Dependent Proteolytic Activity Research Articles

Obesity-associated Adipose Stromal Cells Promote Breast Cancer Invasion Through Direct Cell Contact and ECM Remodeling.

Obesity increases the risk and worsens the prognosis for breast cancer due, in part, to altered adipose stromal cell (ASC) behavior. Whether ASCs from obese individuals increase migration of breast cancer cells relative to their lean counterparts, however, remains unclear. To test this connection, multicellular spheroids composed of MCF10A-derived tumor cell lines of varying malignant potential and lean or obese ASCs were embedded into collagen scaffolds mimicking the elastic moduli of interstitial breast adipose tissue. Confocal image analysis suggests that tumor cells alone migrate insignificantly under these conditions. However, direct cell-cell contact with either lean or obese ASCs enables them to migrate collectively, whereby obese ASCs activate tumor cell migration more effectively than their lean counterparts. Time-resolved optical coherence tomography (OCT) imaging suggests that obese ASCs facilitate tumor cell migration by mediating contraction of local collagen fibers. Matrix metalloproteinase (MMP)-dependent proteolytic activity significantly contributes to ASC-mediated tumor cell invasion and collagen deformation. However, ASC contractility is also important, as co-inhibition of both MMPs and contractility is necessary to completely abrogate ASC-mediated tumor cell migration. These findings imply that obesity-mediated changes of ASC phenotype may impact tumor cell migration and invasion with potential implications for breast cancer malignancy in obese patients.

Polyporus squamosus Lectin 1a (PSL1a) Exhibits Cytotoxicity in Mammalian Cells by Disruption of Focal Adhesions, Inhibition of Protein Synthesis and Induction of Apoptosis.

PSL1a is a lectin from the mushroom Polyporus squamosus that binds to sialylated glycans and glycoconjugates with high specificity and selectivity. In addition to its N-terminal carbohydrate-binding domain, PSL1a possesses a Ca2+-dependent proteolytic activity in the C-terminal domain. In the present study, we demonstrate that PSL1a has cytotoxic effects on mammalian cancer cells, and we show that the cytotoxicity is dependent on the cysteine protease activity. PSL1a treatment leads to cell rounding and detachment from the substratum, concomitant with disruption of vinculin complexes in focal adhesions. We also demonstrate that PSL1a inhibits protein synthesis and induces apoptosis in HeLa cells, in a time- and concentration-dependent manner.

Calpain3 Is Expressed in a Proteolitically Active Form in Papillomavirus-Associated Urothelial Tumors of the Urinary Bladder in Cattle

BackgroundCalpain 3 (Capn3), also named p94, is a skeletal muscle tissue-specific protein known to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Recent experimental studies have hypothesized a pro-apoptotic role of Capn3 in some melanoma cell lines. So far the link between calpain3 and tumors comes from in vitro studies. The objective of this study was to describe Capn3 activation in naturally occurring urothelial tumors of the urinary bladder in cattle.Methods and FindingsHere we describe, for the first time in veterinary and comparative oncology, the activation of Capn3 in twelve urothelial tumor cells of the urinary bladder of cattle. Capn3 protein was initially identified with nanoscale liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS) in a co-immunoprecipitation experiment on E2F3, known to be a transcription factor playing a crucial role in bladder carcinogenesis in humans. Capn3 expression was then confirmed by reverse transcription polymerase chain reaction (RT-PCR). Finally, the Ca2+-dependent proteolytic activity of Capn3 was assayed following ion exchange chromatography. Morphologically, Capn3 expression was documented by immunohistochemical methods. In fact numerous tumor cells showed an intracytoplasmic immunoreactivity, which was more rarely evident also at nuclear level. In urothelial tumors, bovine papillomavirus type 2 (BPV-2) DNA was amplified by PCR and the expression of E5 protein, the major oncogenic protein of BVP-2, was detected by western blotting, immunohistochemistry, and immunofluorescence. E2F3 overexpression and pRb protein downregulation were shown by western blotting.ConclusionThe role of capn3 protein in urothelial cancer of the urinary bladder remains to be elucidated: further studies would be required to determine the precise function of this protease in tumor development and progression. However, we suggest that activated Capn3 may be involved in molecular pathways leading to the overexpression of E2F3, which in turn could be responsible for urothelial tumor cell proliferation also in cattle, though other mechanisms are likely to exist. If further studies corroborate the important role of Capn3 in urothelial tumors of the urinary bladder, cattle with urinary tumors may prove useful as animal model for bladder carcinogenesis.

The Ca2+ binding capacity of epidermal furin is disrupted by H2O2‐mediated oxidation in vitiligo

It was previously established that epidermal keratinocytes and melanosomes express the Ca2+‐dependent precursor convertase furin. All prohormone convertases including furin are regulated by Ca2+. Meanwhile, it is noteworthy that proopiomelanocortin (POMC) cleavage is processed by furin, in addition to the classical PC1 and PC2 convertases, leading to the production of ACTH, β‐LPH and β‐endorphin. Since numerous epidermal peptides and enzymes are affected by H2O2‐mediatedoxidation, including the POMC derived peptides α‐MSH and β‐endorphin, as shown in the epidermis of patients with vitiligo, it was of interest whether furin could also be a possible target for this oxidation mechanism. Confirming the presence of furin in epidermal keratinocytes and melanocytes using immunoflurescence, RT‐PCR, and Western blotting, we further demonstrate significantly decreased in situ immunoreactivity of furin in the epidermis of patients with progressive vitiligo suggesting H2O2‐mediated oxidation. This was substantiated by [45Ca2+] binding studies with human recombinant furin identifying the loss of one Ca2+‐binding site from the enzyme after oxidation with H2O2. Computer simulation supported alteration of one of the two Ca2+‐binding sites on furin. Overall, our results demonstrate that the Ca2+ dependent proteolytic activity of this convertase is a target for H2O2‐mediated oxidation which in turn could contribute to the reduced epidermal expression of the POMC derived peptides α‐MSH and β‐endorphin as documented earlier in patients with vitiligo.

The Ca2+-Binding Capacity of Epidermal Furin Is Disrupted by H2O2-Mediated Oxidation in Vitiligo

The Ca(2+)-dependent precursor convertase furin is abundantly expressed in epidermal keratinocytes and melanocytes. In this context, it is noteworthy that proopiomelanocortin (POMC) cleavage is also processed by furin, leading to ACTH, beta-lipotropin, and beta-endorphin. All prohormone convertases including furin are regulated by Ca(2+). Because numerous epidermal peptides and enzymes are affected by H(2)O(2)-mediated oxidation, including the POMC-derived peptides alpha-MSH and beta-endorphin as shown in the epidermis of patients with vitiligo, we here asked the question of whether furin could also be a possible target for this oxidation mechanism by using immunofluorescence, RT-PCR, Western blotting, Ca(2+)-binding studies, and computer modeling. Our results demonstrate significantly decreased in situ immunoreactivity of furin in the epidermis of patients with progressive vitiligo (n = 10), suggesting H(2)O(2)-mediated oxidation. This was confirmed by (45)Ca(2+)-binding studies with human recombinant furin identifying the loss of one Ca(2+)-binding site from the enzyme after oxidation with H(2)O(2). Computer simulation supported alteration of one of the two Ca(2+)-binding sites on furin. Taken together, our results implicate that the Ca(2+)-dependent proteolytic activity of this convertase is targeted by H(2)O(2), which in turn could contribute to the reduced epidermal expression of the POMC-derived peptides alpha-MSH and beta-endorphin as documented earlier in patients with vitiligo.

Metacaspase 2 of Trypanosoma brucei is a calcium-dependent cysteine peptidase active without processing

Metacaspases are cysteine peptidases that are distantly related to the caspases, for which proteolytic processing is central to their activation. Here, we show that recombinant metacaspase 2 (MCA2) from Trypanosoma brucei has arginine/lysine-specific, Ca 2+-dependent proteolytic activity. Autocatalytic processing of MCA2 occurred after Lys55 and Lys268; however, this was shown not to be required for the enzyme to be proteolytically active. The necessity of Ca 2+, but not processing, for MCA2 enzymatic activity clearly distinguishes MCA2 from the caspases and would be consistent with different physiological roles.

Calpains in muscle: selective and protective?

Calpains in muscle: selective and protective?

Urokinase separation from cell culture broth of a human kidney cell line.

A single step ion-exchange chromatography on a sulfo-propyl (SP)- Sepharose column was performed to separate both the high molecular weight (HMW)- and low molecular weight (LMW)- forms of enzymatically active urokinase type plasminogen activator from human kidney (HT1080) cell culture media. The level of urokinase secreted by the cell line reached to about 145 Plough units/ml culture broth within 48 h of cultivation. The conditioned cell culture media was applied directly to the column without any prior concentration steps. Polyacrylamide gel electrophoresis of the column eluates in the presence of sodium dodecyl sulphate showed that the cell line secretes three forms of two-chain high molecular weight (HMW) urokinase of molecular weights (M(r)) 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW) forms of M(r) 22,000 and 20,000; proteolytic cleavage products of HMW, were also found. The HMW and LMW forms had intrinsic plasminogen dependent proteolytic activity as judged by zymographic analysis. The specific activity of the pooled peak fractions increased (approximately 93-fold) to values as high as 1481 Plough units/ mg protein. Both HMW as well as LMW forms were obtained in significantly high yields.

Metal-dependent hydrolysis of myelin basic protein by IgGs from the sera of patients with multiple sclerosis

Homogeneous IgG fractions were obtained by chromatography of the sera of patients with multiple sclerosis (MS) on Protein G-Sepharose under conditions that remove non-specifically bound proteins. These IgGs contained several chelated metals, the relative amount of which decreases in the order: Fe ≥ Ca > Cu ≥ Zn ≥ Mg ≥ Mn ≥ Pb ≥ Co ≥ Ni. In contrast to homogeneous IgGs of healthy individuals, Abs of MS patients effectively hydrolyzed human myelin basic protein (MBP). A minor metal-dependent fraction was obtained by chromatography of highly purified IgGs from MS patient on Chelex-100. This IgG fraction did not hydrolyze human MBP in the absence of Me 2+ ions but was activated after addition of Me 2+ ions: Mg 2+ > Mn 2+ > Cu 2+ > Ca 2+. Proteolytic activities of IgGs from other MS patients were also activated by other metal ions (Ni 2+, Fe 2+, Co 2+, Zn 2+, Pb 2+, and Co 2+) and especially Ni 2+. Ni 2+-activated IgGs were separated into distinct MBP-hydrolyzing fractions by chromatography on HiTrap™ Chelating Sepharose charged with Ni 2+. Detection of Mg 2+-dependent proteolytic activity in the SDS-PAGE area corresponding only to IgG provided direct evidence that IgG from sera of MS patients possesses metal-dependent human MBP-hydrolyzing activity. Observed properties of MS abzymes distinguish them from other known mammalian metalloproteases and demonstrate their pronounced catalytic diversity. Metal-dependent IgGs from MS patients represent the first example of abzymes with metal-dependent proteolytic activity.

ß2-Agonists and cAMP inhibit protein degradation in isolated chick (Gallus domesticus) skeletal muscle

1. The role of β2-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours. 2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective β2-agonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP. 3. The decrease in the rate of total protein degradation induced by 10−5 m clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10−5 m ICI 118·551, a selective β2-antagonist. 4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10−4 m dibutyryl-cAMP or isobutylmethylxanthine. 5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by β2-adrenoceptors, with the participation of a cAMP-dependent pathway.

Presenilin-dependent Intramembrane Proteolysis of CD44 Leads to the Liberation of Its Intracellular Domain and the Secretion of an Aβ-like Peptide

Alzheimer's disease (AD)-associated gamma-secretase is a presenilin (PS)- dependent proteolytic activity involved in the intramembraneous cleavage of the beta-amyloid precursor protein, Notch, LDL receptor-related protein, E-cadherin, and ErbB-4. This cut produces the corresponding intracellular domains (ICD), which are required for nuclear signaling of Notch and probably ErbB-4, the beta-amyloid precursor protein, E-cadherin, and the LDL receptor-related protein as well. We have now investigated CD44, a cell surface adhesion molecule, which also undergoes an intramembraneous cleavage to liberate its ICD. We demonstrate that this cleavage requires a PS-dependent gamma-secretase activity. A loss-of-function PS1 mutation, a PS1/PS2 knockout, as well as two independent and highly specific gamma-secretase inhibitors, abolish this cleavage. Surprisingly, small peptides similar to the amyloid beta-peptide (Abeta) are generated by an additional cut in the middle of the transmembrane region of CD44. Like Abeta, these CD44 beta-peptides are generated in a PS-dependent manner. These findings therefore suggest a dual intramembraneous cleavage mechanism mediated by PS proteins. The dual cleavage mechanism is required for nuclear signaling as well as removal of remaining transmembrane domains, a general function of PS in membrane protein metabolism.

Mutations in the proteolytic domain of Escherichia coli protease Lon impair the ATPase activity of the enzyme

Conserved residues of the proteolytic domain of Escherichia coli protease Lon, putative members of the classic catalytic triad (H665, H667, D676, and D743) were identified by comparison of amino acid sequences of Lon proteases. Mutant enzymes containing substitutions D676N, D743N, H665Y, and H667Y were obtained by site-directed mutagenesis. The mutant D743N retained the adenosine triphosphate (ATP)-dependent proteolytic activity, thereby indicating that D743 does not belong to the catalytic site. Simultaneously, the mutants D676N, H665Y, and H667Y lost the capacity for hydrolysis of protein substrates. The ATPase activity of these three mutants was decreased by more than an order of magnitude, which suggests a close spatial location of the ATPase and proteolytic active sites and their tight interaction in the process of protein degradation.

The role of plasminogen activators in the development of atherosclerotic and restenotic lesions

The plasminogen activator (PA) system may participate in the pathogenesis of atherosclerosis and of restenosis after angioplasty by modulating the turnover of intimal fibrin and extracellular matrix deposits and by contributing to intimal cell migration. This paper summarise our results on the expression of tissue-type PA (t-PA), urokinase (u-PA), u-PA receptor (uPAR) and PAI-1 in atherosclerotic and restenotic lesions. In atherosclerotic arteries, t-PA, u-PA and PAI-1 were found in intimal smooth muscle cells (SMC) and macrophage-derived foam cells. A particularly high u-PA expression was noted on macrophages localised on the rims of the necrotic core. u-PA-dependent lytic activity was detected in macrophage rich areas of the advanced lesions. The similar distribution pattern of PAI-1 suggests its possible counterbalancing role in the local modulation PA dependent proteolysis within lesions. Time-course analysis of expression of PAs after balloon injury of rat and rabbit arteries have revealed that t-PA and PAI-1 antigen and mRNA are overexpressed by some SMC scattered in the subintimal area after 1–3 days following balloon injury. The neointimal SMC detected after 9 days have exhibited a strong expression of t-PA, PAI-1, u-PA and uPAR antigen and mRNA. PA dependent proteolytic activity was found in the neointimal area, especially after 14 days and it was inhibited in a large extent by anti t-PA antibodies. These results support the existence of a local dynamic process of PA dependent proteolysis in association with the advanced atherosclerotic plaque and with the hyperproliferative response of the SMC of injured arteries..

The plasminogen activation system in bovine milk: Differential localization of tissue-type plasminogen activator and urokinase in milk fractions is caused by binding to casein and urokinase receptor

We have analyzed the occurrence of components of the plasminogen activation system in bovine milk. Zymographic analyses showed that tissue-type plasminogen activator (t-PA) occurred in association with casein micelles, partially as a complex with type-1 plasminogen activator inhibitor (PAI-1), whereas urokinase-type plasminogen activator (u-PA) was confined to milk leukocytes. Whey contained a component with a plasminogen dependent proteolytic activity which was shown to be plasma prekallikrein (PPK). The u-PA in the milk leukocytes was shown to be bound to urokinase receptor (u-PAR). A purification to near-homogeneity of the bovine u-PAR was undertaken. Investigating the novel t-PA binding to casein micelles by ligand blotting and Sepharose immobilized casein, multimeric forms of κ-casein and dimeric α s2-casein were identified as t-PA binding components. The κ-casein gene and the fibrinogen gene are believed to have evolved from a common ancestor. Thus, the recent finding that casein enhances t-PA catalyzed plasminogen activation (Marcus, G., Hitt, S., Harvey, S.R. and Tritsch, G.L. (1993) Fibrinolysis 7, 229–236), and the observed t-PA/casein binding suggests that the casein micelle, which also contains plasminogen, may serve as a matrix for t-PA-catalyzed plasminogen activation in milk.

Plasminogen activators in bovine milk during mastitis, an inflammatory disease

We have investigated the content of plasminogen activators in bovine milk during mastitic inflammation induced by Staphylococcus aureus. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with fibrin agarose zymography and a coupled peptidyl anilide plasminogen activation assay of samples of whey prepared by acidification, we found that the level of tisue-type plasminogen activator (t-PA) in milk was increased immediately after infection and remained elevated during an experimental period of 42 days. The maximal increase was 10 to 20-fold. By zymography, we also demonstrated a strong increase in urokinase-type plasminogen activator (u-PA) associated with the bovine cells in the milk. By ligand blotting, we demonstrated an increase in the level of the urokinase-receptor (u-PAR) on the milk cells during inflammation. Plasma kallikrein was also detected as a plasminogen dependent proteolytic activity by zymography of whey samples. When analyzed in the presence of the t-PA in the milk, the plasma kallikrein lysis zone was strongly increased in mastitic whey, but when analyzed after separation from t-PA, its level was unaffected by mastitis; this could be ascribed to a t-PA dependent stimulation of plasma prekallikrein. These results suggest an important role for plasminogen activators in the inflammatory response during bovine mastitis. Using an enzyme-linked immunosorbent assay we measured the plasminogen/plasmin level during the inflammation, but found a less than 2-fold increase during the experimental period.

Calpain activity in patients with thrombotic thrombocytopenic purpura is associated with platelet microparticles

Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium- dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.

The calcium-dependent proteolytic system calpain-calpastatin in Drosophila melanogaster.

Ca2+-dependent proteolytic activity was detected at pH 7.5 in head extracts of the fruit fly Drosophila melanogaster. This activity was abolished by iodoacetate, but was unaffected by phenylmethanesulphonyl fluoride. These properties resemble those of the Ca2+-dependent thiol-proteinase calpain. The activity appeared at Mr 280,000 on Sepharose CL-6B gel chromatography. DEAE-cellulose chromatography revealed two activity peaks, with elution positions corresponding to vertebrate calpains I and II. The fly head enzymes were inhibited by a heat-stable and trypsin-sensitive component of the fly head extract, which also inhibited calpains from rat kidney. The inhibitor emerged from Sepharose CL-6B columns at Mr 310,000 and from DEAE-cellulose at a position corresponding to the protein inhibitor calpastatin from other sources. It is concluded that Drosophila heads comprise the Ca2+-dependent calpain-calpastatin proteolytic system.

Increased phosphorylation in red cell membranes of subjects affected by essential hypertension

In hemolysates of red cells from hypertensive patients the proteolytic activity of calpain is expressed at a rate approximately three fold higher than in red cells of normotensive subjects. Susceptibility to lysis upon exposure to ionophore A23187 and calcium, conditions that increase intracellular calpain activity, is also significantly enhanced in erythrocytes of hypertensive patients. In inside-out vesicles prepared from erythrocytes of these patients band 3 region undergoes a high extent of phosphorylation which is 1.5 fold higher than that occurring in control red cells from normotensive subjects. This increased phosphorylation can be reproduced in inside-out vesicles from erythrocytes of normal subjects following pretreatment with calpain. Taken together, these results suggest that the presence in erythrocytes of hypertensive subjects of an unregulated calpain dependent proteolytic activity may affect the structure of plasma membranes and determine an increased phosphorylation of intrinsic membrane proteins.

Regional distribution of soluble calcium activated proteinase activity in neonatal and adult rat brain

Calcium dependent proteolytic activity in the soluble fraction of various rat brain regions was assayed using 14C-radiolabelled denatured casein as a substrate. Two forms of activity, distinguishable by their calcium requirement for half maximal activation (5 and 80 μM), were found; both were blocked by sulfhydryl alkylating agents and thiol proteinase inhibitors. Preincubation at 58 °C for 10 min also eliminated the high threshold activity. These characteristics are identical to those reported for calcium activated neutral proteinases (‘calpains’) found in other tissues. Calpain activity varied markedly across brain regions. The greatest values for the high threshold enzyme were found in pons-medula followed by cerebellum/mesencephalon and finally the telencephalon. The low threshold enzyme had low levels of activity throughout the brainstem and diencephalon and was barely detectable in telencephalic structures. In contrast, a previously described endogenous inhibitor of calpain, ‘calpastatin’, was found not to vary in its activity across brain regions. Calpain activity was high in the prenatal brain, but while the hindbrain maintained high levels of activity into adulthood, the activity in the forebrain dropped 80% during the early postnatal period. The differences between forebrain and hindbrain activity levels were evident during the first 5 days of the neonatal period, suggesting that glial cell differentiation is not responsible for the regional variations found in the adult. These results are discussed with regard to the possibility that the turnover of anatomical structures differs between brain regions.

Ca2+-dependent proteolytic activity in crab claw muscle. Effects of inhibitors and specificity for myofibrillar proteins.

The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. We describe here the role of Ca2+-dependent proteinases in the turnover of myofibrillar protein in normal anecdysial (intermolt) claw muscle. Crab Ca2+-dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca2+-dependent proteinase activity in whole homogenates and 90,000 X g supernatant fractions from muscle homogenates has been characterized with respect to Ca2+ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The Ca2+-dependent proteinase is a sarcoplasmic cysteine proteinase that shows maximal activation at 1 mM Ca2+ at neutral pH. Since approximately 28% of the activity remains at 1.5 microM Ca2+, the enzyme is partially active at physiological Ca2+ concentrations. The specificity of crab Ca2+-dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of 125I-myosin occurs in two phases, both Ca2+-dependent: 1) heavy chain (Mr = 200,000) is cleaved into four large fragments (Mr = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (Mr = 18,000) is cleaved to a 15,000-Da fragment; 2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca2+-dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca2+-dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy.