Cathepsin B-like Protease Research Articles

Cloning, Characterization, and Expression of the Human TIN-ag-RP Gene Encoding a Novel Putative Extracellular Matrix Protein

The human gene encoding a novel tubulointerstitial nephritis antigen (TIN-ag)-related protein (TIN-ag-RP) was isolated, and its genomic organization was determined. BLAST searches revealed the highest degree of homology to several mammalian TIN-ag orthologues, and a weak homology to cathepsin B-like proteases. The 12 kb gene was mapped by fluorescence in situ hybridization to chromosome 1p34.2-3, a locus neither related to that of the human TIN-ag (6p11.2-12) nor to that of cathepsin B (8p22-23.1). The TIN-ag-RP is encoded in ten exons with introns ranging from 83 bp to 4 kb. In addition, the gene contained one exon in the 5′UTR, but none in the 3′UTR. Five of the 10 splice sites of the TIN-ag-RP gene were fully conserved when compared to a related gene of C. elegans, whereas only one splice site was identical to those found in cathepsin B genes. Furthermore, human TIN-ag-RP tagged with the T7-epitope, was expressed in HeLa cells, and was found to be localized in vesicular compartments as well as secreted into the medium suggesting the involvement of the endosomal trafficking pathway. Based on the high degree of homology of the amino acid sequences and genomic organization between TIN-ag-RP and TIN-ag, we suggest that both molecules may form a distinct group or family of TIN-ag-like proteins.

Isolation and characterization of a cDNA clone encoding a thiol proteinase of Sarcocystis muris cyst mero' zoites (Apicomplexa).

A cDNA library of Sarcocystis muris cyst merozoites was screened using a digoxigenin-labeled probe. This probe was derived from a 504-bp polymerase-chain-reaction fragment representing part of a thiol proteinase. Several cDNA clones were isolated, one of which (PH08) consists of a nucleotide sequence of 1694 bp and encodes the complete prepropolypeptide of a cathepsin L-like proteinase. PH08 contains an open reading frame of 394 amino acid (aa) residues with a 46-residue signal sequence, which is followed by a 129-residue propeptide and 219 aa residues of the mature enzyme. Two potential glycosylation sites and a putative polyadenylation signal were also identified. The occurrence of the highly conserved interspersed ERFNIN aa motif, not found in cathepsin B-like proteinases, suggests the classification of the enzyme as a cathepsin L-like proteinase. Results worked out in this study will enable production of the recombinant thiol proteinase of S. muris cyst merozoites necessary for study of the substrate specificity as well as other biochemical parameters of this enzyme.

Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km = 3740±413 M-1·s-1 versus 472±72.4 M-1·s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki = 208200±36000 M-1·s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-ʟ-isoleucyl-L-proline, kinact/Ki = 199200±32900 M-1·s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km=3740+/-413 M-1.s-1 versus 472+/-72.4 M-1.s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki=208200+/-36000 M-1. s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l- prolin e, kinact/Ki=199200+/-32900 M-1.s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

Mosquito Cathepsin B-like Protease Involved in Embryonic Degradation of Vitellin Is Produced as a Latent Extraovarian Precursor

Here we report identification of a novel member of the thiol protease superfamily in the yellow fever mosquito, Aedes aegypti. It is synthesized and secreted as a latent proenzyme in a sex-, stage-, and tissue-specific manner by the fat body, an insect metabolic tissue, of female mosquitoes during vitellogenesis in response to blood feeding. The secreted, hemolymph form of the enzyme is a large molecule, likely a hexamer, consisting of 44-kDa subunits. The deduced amino acid sequence of this 44-kDa precursor shares high similarity with cathepsin B but not with other mammalian cathepsins. We have named this mosquito enzyme vitellogenic cathepsin B (VCB). VCB decreases to 42 kDa after internalization by oocytes. In mature yolk bodies, VCB is located in the matrix surrounding the crystalline yolk protein, vitellin. At the onset of embryogenesis, VCB is further processed to 33 kDa. The embryo extract containing the 33-kDa VCB is active toward benzoyloxycarbonyl-Arg-Arg-para-nitroanilide, a cathepsin B-specific substrate, and degrades vitellogenin, the vitellin precursor. Both of these enzymatic activities are prevented by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a thiol protease inhibitor. Furthermore, addition of the anti-VCB antibody to the embryonic extract prevented cleavage of vitellogenin, strongly indicating that the activated VCB is involved in embryonic degradation of vitellin.

Target genes and regulatory domains of the GAMYB transcriptional activator in cereal aleurone.

GAMYB is an MYB transcription factor which is expressed in cereal aleurone cells in response to gibberellin (GA). HvGAMYB binds to the TAACAAA box of a high-pl alpha-amylase gene promoter and transcriptionally activates its expression. In this study, we examined the role of HvGAMYB in activating expression of other GA-regulated genes encoding hydrolytic enzymes. In transient expression experiments, HvGAMYB transactivated expression of reporter genes fused to a low-pl alpha-amylase gene promoter, an EII (1-3, 1-4)-beta-glucanase gene promoter and a cathepsin B-like protease promoter. HvGAMYB DNA binding specificity was determined using a PCR-based random site selection using HvGAMYB fusion protein isolated from E. coli. The deduced consensus closely resembled gibberellin response elements in alpha-amylase promoters. Functional analysis of HvGAMYB by transient expression of C terminal HvGAMYB deletions in barley aleurone cells identified two transcriptional activation domains (TADs) which function in transcriptional regulation of both high- and low-pl alpha-amylase promoters. The same TADs were identified using a heterologous yeast expression system. Together, these results indicate that HvGAMYB has two TADs. These domains are C-terminal to its DNA-binding domain.

Cloning and sequencing of tccb, a gene encoding a Trypanosoma cruzi cathepsin B-like protease

Cloning and sequencing of tccb, a gene encoding a Trypanosoma cruzi cathepsin B-like protease

Binding of the Cysteine Proteinases Papain and Cathepsin B-like to Coated Laminin: Use of Synthetic Peptides from Laminin and from the Laminin Binding Region of the β1Integrin Subunit to Characterize the Binding Site

Cysteine proteinases of the papain superfamily, i.e., papain and cathepsin B-like proteinase, were found to be able to bind to laminin-coated wells. When papain and cathepsin B-like proteinase were used, saturable binding curves were found. The characterization of the binding site was carried out using synthetic peptides which corresponded to the most relevant functional sites of laminin and an octapeptide from the laminin binding region of the β1integrin subunit. In binding experiments, the decapeptide RNIAEIIKDI and the pentapeptide YIGSR were able to displace papain and cathepsin B-like proteinase from coated laminin. Nevertheless, the integrin β1peptide DLYYLMDL was the most powerful in the same experimental system. From these results, the C-terminal region of this cross-shaped protein, i.e., the end of the long arm, and the region including the YIGSR sequence of the short arm of the β chain would be the cysteine proteinase binding site. This binding site is probably the result of the network organization of laminin which brings two regions, separated on a single laminin molecule, into proximity. In previous work, digestion of basement membranes has been found to be associated with the binding of cysteine proteinases to these supramolecular structures [N. Guinec, V. Dalet-Fumeron, and M. Pagano (1992)FEBS Lett.308, 305–308]. The present report demonstrates that laminin is the cysteine proteinase binding protein of basement membranes. This property of laminin could be associated with tumor invasion and other tissue remodeling processes linked to proteolysis of basement membranes and extracellular matrices.

Affinity Isolation and Characterization of the Cathepsin B-like Proteinase Sj31 from Schistosoma japonicum

The major cathepsin B-like proteinase of adult Schistosoma japonicum has been isolated for the first time. Affinity chromatography with the mammalian cathepsin B inhibitor glycyl-phenylalanyl-glycine-semicarbazone purified a protein that was identified by N-terminal sequencing as Sj31. Sensitivity of Sj31 to PNGase F demonstrated the presence of asparagine-linked N-glycan. Marked resistance to the action of Endo-beta-glycosidase H indicated that most of the N-glycan chains are of the complex type. Binding of horseradish peroxidase-conjugated lectins demonstrated the presence of N-mannose, N-acetylglucosamine, and N-acetyllactosamine type 2 in the N-glycan. Fucose was not detected, and the presence of sialic acid remained questionable. Sj31 degraded the fluorogenic substrates Z-Phe-Arg-NMec and Z-Arg-Arg-NMec with an optimum between pH 5.0 and 6.0. The specific activity was 18-21-fold higher with the Phe-Arg substrate compared with the Arg-Arg substrate, whereas this value was 4-6-fold for bovine spleen cathepsin B, thus suggesting differences in the S2 subsite between parasite and host proteinases. Quantitative purification of Sj31 led to the conclusion that cathepsin B-like activity predominates over cathepsin L-like activity in S. japonicum. Because Sj31 degraded hemoglobin in vitro and was localized in the parasite gut, the proteinase may degrade ingested proteins in vivo.

A unique cathepsin-like protease isolated from CV-1 cells is involved in rapid degradation of retinoblastoma susceptibility gene product, RB, and transcription factor SP1

The regulation of transcription factors by kinase or phosphatase has been well-described. However, little is known about the inactivation of transcription factors or the nuclear regulators by proteolytic degradation. In this report, we purified a specific protease, SPase, from nuclear extracts of the green monkey kidney cell line, CV-1. Studies of biochemical characteristics and substrate specificity indicated that SPase is a cathepsin B-like cysteinyl protease. However, the two tryptic peptide sequences derived from the purified SPase are either identical or highly homologous to those of human cathepsin L, and furthermore, SPase shares immunoreactivity with both anti-human cathepsin L and anti-mouse cathepsin L antibody. The SPase was shown to be localized in both cytoplasm and nucleus when subcellular compartments of CV-1 cells were fractionated. Transcription factor, SP1, and retinoblastoma susceptible gene product, RB, are substrates of SPase while other nuclear factors such as c-Jun and c-Fos are not. These results implied that SPase plays an integral role in regulating a set of proteins in the nuclei. In vivo treatment of CV-1 cells with cysteinyl protease inhibitor, E-64d, protected RB from degradation. SPase failed to degrade underphosphorylated RB present in TPA induced terminally differentiated HL-60 or U937 cells. Phosphorylation of RB may cause conformational changes, thus facilitating proteolytic digestion. These observations suggest that an alternative pathway inactivates the function of RB in controlling cell growth. Therefore, a possible role of SPase may be to affect the stability of important regulators involved in controlling cellular proliferation and differentiation.

Trichuris suis:Thiol Protease Activity from Adult Worms

Trichuris suis,the whipworm of swine, causes anemia, weight loss, anorexia, mucohemorrhagic diarrhea, and death in heavy infections. A zinc metalloprotease has been suggested to play a role in the severe enteric pathology associated with infection and the infiltration of opportunistic bacteria into deeper tissues in the swine colon. In this study, a thiol protease from gut extracts of adultT. suisand from excretory/secretory components (E/S) of adult worms was characterized using fluorogenic peptide substrates and protein substrate gels. The protease cleaved the fluorogenic substrate Z-Phe-Arg-AMC, and this cleavage was completely inhibited by the thiol protease inhibitors E-64, leupeptin, Z-Phe-Ala-CH2F, and Z-Phe-Arg-CH2F. Gelatin substrate gels and fluorescence assays using both the gut and the stichosome extracts and E/S revealed enhanced activity when 2 mMdithiothreitol or 5 mMcysteine was included in the incubation buffer, and optimal activity was seen over a pH range of 5.5 to 8.5. Incubation of gut extracts or E/S material with inhibitors of aspartic, serine, or metalloproteases had no effect on the cleavage of Z-Phe-Arg-AMC. Thiol protease activity was found in extracts of gut tissue but not in the extracts of stichocytes of adult worms. N-terminal amino acid sequencing of the protease revealed sequence homologies with cathepsin B-like thiol protease identified from parasitic and free-living nematodes.

Structural analysis of the catalytic site of AcCP-1, a cysteine proteinase secreted by the hookworm Ancylostoma caninum

Previously, we have reported that hookworms secrete cysteine proteinase activity that is capable of cleaving the cathepsin L-specific substrate Z-Phe-Arg-AMC. We have also reported the gene sequences of novel cathepsin B-like proteinases from hookworms, but have been unable to locate cathepsin L-like genes that could account for the presence of the cathepsin L activity in these parasites. Here we present an homology model for the secreted hookworm cysteine proteinase AcCP-1 based upon the crystal structure co-ordinates of human cathepsin B. The model predicts that substrate binding and specificity differs between AcCP-1 and cathepsin B, and demonstrates that AcCP-1 would preferentially cleave Phe-Arg over Arg-Arg. This thereby provides an explanation for our previous observations that the hookworm proteinase, while structurally cathepsin B-like, displays a cathepsin L-like substrate specificity.

Occurrence of a cathepsin B-like acid cysteine proteinase in the eggs of silkworm moth, Antheraea pernyi

In the crude extract of mature eggs of the pernyi silkworm moth, Antheraea pernyi, proteolytic activity was detected in the acidic pH region, with maximal activity at pH 3.5., however, no appreciable activity was observed in the neutral to alkaline pH region. The enzyme activity was strongly inhibited by cysteine proteinase inhibitors, such as iodoacetic acid (IAA) and N-[N-(L-3-trans-carboxyoxirane-2-carbony)- l-leucyl] agmatine (E-64); whereas, pepstatin, diisopropyl fluorophosphate (iPr 2P-F), and EDTA were without effect, suggesting the enzyme to be a cysteine proteinase. From the substrate specificity, major proteinase in the eggs is likely to be a cathepsin B-like proteinase. When the crude extract of eggs was incubated at pH 4.0, enzymatic activity appeared after a few minutes lag period, indicating that the enzyme was stored in a latent form in the eggs and thus was activated during the incubation. Pepstatin completely inhibited the activation but had no effect on the activated enzyme, suggesting that aspartic protease (s) was involved in this activation. The proteinase was found in vitro to readily degrade yolk polypeptides, as well as hemoglobin and other substrates. In the pupal ovary, this activity gradually increased during embryogenesis, reaching a maximum at days 10 after pupal ecdysis. After reaching a maximum, the level of cysteine proteinase remained approximately constant and the high activity was maintained during embryogenesis. The results altogether suggest that an acid cysteine proteinase (ACP) is stored as a latent form in the eggs and is implicated in yolk protein degradation of Antheraea pernyi eggs during embryogenesis.

Processing of procathepsin from Musca domestica eggs

The major source of amino acids for insect embryos are yolk proteins which accumulate in developing oocytes and are hydrolyzed during embryogenesis. Studies on Musca domestica embryogenesis indicated that a cathepsin B-like proteinase is responsible for yolk protein degradation (Ribolla et al 1993). In this study, we report the purification of mature cathepsin and show that it is made up of a single 41 kDa polypeptide chain. The Musca domestica cathepsin NH 2-terminal 11-residue sequence was determined (Ala-Pro-Lys-Tyr-Val-Asp-Tyr-Gly-Glu-Asn-Glu) and reveals homology with other cathepsins of the papain family. Experiments using serum anti-cathepsin show that the enzyme is stored in oocytes as a 55 kDa zymogen. The activation of the zymogen occurs in vitro only at low pH. In vitro activation in the presence of cysteine protease inhibitors is blocked at an intermediary polypeptide of 48 kDa. Kinetic studies of this activation process at pH 3.5 and 4.6 show that the zymogen is processed in a manner similar to that of pepsin (Foltmann, 1986) and papain (Vernet et al 1991). We propose that Musca domestica cathepsin zymogen activation occurs in two steps. First, an intramolecular cleavage of the procathepsin polypeptide chain (55,000), induced by low pH gives rise to an intermediary polypeptide (48,000) which then undergoes autolysis to produce the mature enzyme (41,000).

Clinical relevance of cathepsin B-like enzyme activity and cysteine proteinase inhibitor in melanocytic tumours.

We studied the relationship between cathepsin B (CB)-like enzyme activities and cysteine proteinase inhibitor (CPI) activities in lesions of human pigmented naevi (PN), primary melanomas (PM) and metastases/metastatic melanomas (MM). The CB-like enzyme activities in PM and MM were 2.5 and 6.8 times higher than in PN, respectively. The CB-like/CPI ratios in PM and MM were 1.9 and 11.6 times higher than in PN, respectively. CPI had the highest activity in PM and the lowest activity in MM. The disease-free interval of patients with a high CB-like enzyme activity (> or = 80 U/mg protein) and/or a high CB-like/CPI ratio (> or = 7) was shorter than that of patients with a low CB-like enzyme activity (< 80 U/mg protein) and/or a low CB-like/CPI ratio (< 7). Analysis of CB-like enzyme activity and/or the CB-like/CPI ratio may be useful in predicting the prognosis of human malignant melanoma.

A study on cathepsin B-like substance in cancer of the urinary tract

Cathepsin B, a cysteine proteinase, has been shown to be increased in tumor tissue and to be responsible for the process of invasion and metastasis. To study the biochemical characteristics of cathepsin B-like substance in the tissue of a non-cancerous kidney and some urologic cancer tissues, we extracted cathepsin B-like substances from a non-cancerous human kidney tissue, human renal tumor, human renal pelvic tumor and human bladder tumor. The cathepsin B-like substance extracted from the non-neoplastic human kidney has a molecular weight of approximately 28,000. On the result of western blotting with anti-human liver cathepsin B antibody, two types of protein were observed in the fraction extracted from urologic cancer tissues. The molecular weights of them were 28,000 and 35,000, respectively. Both types of cathepsin B-like substances had the same activities for benzyloxy-carbonyl-L-arginine-L-arginine-4-methyl-7-coumarylamide, and both activities were inhibited by (L-3-transcarboxyoxiran-2-carbonyl)-L-leucyl-agmatin. These findings suggest that both cancer cells of the renal parenchyma and urothelium secrete cathepsin B-like protease which express not only a normal cathepsin B but also a higher molecular weight of cathepsin B-like protease and that a higher one plays an important role in a process of invasion.

Further characterization of platelet‐aggregating cysteine proteinase activity in thrombotic thrombocytopenic purpura

Circulating platelet-aggregating activities have been described by several authors. In a previous study we have confirmed the presence in serum and plasma from patients with thrombotic thrombocytopenic purpura (TTP) of a platelet aggregating cysteine proteinase (PACP). This activity differed in that, among the class of thiol enzymes, it showed characteristics of a lysosomal cathepsin rather than of a cytoplasmic calpain. To further investigate the enzymatic properties of this PACP we have designed a study to evaluate the dependence of the activity on Ca++. Calcium-dependence is a property of calpains but not of cathepsins. A proteolytic assay was set up and conducted with and without Ca++. The release of acid-soluble peptide from denatured human globin promoted by TTP samples and standards was fluorimetrically measured. The results show that TTP samples were equally active with and without Ca++ similar to standard papain (the cathepsin B-like proteinase), whereas standard calpain only acted with Ca++. An inhibition study performed by the proteolytic assay confirmed the same pattern of sensitivity of PACP to a series of specific cystein proteinase inhibitors previously shown by the proaggregating assay. The enzymatic behaviour of PACP of TTP resembled a lysosomal (cathepsins) rather than a cytoplasmic (calpains) cysteine proteinase.

The detection, quantification and partial characterisation of cathepsin B-like activity in human pathological synovial fluids.

In this study, the levels of the cysteine proteinase--cathepsin B were measured in diseased synovial fluids using a steady state fluorometric assay. Cathepsin B-like activity was shown to be present in all the samples analysed, with the rheumatoid arthritic synovial fluids possessing significantly higher concentrations (mean value ca. 416 mg/l) than the osteoarthritic fluids (mean value ca. 142.4 mg/l). In addition, upon treatment with pepsin, all of the rheumatoid arthritis samples were shown to possess additional cathepsin B-like activity, suggesting the presence of a reservoir of latent precursor molecules. By utilising a recently developed biotinylated affinity label for cathepsin B-like proteinases and sheep anti-(human cathepsin B) antibodies, used in combination with SDS-PAGE and Western blotting, the rheumatoid arthritic synovial cathepsin B was shown to exist in two forms with apparent molecular masses of M(r) 29,000 and 42,000. We propose that the former is a functionally active proteinase, whereas the latter is a pepsin activatable proform which, when cleaved by this aspartyl proteinase, is converted into a catalytically competent species of M(r) 20,000.

Two distinct gene subfamilies within the family of cysteine protease genes.

A cDNA clone for a physiologically regulated Tetrahymena cysteine protease gene was sequenced. The nucleotide sequence predicts that the clone encodes a 336-amino acid protein composed of a 19-residue N-terminal signal sequence followed by a 107-residue propeptide and a 210-residue mature protein. Comparison of the deduced amino acid sequence of the protein with those of other cysteine proteases revealed a highly conserved interspersed amino acid motif in the propeptide region of the protein, the ERFNIN motif. The motif was present in all of the cysteine proteases in the data base with the exception of the cathepsin B-like proteins, which have shorter propeptides. Differences in the propeptides and in conserved amino acids of the mature proteins suggest that the ERFNIN proteases and the cathepsin B-like proteases constitute two distinct subfamilies within the cysteine proteases.

Cathepsin B and acid phosphatase activities during Musca domestica embryogenesis

During Musca domestica embryogenesis, we detected a proteolytic activity that is probably involved in yolk protein degradation. This was characterized as a cathepsin B-like proteinase, with a pH optimum of 4.6 and a molecular weight of 25,000 ± 5000. An acid phosphatase activity was also found during M. domestica embryogenesis. This enzyme has a molecular weight equal to 84,300 ± 3000, is inhibited by fluoride and tartrate and therefore may be of lysosomal origin. The activity patterns of both enzymes are similar during embryogenesis. Acid phosphatase inhibition does not have any effect on the cathepsin proteolytic action against yolk protein, indicating that the two enzymes do not have a cooperative action for initiation of vitellin hydrolysis.