In vitro Proteolytic Degradation of Bovine Brain Calcineurin by m-Calpain

A major cause of neuronal dysfunction is due to altered Ca2+ regulation. An increase in Ca2+ influx can activate Ca2+-dependent enzymes including calpains, causing the proteolysis of its specific substrates. In the present study, calcineurin (CaN) was found to be proteolysed by a Ca2+-dependent cysteine protease, m-calpain. In the presence of Ca2+, the 60 kDa subunit (CaN A) was degraded to a 46 kDa immunoreactive fragment, whereas in the presence of Ca2+ /calmodulin (CaM) immunoreactive fragments of 48 and 54 kDa were observed. The beta-subunit (CaN B) was not proteolysed in either condition. The proteolysis of CaN A increased its phosphatase activity and rendered it totally CaM-independent after 10 min of proteolysis. The molecular weight of the proteolytic fragments suggested that the m-calpain cleaved CaN A in the CaN B binding domain. A CaM-overlay experiment revealed that the CaM-binding site was present only in the 54 kDa fragment produced by CaN A proteolysis in the presence of Ca2+ /CaM. Thus, the increase in CaN A phosphatase activity observed in many neuronal disorders, may be due to the action of calpain.     

Autophosphorylation of the type II calmodulin-dependent protein kinase is essential for formation of a proteolytic fragment with catalytic activity. Implications for long-term synaptic potentiation

Autophosphorylation plays an essential role in proteolytic activation of the type II calmodulin-dependent protein kinase (CaM kinase II). Limited proteolysis of CaM kinase II by trypsin, alpha-chymotrypsin, and Ca2+-stimulated neutral protease (calpain) yielded a catalytically active kinase fragment only when the holoenzyme was autophosphorylated prior to proteolysis. Slightly larger, inactive fragments were obtained from nonphosphorylated CaM kinase II, regardless of whether Ca2+/calmodulin or Mg2+/ATP were present or absent. The active fragment exhibited Ca2+/calmodulin-dependent kinase activity with kinetic parameters identical with those of the activated holoenzyme. The key autophosphorylation site of CaM kinase II was absent from the active fragment which indicates that proteolysis can effectively uncouple the activation state and Ca2+/calmodulin independence of the kinase from the action of phosphoprotein phosphatases. Because autophosphorylation exerts such a tight control over this irreversible process, proteolytic activation of CaM kinase II by intracellular proteases offers an attractive mechanism for prolonging the effects of Ca2+ at the synapse.     

Fragmentation of fibronectin in cystic fibrosis]

Fibronectin (FN) plays an important role in mediating cell-matrix interactions and also as an opsonin in the phagocytosis of some microorganisms. Due to its domain structure FN is easily attacked by proteolytic enzymes and especially by elastases. Some of the fragments possess original properties as potentiation of viral transformation or proteolytic activity absent in the intact molecule. Cystic fibrosis is frequently accompanied by infection with protease generating microorganisms, such as Pseudomonas aeruginosa. Polyacrylamide gel electrophoresis and immunoblotting revealed the presence of FN fragments in the plasma of patients with molecular weight between 30 and 100 kD. Purified plasma FN was rapidly hydrolyzed in fragments by the sputum of patients as well as by purified Pseudomonas elastase. The comparison of fragments detected in patients' plasma with those produced by in vitro proteolysis confirms the probability of in vivo fragmentation of FN in cystic fibrosis and suggests that several proteolytic enzymes, endogenous and of bacterial origin, might be involved.     

Acid protease from Trichoderma reesei: limited proteolysis of fungal carbohydrases

Mechanisms regulating post-secretory limited proteolysis, carried out by the acid protease from Trichoderma reesei, were studied by following the release of α-galactosidase and multiple forms of cellobiohydrolase from this species. Both the rate of the proteolysis and the mode of action of the protease were affected by the pH of the culture medium, and only weakly depended on the amount of the enzyme. At pH between 2.7 and 3.5 the proteolytic reaction was limited, while at lower pH proteins were completely digested. Proteolysis depended on the degree of glycosylation of secreted enzymes. Inhibition of post-secretory deglycosylation decreased the rate of limited proteolysis in the culture medium in the course of fungal growth. Glucose and cellobiose, the main products of cellulose degradation carried out by the fungal cellulolytic complex, inhibited the proteolysis of the cellobiohydrolase in a concentration-dependent manner. A 32-kDa aspartic protease (EC 3.4.23.18) secreted by T. reesei was purified to homogeneity. The acid protease cleaved α-galactosidase and cellobiohydrolase into the same proteolytic fragments that had been isolated from the culture medium. Received: 4 December 1998 / Received revision: 22 February 1999 / Accepted: 5 March 1999     

Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.     

Regulation of fungal proteolysis on cyclic AMP-dependent protein kinase,cyclic AMP phosphodiesterase,glycogen synthase and histones

Limited proteolysis of catalytic and regulatory subunits of cyclic AMP-dependent protein kinase (A-pk), cyclic AMP phosphodiesterase, glycogen synthase, and histones by fungal protease (type XIX) was analyzed by the digested peptide bands in SDS polyacrylamide gel electrophoresis. The modulatory effects on proteolysis by nucleotides, polypeptides, and phospholipids may greatly depend on the intrinsic nature of substrates. The proteolysis of the regulatory subunit of A-pk and glycogen synthase was not regulated by nucleotides and nucleic acids. In comparison, phosphatidyl serine, cardiolipin, and pepstatin A stimulated the proteolysis of the catalytic subunit of A-pk. Whereas, DNA (Hind III digest), t-RNA, GTP, phosphatidyl serine, sphingosine inhibited the proteolysis of cyclic AMP phosphodiesterase. Moreover, MS2 RNA, DNA, t-RNA, dGTP, Phosphatidyl serine, phosphatidyl inositol, antipain, and chymostatin exerted inhibitory proteolytic effect on histone VIII-S. Some of these agents also had similar inhibitory effect on other types of histones (types III-S and VII-S). The inhibitory effect of phosphatidyl serine on proteolysis of histone may be due to their interaction which was monitored by the drastic increase of uv absorbance.     

Activation of a calmodulin-dependent phosphatase by a Ca2+-dependent protease

A calmodulin-dependent protein phosphatase (calcineurin) was converted to an active, calmodulin-independent form by a Ca2+-dependent protease (calpain I). Proteolysis could be blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, leupeptin, or N-ethylmaleimide, but other protease inhibitors such as phenylmethanesulfonyl fluoride, aprotinin, benzamidine, diisopropyl fluorophosphate, and trypsin inhibitor were ineffective. Phosphatase proteolyzed in the absence of calmodulin was insensitive to Ca2+ or Ca2+/calmodulin; the activity of the proteolyzed enzyme was greater than the Ca2+/calmodulin-stimulated activity of the unproteolyzed enzyme. Proteolysis of the phosphatase in the presence of calmodulin proceeded at a more rapid rate than in its absence, and the proteolyzed enzyme retained a small degree of sensitivity to Ca2+/calmodulin, being further stimulated some 15-20%. Proteolytic stimulation of phosphatase activity was accompanied by degradation of the 60-kilodalton (kDa) subunit; the 19-kDa subunit was not degraded. In the absence of calmodulin, the 60-kDa subunit was sequentially degraded to 58- and 45-kDa fragments; the 45-kDa fragment was incapable of binding 125I-calmodulin. In the presence of calmodulin, the 60-kDa subunit was proteolyzed to fragments of 58, 55 (2), and 48 kDa, all of which retained some ability to bind calmodulin. These data, coupled with our previous report that the human platelet calmodulin-binding proteins undergo Ca2+-dependent proteolysis upon platelet activation [Wallace, R. W., Tallant, E. A., & McManus, M. C. (1987) Biochemistry 26, 2766-2773], suggest that the Ca2+-dependent protease may have a role in the platelet as an irreversible activator of certain Ca2+/calmodulin-dependent reactions.     

A triglobular model for the polypeptide chain of aspartokinase I-homoserine dehydrogenase I of Escherichia coli

Limited proteolysis of aspartokinase I-homoserine dehydrogenase I from Escherichia coli by type VI protease from Streptomyces griseus yields five proteolytic fragments, three of which are dimeric, the other two being monomeric. One of the monomeric fragments (27 kilodaltons) exhibits residual aspartokinase activity, while the second one (33 kilodaltons) possesses residual homoserine dehydrogenase activity. The smallest of the dimeric species (2 X 25 kilodaltons) is inactive; the two other dimers exhibit either only homoserine dehydrogenase activity (2 X 59 kilodaltons) or both activities (hybrid fragment, 89 + 59 kilodaltons). This characterization of the proteolytic species in terms of molecular weight, subunit structure, and activity leads to the proposal of a triglobular model for the native enzyme. In addition, the time course of the formation of the various fragments was followed by measuring enzymatic activity and performing gel electrophoretic analysis of the protein mixture at defined time intervals during proteolysis. On the basis of the results of these studies, a reaction scheme describing the succession of events during proteolysis is given.     

Differential expression of protease activity in serum samples of prostate carcinoma patients with metastases

We present here the results from MS peptide profiling experiments of prostate carcinoma patients and controls with a specific focus on protease activity‐related protein fragments. After purification with surface‐active magnetic beads, MALDI‐TOF profiling experiments were performed on tryptic digests of serum samples of prostate cancer patients with metastases (n=27) and controls (n=30). This resulted in the reproducible detection of eight differentially expressed peptides, which were then identified by nanoLC‐MALDI‐TOF/TOF and confirmed by MALDI‐FTMS exact mass measurements. All differentially expressed peptides are derived from two homologous parts of human serum albumin; two of the eight peptides were tryptic and six were nontryptic. The presence of the nontryptic fragments indicates that a proteolysis process occurs which is not mediated by trypsin. Since the nontryptic fragments were found at significantly higher levels in control samples compared with metastases samples, it is proposed that a specific proteolytic inhibition process is in effect in the serum of prostate cancer patients. Experiments using synthetic peptides showed that this proteolytic activity occurs ex vivo and is sequence specific. Importantly, the observed prostate carcinoma‐related inhibition of the proteolysis was reproduced ex vivo using synthetic peptides.     

Separation of various calmodulins, calmodulin tryptic fragments, and different homologous Ca2+-binding proteins by reversed-phase, hydrophobic interaction, and ion-exchange high-performance liquid chromatography techniques

Reversed-phase, hydrophobic interaction, and ion-exchange high-performance liquid chromatography techniques have been used to separate different Ca2+-binding proteins and their proteolytic fragments. An alkali-stable ion-exchange column permitted the baseline separation of calmodulin fragments which differed only by one to three charged amino acids. The new hydrophobic interaction chromatography system displayed a high-resolution power separating calmodulins from different sources and calmodulin fragments obtained by trypsin proteolysis. The properties and advantages of the different systems are discussed in detail.     

Intensity and anisotropy decays of the tyrosine calmodulin proteolytic fragments, as studied by GHz frequency-domain fluorescence

Frequency-domain fluorescence measurements to 2 GHz were able to recover and account for essentially all of the intrinsic tyrosine anisotropy of calmodulin and its proteolytic fragments containing one or two tyrosine residues. Low-temperature measurements have detected a very rapid initial anisotropy decay in the 2-tyrosine species which may be attributed to radiationless energy transfer between the two tyrosines. The observed values of the rotational correlation times indicate that both tyrosines of calmodulin possess considerable mobility, which decreases in the presence of Ca2+ and at low temperatures.     

Relationship Among Calmodulin-, Forskolin-, and Guanine Nucleotide-Dependent Adenylate Cyclase Activities in Cerebellar Membranes: Studies by Limited Proteolysis

Adenylate cyclase activity in bovine cerebellar membranes is regulated by calmodulin, forskolin, and both stimulatory (Ns) and inhibitory (Ni) guanine nucleotide-binding components. The susceptibility of the enzyme to chymotrypsin proteolysis was used as a probe of structure-function relationships for these different regulatory pathways. Pretreatment of membranes with low concentrations of chymotrypsin (1-2 micrograms/ml) caused a three- to fourfold increase in basal adenylate cyclase activity and abolished the Ca2+-dependent activation of the enzyme by calmodulin. In contrast, the stimulation of the enzyme by GTP plus isoproterenol was strongly potentiated after protease treatment, an effect that mimics the synergistic activation of adenylate cyclase by Ns and calmodulin in unproteolyzed membranes. Limited proteolysis revealed low- and high-affinity components in the activation of adenylate cyclase by forskolin. The low-affinity component was readily lost on proteolysis, together with calmodulin stimulation of the enzyme. The activation via the high-affinity component was resistant to proteolysis and nonadditive with the Ns-mediated activation of the enzyme, suggesting that both effectors utilize a common pathway. The inhibitory effect of low concentrations (10(-7) M) of guanyl-5'-yl imidodiphosphate [Gpp(NH)p] on forskolin-activated adenylate cyclase was retained after limited proteolysis of the membranes, indicating that the proteolytic activation does not result from an impairment of the Ni subunit. Moreover, in the rat cerebellum, proteolysis as well as calmodulin was found to enhance strongly the inhibitory effect of Gpp(NH)p on basal adenylate cyclase activity. Our results suggest that calmodulin and Ns/Ni interact with two structurally distinct but allosterically linked domains of the enzyme. Both domains appear to be involved in the mode of action of forskolin.     

PROTEOLYTIC MICRODISSECTION OF SMOOTH-SURFACED VESICLES OF LIVER MICROSOMES

Digestion of rabbit liver microsomal smooth vesicles with Bacillus subtilis protease released proteins and peptide fragments from the vesicles, without solubilizing phospholipids and cholesterol. The proteolysis was, however, limited when about 30% of the protein had been solubilized. The same limitation was observed when the vesicles were treated with trypsin, chymotrypsin, or their combinations with the bacterial protease. The limited proteolysis was accompanied by selective solubilization of cytochrome b₅ and microsomal NADPH-specific flavoprotein, leaving the CO-binding hemoprotein and some other enzymes still attached to the vesicular membranes. Sucrose density gradient centrifugation of protease-treated vesicles indicated that all the vesicles had been attacked by the protease to similar extents. The behavior of intact and digested vesicles in dextran density gradient centrifugation suggested that the vesicles, even after proteolytic digestion, existed in the form of closed sacs which were impermeable to macromolecules such as dextran and proteases. It was concluded that only the outside surface of the vesicles is susceptible to the proteolytic action and that cytochrome b₅ and the NADPH-specific flavoprotein are located in the susceptible area.     

Limited digestion of calmodulin with trypsin in the presence or absence of various metal ions

The limited proteolysis of bovine brain calmodulin with trypsin in the presence or absence of various metal ions was reinvestigated in detail by HPLC. With metal ion-free calmodulin, limited proteolysis occurred at Arg 37 and Arg 106 with a cleavage ratio of 1 to 5, resulting in fragments consisting of residues 1-37, 38-148, 1-106 and 107-148. Fragments 1-37 and 107-148 accumulated under metal ion-free conditions. In the presence of calcium ions, the susceptibility of these sites to trypsin decreased and limited proteolysis occurred at Lys 77 as already reported by other workers. Fragment 78-148 accumulated, whereas fragment 1-77 was unstable under calcium-bound conditions, giving smaller peptides. Upon binding of manganese ions, calmodulin underwent a change of susceptibility to trypsin, resulting in cleavage at Lys 77, as observed for calcium-bound calmodulin. In the presence of zinc or magnesium ions, calmodulin was cleaved at the same sites as metal ion-free calmodulin under conditions where calmodulin would be expected to bind the respective ions.     

Structural and functional analysis of a bacterial cellulase by proteolysis

CenA is an endo-beta 1,4-glucanase from the cellulolytic bacterium Cellulomonas fimi. It is a bifunctional enzyme comprising an amino-terminal cellulose-binding domain and a carboxyl-terminal catalytic domain joined by a short sequence of prolyl and threonyl residues (the Pro-Thr box). Additional structural and functional information was revealed by a detailed analysis of the products generated by proteolytic cleavage of a nonglycosylated form of CenA. An extracellular C. fimi protease attacked nonglycosylated CenA at the junctions between the Pro-Thr box and the two functional domains. A stable "core" peptide (p30), corresponding to the catalytic domain, remained after extensive proteolysis. p30 was resistant to further attack even in the presence of 2-mercaptoethanol plus urea or dithiothreitol, but treatment in the presence of sodium dodecyl sulfate allowed complete fragmentation to small peptides. Stable peptides, identical, or closely related to p30, were generated by alpha-chymotrypsin or papain. These results indicated that the catalytic domain adopts a tightly folded conformation affording protection from proteolytic attack. In contrast, the cellulose-binding domain showed a relatively loose conformation. Progressive proteolytic truncation from the amino terminus was apparent during incubation with alpha-chymotrypsin or papain, or with C. fimi protease under reducing conditions. Affinity for cellulose was retained by products missing up to 64 amino-terminal amino acids. The remaining carboxyl-proximal region of the cellulose-binding domain with affinity (47 amino acids) contained sequences highly conserved in analogous domains from other bacterial endo-beta 1,4-glucanases. By analogy with other systems, the properties of the Pro-Thr box are consistent with an elongated conformation. The results of this investigation suggest that CenA has a tertiary structure which resembles that of certain fungal cellulases.     

The N-terminal region of the plasma membrane Ca(2+) pump does not separate from the main catalytic fragments after proteolysis

The purified plasma membrane Ca(2+) pump (PMCA) was digested with trypsin, and the proteolytic products were identified by immunoblotting with monoclonal antibodies JA9 or 5F10 directed against the extreme N-terminal segment and the central portion of the molecule, respectively. After a short treatment with low concentrations of the protease, JA9 reacted predominantly with a peptide of 35 kDa whereas 5F10 detected a peptide of 90 kDa. The trypsin cut leading to the production of these fragments had no effect on the maximal activity of the enzyme. At higher concentrations of trypsin, JA9 detected a main fragment of 33 kDa and smaller fragments of 19 and 15 kDa. The persistence of fragments reacting with JA9 indicates that the N-terminal region containing its epitope (residues 51-75) was not easily accessible to the protease in the native PMCA. However, the reactivity with JA9 was rapidly lost during proteolysis of the denatured protein. The passage of the mixture of PMCA fragments through a calmodulin-Sepharose column resulted in the retention of the N-terminal 35 kDa fragment together with that of 90 kDa, despite the fact that only the latter binds calmodulin. The ethylenediaminetetraacetic acid (EDTA) eluate, which contained about equal amounts of both fragments, had a Ca(2+) ATPase activity similar to that of the intact enzyme. The tight association between the two peptides was evidenced by the fact that concentrations of polyoxyethylene 10 lauryl ether (C(12)E(10)), sodium dodecyl sulfate (SDS) high enough for inactivating the enzyme and dissociate the pump from calmodulin were unable of breaking the interaction between the 35 and 90 kDa fragments. Altogether, these results show that after digestion with trypsin, the N-terminal portion of the PMCA, including the extreme N-terminal segment, remains part of a fully functional catalytic complex.     

Calpain I activates Ca2+ transport by the reconstituted erythrocyte Ca2+ pump

Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca²⁺ transport function of the Ca²⁺-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca²⁺ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca²⁺/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca²⁺-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca²⁺ translocation function of the Ca²⁺-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain.     

Human seminal vesicle-specific antigen is a substrate for prostate-specific antigen (or P-30)

Human seminal vesicle and prostatic fluids were obtained separately and reconstituted in vitro to test the hypothesis that proteolytic enzymes of prostatic origin would degrade seminal vesicle-specific antigen (SVSA). Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis to follow the fate of SVSA over time, we found that upon mixing the two secretions, SVSA was converted to forms of intermediate and low molecular weight identical to transformations seen in normal liquefied ejaculates. Diisoproprylfluorophophate, a serine protease inhibitor, prevented this degradation, indicating serine protease involvement in the proteolysis of SVSA. Prostate-specific antigen (PSA; also known as P-30), recently identified as a serine protease, was examined for its ability to mimic the effects of prostatic fluid on SVSA. Purified PSA catalyzed degradation of SVSA to produce proteolytic fragments that comigrated and were immunologically related to SVSA fragments produced by prostatic fluid. Purified PSA in the presence of serine protease inhibitors was unable to degrade SVSA. These results demonstrate that SVSA is a substrate for PSA during human semen liquefaction.     

Fibronectin cleavage fragments provide a growth factor-like activity for the differentiation of Trypanosoma cruzi trypomastigotes to amastigotes.

The morphological and biochemical events following Trypanosoma cruzi trypomastigote-fibronectin (Fn) interactions have been studied. Adhesion of trypomastigotes to Fn-coated surfaces is followed by Fn degradation. The proteolytic cleavage of Fn was demonstrated by qualitative and quantitative measurement of Fn degradation after its exposure to trypomastigotes as well as polyacrylamide gel analysis of Fn proteolysis by a parasite protease (s). The released Fn peptide fragments stimulated the transformation of trypomastigotes to amastigotes. The gelatin (45 kDa) and heparin (40 kDa) binding fragments were shown to be able to promote trypomastigote differentiation. In contrast, native Fn and the 120 kDa fragment (cell attachment domain) were inactive. Complementary investigations showed that the gelatin and heparin binding fragments stimulated parasite RNA synthesis and protein synthesis and phosphorylation but not DNA replication and increased parasite intracellular cAMP concentrations. These findings suggest that the proteolysis of Fn by parasite proteases, which occurs under physiological conditions, might facilitate invasion of target cells by trypomastigotes. The Fn peptides released during this process may act as "growth factor-like" substances.     

Sequential Limited Proteolysis of Myelin Basic Protein by Neutral Protease Activities of Bovine Brain

Acid extracts of delipidated white matter of bovine brain were prepared, and their proteolytic activities toward myelin basic protein (MBP) were evaluated at pH 3 and pH 7. This was done by measuring changes in total protein using a selective dye-binding assay, and by evaluating peptide patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. At pH 7 greater than 50% of total protein and about 75% of MBP were degraded after 48 h, whereas at pH 3 it was less than 20% altogether. Neutral proteolysis of MBP entailed up to 12 different proteolytic peptide fragments in the molecular weight range of 17.5 to 6 kd. Its enzymatic nature was verified using protease inhibitors, including N-ethylmaleimide, phenylmethylsulfonyl fluoride, o-phenanthroline, and EDTA, as well as pepstatin A and alpha 2-macroglobulin. Both transient changes in percentages of some intermediate peptides and differential effects of individual inhibitors on electrophoretic peptide patterns strongly suggest a sequential type of limited proteolysis. The results also indicate that acid extracts contained several endopeptidases of which a cysteine protease appears to initiate the breakdown of MBP.