-Leu-Tyr diazomethylketone (ZLLY-CHN2), on the serum-stimulated growth of WI-38 human fibroblasts has been investigated. Only cell permeant protease inhibitors with activity against calpains prevented progression into S-phase. Protein blotting experiments indicated that p53 immunoreactivity increased in late G1 cells treated with ZLLY-CHN2. The content of p21Waf1/Cip1 CDK inhibitor also increased, providing a mechanism for the observed failure to enter S-phase. Further studies indicated that p53 could be degraded by a ZLLY-CHN2-sensitive protease immediately prior to S-phase, but that proteolysis did not occur after this critical time point. Chelation of extracellular Ca2+ by addition of EGTA inhibited the p53 degradation. Consistent with proteolysis of p53 in late G1 phase, mu-calpain immunoreactivity transiently accumulated in cell nuclei at this time. ZLLY-CHN2 did not appear to increase p53 mRNA in WI-38 cells. Purified mu-calpain required only 1 to 3 microM Ca2+ to proteolyze p53 in WI-38 cell lysates. These results indicate that ZLLY-CHN2 inhibits progression of WI-38 cells into S-phase by inactivating a calpain-like protease that is responsible for proteolysis of constitutively expressed p53 in late G1.

Regulation of cell migration by the calcium-dependent protease calpain.

Integrin receptors play an important role during cell migration by mediating linkages and transmitting forces between the extracellular matrix and the actin cytoskeleton. The mechanisms by which these linkages are regulated and released during migration are not well understood. We show here that cell-permeable inhibitors of the calcium-dependent protease calpain inhibit both beta1 and beta3 integrin-mediated cell migration. Calpain inhibition specifically stabilizes peripheral focal adhesions, increases adhesiveness, and decreases the rate of cell detachment. Furthermore, these inhibitors alter the fate of integrin receptors at the rear of the cell during migration. A Chinese hamster ovary cell line expressing low levels of calpain I also shows reduced migration rates with similar morphological changes, further implicating calpain in this process. Taken together, the data suggest that calpain inhibition modulates cell migration by stabilizing cytoskeletal linkages and decreasing the rate of retraction of the cell's rear. Inhibiting calpain-mediated proteolysis may therefore be a potential therapeutic approach to control pathological cell migration such as tumor metastasis.

Calpain inhibitors and serine protease inhibitors can produce apoptosis in HL-60 cells.

Recent investigations indicate that proteolysis is an important event in generation of the apoptosis phenotype. Although various proteases have been suggested to be candidates for this proteolysis, the results from different laboratories are inconsistent. In the present studies, HL-60 cells were treated with cycloheximide to investigate proteases involved in apoptosis. The calpain inhibitors benzyloxycarbonyl-Leu-Leu-Tyr diazomethylketone and acetyl-Leu-Leu-Nle aldehyde were not capable of preventing apoptosis induced by cycloheximide. In the absence of cycloheximide, these two inhibitors could initiate apoptosis in HL-60 cells. The thiol protease inhibitor benzyloxycarbonyl-Leu-Val-Gly diazomethylketone neither prevented nor produced apoptosis. The serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and tosyl-Phe chloromethylketone (TPCK) also induced apoptosis in the absence of cycloheximide. On the other hand, the latter two inhibitors decreased cycloheximide-induced apoptosis, assessed either by cell morphologic changes or DNA ladder generation. Benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone and iodoacetamide, inactivators of interleukin 1beta-converting enzyme (ICE)-like proteases, did not produce apoptosis and inhibited the induction of apoptosis by cycloheximide, calpain inhibitors, or serine protease inhibitors. These results are consistent with the ICE-like proteases having a central role in proteolysis during apoptosis, while calpain-like proteases and the serine proteases sensitive to DCI or TPCK are not required for generation of the apoptosis phenotype in HL-60 cells.

Calpain subunits remain associated during catalysis.

The Ca2+ dependent cysteine proteases, calpains are heterodimers containing a large (ca. 80 kDa) catalytic subunit and a 25-30 kDa small subunit. Whether calpains remain dimers while catalyzing hydrolysis of protein substrates has been controversial. Now by doing subunit co-immunoprecipitation, we provide direct evidence to resolve this argument. In the presence of Ca2+ concentrations which permit catalytic activity, both subunits of either m- or mu-calpain are co-immunoprecipitated by monoclonal antibodies directed against a single subunit. Furthermore, both subunits can be co-immunoprecipitated during calpain-catalyzed proteolysis of the substrate casein. These results indicate that both major calpain isozymes maintain their heterodimeric form during the catalytic cycle. Thus, small subunit might have a direct role in regulating the physiologic function of either major calpain isozyme.

The major calpain isozymes are long-lived proteins. Design of an antisense strategy for calpain depletion in cultured cells.

Calpains are intracellular Ca2+-dependent proteases that are thought to participate in Ca2+-associated signal transduction pathways. It has been proposed that calpains are activated by an autoproteolytic mechanism. If this is true one would expect a relatively short half-life for calpain protein in cells. To test this hypothesis, WI-38 human diploid fibroblasts were pulse-labeled with [35S]methionine, and calpain was immunoprecipitated at various times after chasing with nonradioactive methionine to determine residual radioactivity. The results demonstrated that the two major calpain isozymes, m-calpain and micro-calpain, had metabolic half-lives of approximately 5 days. Calpains were long-lived proteins in several human cell lines, A-431, HeLa, VA-13, C-33A, and TE2 cells. In addition, calpastatin, the calpain-specific inhibitor protein, also had a long metabolic half-life. These observations suggest that the model for calpain activation by autoproteolysis requires re-investigation. Based on a knowledge of calpain metabolic stability, a protocol was devised for chronic exposure of WI-38 cells and HeLa cells to a calpain small subunit antisense oligodeoxyribonucleotide. Depletion of calpain small subunit after 5 or more days of treatment led to inhibition of cell proliferation that could be reversed by removal of antisense oligodeoxyribonucleotide from the culture medium. Together with previous studies, these results indicate a requirement for calpains in mammalian cell proliferation.

Isolation of a Chinese hamster ovary cell clone possessing decreased mu-calpain content and a reduced proliferative growth rate.

A Chinese hamster ovary cell line (CHOp) was cultured in the presence of benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone (ZLLY-CHN2), to select for resistance to this cell-permeant calpain inhibitor. A clone isolated after several courses of exposure (SHI cells) demonstrated decreased sensitivity to ZLLY-CHN2 toxicity and a decreased growth rate. SHI cells also possessed less mu-calpain isozyme relative to CHOp cells, as determined by activity measurement or by protein immunoblotting. Activities of m-calpain, calpastatin, cathepsin B, cathepsin L, and glycogen phosphorylase were not altered. SHI mu-calpain was partially purified by sequential chromatography on Bio-Gel A-1.5m and DEAE-Sepharose. Its chromatographic behavior in either system was the same as for CHOp mu-calpain. Further studies with the partially purified SHI and CHOp mu-calpain fractions failed to distinguish any difference in Ca2+ requirement or in sensitivity to inhibition by calpastatin or ZLLY-CHN2 for these enzymes. These experiments suggest that SHI cells underproduce a form of mu-calpain which is very similar to, if not identical with, CHOp mu-calpain. SHI cells displayed a population doubling time of 29 h compared with 19 h for CHOp cells. The decreased growth rate of SHI cells was the result of a prolonged G1 phase. Introduction of purified human mu-calpain into SHI cells by electroporation transiently restored the growth rate and also increased toxicity associated with exposure to ZLLY-CHN2. SHI cells should be a valuable model in further studies to delineate the function of mu-calpain in cell proliferative growth.

Inhibition of growth of human TE2 and C-33A cells by the cell-permeant calpain inhibitor benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone.

Calpains are Ca(2+)-requiring, nonlysosomal proteases which are thought to participate in some aspects of intracellular Ca(2+)-signal transduction. However, their exact physiologic function has not yet been established. Addition of the cell-permeant, irreversible calpain inhibitor, ZLLY-CHN2, to human TE2 or C-33A cells inhibited growth, as assessed either by mitochondrial MTT reductase activity or by direct cell counting. Inhibition of growth produced by a 24-h exposure to 50 microM ZLLY-CHN2 was reversed upon substituting growth medium without inhibitor. Homogenates produced from cells cultured in the presence of ZLLY-CHN2 displayed decreased calpain and Ca(2+)-independent proteolytic activities. Protein immunoblot analysis showed that cell cultures which had lost 80% of their calpain activity still retained full calpain immunoreactivity. Therefore, inhibition by ZLLY-CHN2 appeared to result in accumulation of irreversibly inactivated calpain within the cells. Homogenates from cells cultured in the presence of 20 or 50 microM ZLVG-CHN2, a cell-permeant inhibitor with little activity against calpains, had decreased Ca(2+)-independent proteolytic activity, but demonstrated no decrease in calpain activity. ZLVG-CHN2 did not inhibit cell growth under these conditions. Growth of Saccharomyces cerevisiae cells, which do not appear to express calpain-like proteases, was not inhibited by including 50 microM ZLLY-CHN2 in the culture medium. These results indicate that calpains participate in the social regulation of cell growth in multicellular organisms.

Selective nuclear transport of mu-calpain.

To study nuclear transport of purified calpains in an in vitro system, A431 cells were permeabilized with digitonin, and fluorescein-labeled calpains were introduced under conditions known to facilitate energy-dependent nuclear transport of proteins. Fluorescein-mu-calpain was transported into nuclei in an ATP-dependent fashion. The calpain-specific inhibitor protein, calpastatin, could not block mu-calpain translocation. Fluorescein-calpastatin and fluorescein-m-calpain were poorly transported at best. In the presence of rat liver cytosolic factors, accumulation of nuclear mu-calpain was maximum at approximately 1 microM Ca2+, and no transport was observed at 0.3 microM Ca2+. Rat erythrocyte and HeLa cell extracts supported transport in the absence of Ca2+.

Different sensitivities of native and oxidized forms of Na+/K(+)-ATPase to intracellular proteinases.

Inactivation of Na+/K(+)-ATPase by partially reduced oxygen metabolites has been implicated in ischemia-reperfusion injury to heart and other organs. Because oxidation of many proteins makes them more susceptible to degradation by intracellular proteinases, we studied the effects of several such proteinases on native and H2O2-oxidized preparations of Na+/K(+)-ATPase from canine kidney (containing alpha 1 isoform of the catalytic subunit) and rat axolemma (containing alpha 2 and alpha 3 isoforms). Lysosomal cathepsin D degraded the native and the oxidized preparations at acid pH, but it was significantly more effective against the oxidized forms. m-Calpain had little or no effect on the native Na+/K(+)-ATPase preparations, but it digested the oxidized alpha-subunits of the axolemma and the kidney enzymes. mu-Calpain's effects were similar to those of m-calpain. Multi-catalytic proteinase which is known to degrade a large number of oxidized proteins, did not affect the native or the oxidized forms of Na+/K(+)-ATPase. The findings suggest that (a) during oxidative stress there may be accelerated degradation of the oxidatively damaged Na+/K(+)-ATPase, either through internalization and transport to lysosomes, or by the action of calpains at the membrane; and (b) those isoforms of the enzyme that are more sensitive to oxidants are more susceptible to degradation by the above processes.

Distinction between places and paths in rats' spatial representations.

Rats were trained on a 3-dimensional, 4-arm radial maze. In Experiment 1, Ss trained to climb to the single goal platform chose fewer novel routes to the goal than Ss trained to climb to the 4 spatially distinct platforms. In Experiment 2 a reinforcement contingency was imposed, requiring a novel route choice on each trial to receive reinforcement. Learning to associate route choice with reinforcement outcome was much more difficult for Ss tested with the single goal than for Ss tested with the 4 distinct goals. In Experiment 3 a partitioned central platform group learned the reinforcement contingency as quickly as the Ss given 4 spatially distinct platforms. In Experiment 4, distinctive floor inserts did not affect performance relative to no inserts.

Inhibition of nonlysosomal calcium-dependent proteolysis by glycine during anoxic injury of rat hepatocytes.

BACKGROUND/AIMS: The mechanism by which glycine protects against hepatocyte death during anoxia remains unclear. Nonlysosomal proteolysis, including calpain proteolysis, has been implicated as a mechanism of lethal cell injury. However, the effect of glycine on nonlysosomal proteolysis is unknown. The aim of this study was to ascertain if glycine cytoprotection is associated with inhibition of nonlysosomal proteolysis. METHODS: Rat hepatocyte suspensions were rendered anoxic using an anaerobic chamber. Cell viability was measured by propidium iodide fluorometry. Nonlysosomal protease activity was quantitated by the release of trichloroacetic acid-soluble free amines or tyrosine. Calpain protease activity was measured using a fluorogenic substrate. RESULTS: Glycine and alanine (but not valine) markedly improved cell viability during anoxia in a concentration-dependent manner. During anoxia, the majority of nonlysosomal proteolysis (60%) was dependent on extracellular Ca2+. Glycine only inhibited that portion of nonlysosomal proteolysis that was dependent on extracellular Ca2+. Amino acids inhibited the anoxia-stimulated increase in calpain protease activity with the same specificity and concentration-dependence observed for cytoprotection. Glycine was more potent in directly inhibiting purified m-calpain as compared with mu-calpain protease activity. CONCLUSIONS: Glycine may exert its cytoprotective activity during lethal anoxic hepatocyte injury, in part by inhibiting Ca(2+)-dependent degradative, nonlysosomal proteases, including calpains.

m-Calpain requires DNA for activity on nuclear proteins at low calcium concentrations.

m-Calpain (calpain II, m-CANP), which normally requires millimolar Ca2+ for activity in vitro, was capable of proteolyzing a number of matrix proteins in isolated rat liver nuclei at Ca2+ concentrations as low as 3 microM (Mellgren, R. L. (1991) J. Biol. Chem. 266, 13920-13924). Treatment of nuclei with deoxyribonuclease I eliminated the activity of m-calpain at low Ca2+ concentrations, while ribonuclease A and phospholipase C had no effect. Addition of DNA to DNase-treated nuclei restored m-calpain activity at low Ca2+. RNA had little if any effect. Eukaryotic and prokaryotic DNA were equally effective, and synthetic polydeoxyribonucleotides were also activators. m-Calpain did not bind to a DNA-cellulose column in the presence of 200 microM Ca2+, and m-calpain preincubated in the presence of DNA and 200 microM Ca2+ was not activated at low Ca2+ concentrations following removal of the DNA. DNA did not alter the Ca2+ requirement for m-calpain-catalyzed cleavage of casein. These results demonstrate that the Ca2+ requirement for proteolysis of nuclear matrix proteins by m-calpain can be dramatically decreased in the presence of DNA. Activation did not seem to be a result of DNA binding directly to calpain but appeared to require interaction of DNA, calpain, and calpain substrates in the nuclear matrix.

A comparison of the intracellular distribution of mu-calpain, m-calpain, and calpastatin in proliferating human A431 cells.

Little is known about the relative intracellular localizations of the calcium-dependent proteases, calpains, and their naturally occurring inhibitor, calpastatin. In the present study, the intracellular localization of mu-calpain, m-calpain, and calpastatin was studied at the light microscopic level in proliferating A431 cells. Highly specific antibodies against the three antigens revealed distinct staining patterns in interphase and mitotic cells. Most notably, calpastatin in interphase cells was localized near the nucleus in tube-like, or large granular structures, while the calpains were more uniformly distributed through the cytoplasm in either a fibrillar form (mu-calpain) or a diffuse or fine granular form (m-calpain). The distribution patterns of the two calpain isozymes were distinctly different during mitosis. m-Calpain was concentrated at the mitotic spindle poles and midbody, while mu-calpain appeared to accumulate at the cell membrane and the spindles. Four other human cell lines as well as normal human monocytes were examined to determine if the calpains-calpastatin segregation patterns are common to other cells or are unique to the A431 line. With the exception of abundant nuclear mu-calpain in the C-33A cervical carcinoma, the segregation of the proteins was similar to that of A431. These studies indicate that calpains may be localized at regions which are relatively poor in calpastatin content. Proteins at these sites may be susceptible to calpain-catalyzed cleavage.

Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction.

The Ca2+ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m)[125I]iodophenyl) diazirine and then isolated and proteolysed with calpain II, the [125I] was traced from the 565 kDa parent to Mr 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa fragment was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin.

Proteolysis of nuclear proteins by mu-calpain and m-calpain.

Purified calpains are capable of proteolyzing several high Mr nuclear proteins and solubilizing a histone H1 kinase activity from rat liver nuclei upon exposure to 10(-6) - 10(-5) M Ca2+. Major nuclear substrates displayed apparent molecular masses of 200, 130, 120, and 60 kDa on Coomassie Blue-stained SDS-PAGE gels. The nuclear proteins and the H1 kinase were released from Triton-treated nuclei following incubation with buffer containing 0.5 M NaCl. They therefore appeared to be internal nuclear matrix proteins. The nuclear H1 kinase activity solubilized by incubation with m-calpain was eluted in the void volume of a Bio-Gel A-1.5m column, indicating an apparent mass greater than 1,500 kDa. Treatment of the calpain-solubilized kinase with 0.5 M NaCl dissociated it to a form having an apparent mass of 300 kDa (Stokes radius = 5.6 nm), suggesting that the 300-kDa (Stokes radius = 5.6 nm), nuclei by calpain treatment as a large complex containing other internal matrix proteins. Purified human erythrocyte mu-calpain was capable of proteolyzing the nuclear matrix proteins at 10(-6) M Ca2+. In contrast, human erythrocyte multicatalytic protease complex produced little cleavage of the nuclear proteins. Proteolysis of nuclear proteins by either mu-calpain or m-calpain was inhibited by calpastatin. These experiments suggest a physiologic role for the calpains in the turnover of nuclear proteins.

Interaction of human erythrocyte multicatalytic proteinase with polycations.

The multicatalytic proteinase from human erythrocytes (macropain, proteasome) is a large enzyme composed of at least six distinct subunits ranging in molecular masses from 20 to 30 kDa. As its name implies, this proteinase appears to contain multiple catalytic sites with differing specificities toward peptide substrates. Several polycationic substances, including polylysines, polyarginine, protamine and histone H1 markedly stimulated caseinolytic activity of the proteinase. Activation was instantaneous, and involved increasing the Vmax of the proteinase for casein. Prolonged preincubation with polylysine at 37 degrees C resulted in autolytic inactivation of the proteinase. The polylysine concentrations required for half-maximal activation or autolytic inactivation were the same. A 23 kDa subunit of the proteinase disappeared at the same rate as loss of catalytic activity, and with the same pH dependence and polylysine concentration dependence. These results suggest that polylysine perturbs the structure of the multicatalytic proteinase, resulting in increased catalytic activity toward substrates; and, with prolonged exposure, allowing autoproteolytic inactivation to occur. The 23 kDa subunit appeared to be required for expression of caseinolytic activity, and may therefore be a catalytic subunit of the complex having activity against casein.