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At least 127 records · Page 7Linked to original sources
PubMed1987

Characterization of the receptor for protease nexin-I:protease complexes on human fibroblasts.

Fibroblasts as well as several other cell types, secrete a number of protease inhibitors into their culture media. Among these inhibitors are the protease nexins, a class of proteins which covalently bind serine proteases, thereby inactivating their specific targets. Protease nexin-I, first discovered in human foreskin fibroblasts, binds thrombin, plasmin, and urokinase with high affinity, forming covalently linked complexes. Human fibroblasts bind complexes of protease nexin-I and its target protease via a cell-surface, high-affinity receptor. We have analyzed a number of characteristics of this receptor, and found them to be typical of class II receptors in general. At 4 degrees C binding of PN-I:protease complexes was competed by heparin. In addition, binding was independent of the particular protease bound to the PN-I; purified complexes of PN-I with thrombin or urokinase competed equipotently for [125]I-thrombin:PN-I binding. As the pH of the binding buffer was lowered, binding to cells increased. A twofold increase in binding was attained by lowering the pH from 7.5 to 4.5. This phenomenon was not due to irreversible, pH-induced changes to either the cell surface or the labeled complexes. At 37 degrees C, the removal of labeled complexes from culture medium was rapid; approximately 80% was removed by 4 hours under given conditions. The internalization of complexes was also very rapid, with an estimated ke (endocytic rate constant) of 1.0 min-1. At neutral pH, fibroblasts bind complexes in a saturable manner. Scatchard analysis yields a receptor number of 250,000 per cell and a Kd of 1 nM.

Amyloid beta-Protein Precursor↗
PubMed1999

Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization.

Egg activation in cross-fertilization between Xenopus eggs and Cynops sperm may be caused by a protease activity against Boc-Gly-Arg-Arg-MCA in the sperm acrosome. To determine the role of the sperm protease in fertilization, the protease was purified from Cynops sperm using several chromatographic techniques. We found that purified sperm protease readily hydrolyzes Boc-Gly-Arg-Arg-MCA and Z-Arg-Arg-MCA, that protease activity was inhibited by the trypsin inhibitors aprotinin and leupeptin, and that not only the purified protease, but also cathepsin B, induces activation in Xenopus eggs. We inseminated unfertilized Xenopus eggs with homologous sperm in the presence of various peptidyl MCA substrates or protease inhibitors and demonstrated that trypsin inhibitors or MCA substrates containing Arg-Arg-MCA reversibly inhibited fertilization of both fully jellied and denuded eggs. Sperm motility was not affected by the reagents. An extract obtained from Xenopus sperm showed hydrolytic activity against Boc-Gly-Arg-Arg-MCA, Z-Arg-Arg-MCA, and Arg-MCA. These results suggest that the tryptic protease in Xenopus sperm is involved in fertilization, most likely by participating in egg activation.

Animals↗
PubMed1995

Inhibition of neutrophil chemotaxis by protease inhibitors. Differential effect of inhibitors of serine and thiol proteases.

Previous studies have demonstrated that neutrophils possess an active serine protease(s) which may be involved in the process of chemotaxis but the precise identity of this enzyme(s) remains to be determined. In this study fourteen different protease inhibitors were tested over a wide concentration range for their ability to inhibit unstimulated neutrophil movement and chemotaxis to C5a, fMLP and IL-8. Pretreatment of neutrophils with aspartyl or metallo-protease inhibitors had no effect on either chemotaxis or random cell movement. The thiol protease inhibitors E-64 and cystatin, as well as the thiol/serine inhibitors antipain and leupeptin, diminished only C5a-induced chemotaxis. Pretreatment of neutrophils with the serine protease inhibitors PMSF or 3,4-DCI significantly reduced chemotaxis to C5a, fMLP and IL-8. The inhibitor of trypsin-like serine proteases, TLCK, and the neutrophil elastase inhibitor MeO-Suc-AAPV-CMK had no inhibitory effect on cell movement. However, two different inhibitors of chymotrypsin-like serine proteases, TPCK and chymostatin, significantly inhibited movement to any chemoattractant. These results suggest that an active chymotrypsin-like serine protease is essential for neutrophils to respond to chemotactic stimuli.

Chemotaxis, Leukocyte↗
PubMed1999

Stratum corneum thiol protease (SCTP): a novel cysteine protease of late epidermal differentiation.

Proteolytic enzymes play crucial roles in the formation of the stratum corneum barrier tissue and in its subsequent maturation. Despite this, the proteases involved in stratum corneum physiology are not well characterized. Hence, studies were performed to identify these proteolytic enzymes present in the peripheral layers of this tissue using a combination of tape stripping and zymography. Using this approach, a novel human cysteine protease was identified and characterized, and named stratum corneum thiol protease (SCTP). Gelatin zymography revealed that SCTP is composed of two variants with apparent molecular weights of 34 and 35 kDa which do not correspond to any previously described stratum corneum protease. Mechanistically SCTP belongs to the cysteine proteinase class as shown by: (1) acid protease activity, (2) a requirement for mild reducing conditions, and (3) the specific inhibition of activity by E64 and Z-phe-ala-diazomethylketone. Further analysis using concanavalin A affinity chromatography demonstrated that the two 34 and 35 kDa variants are both glycoproteins, which, after removal of the oligosaccharide sidechains with the specific enzyme N-glycopeptidase F, reveal a single active core protease of 32 kDa. SCTP did not crossreact with antibodies raised against the lysosomal cysteine proteases cathepsins B, H or L, thereby distinguishing it from the classical cysteine cathepsins. Localization studies revealed that SCTP is present at all depths in the stratum corneum, thereby precluding microbial contamination as the enzyme source. Moreover, it was also present at all body sites investigated, except for the hyperkeratotic palmoplantar stratum corneum. SCTP was found to be a product of late differentiation in cultured human keratinocytes; the enzyme was synthesized by differentiated calcium-switched cells and secreted into the medium, whereas nondifferentiated basal keratinocytes did not produce this protease. Moreover, human fibroblast cultures did not produce the enzyme, suggesting that SCTP is not produced by the dermis and hence is epidermal specific. The function of SCTP is unknown, but the observed gelatinolytic activity coupled with its secretion into the medium by cultured keratinocytes indicates that physiologically it is responsible for the degradation of extracellular structural proteins. Furthermore, the optimal activity at acid pH suggests that it can function in the acidic environment of the stratum corneum. It remains to be elucidated whether this enzyme has a role in desquamation.

Blotting, Western↗
PubMed1978

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.

Edetic Acid↗
PubMed1986

Endogenous inhibitor of nonlysosomal high molecular weight protease and calcium-dependent protease.

An endogenous inhibitor of high molecular weight protease was purified from human erythrocytes and partially characterized. The inhibitor was isolated by DEAE-Sephacel ion-exchange chromatography followed by separation on a Bio-Gel A-0.5m column. The inhibitor displayed a native Mr of 240,000 and contained a single subunit of Mr 40,000 after NaDodSO4/polyacrylamide gel electrophoresis. The Mr 240,000 hexamer inhibited high molecular weight protease noncompetitively (Ki = 8.3 X 10(-8) M) and showed marked susceptibility to proteolytic digestion and heat treatment. The purified factor was also a potent inhibitor of calcium-dependent protease (Ki = 2.8 X 10(-8) M), whereas it had no effect on trypsin, chymotrypsin, or papain. Heat treatment (50-70 degrees C X 10 min) caused loss of inhibition against high molecular weight protease; however, inhibition of calcium-dependent protease was stable under the same conditions. This result is consistent with different domains on the inhibitor that interact with high molecular weight protease and calcium-dependent protease. Together with earlier studies in which repression of inhibitor by an ATP-ubiquitin-dependent process was proposed, the present results suggest a general mechanism for regulation of multiple nonlysosomal proteases that are complexed with endogenous inhibitors.

Adenosine Triphosphate↗
PubMed1987

A serine protease activity in C3H/10T1/2 cells that is inhibited by anticarcinogenic protease inhibitors.

Several different protease inhibitors have the ability to suppress transformation in vitro and carcinogenesis in vivo. The mechanism(s) by which protease inhibitors suppress carcinogenesis, however, is not fully understood. Presumably, these agents inhibit one or more intracellular proteases whose functions are essential for the induction and/or expression of the transformed phenotype. We have isolated an endopeptidase activity capable of hydrolyzing the substrate Boc-Val-Pro-Arg-MCA (Boc = butoxycarbonyl; MCA = 7-amino-4-methylcoumarin) from C3H/10T1/2 mouse embryo fibroblast cells. This intracellular protease was inhibited by the soybean-derived Bowman-Birk inhibitor (BBI), chymostatin, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, all of which have anticarcinogenic activity, but was unaffected by soybean trypsin inhibitor, which lacks anticarcinogenic activity. Other protease inhibitors affected the proteolytic activity to an extent that correlates with their relative ability to suppress transformation in vitro. The enzyme has a mass of about 70 kDa, contains a single subunit, and exhibits maximal activity at pH 7.0. Diisopropyl fluorophosphate covalently binds to this enzyme and blocks its activity, indicating that the enzyme is a serine protease. We have previously demonstrated that several protease inhibitors are effective suppressors of radiation-induced transformation of C3H/10T1/2 cells. Since these agents reduce the Boc-Val-Pro-Arg-MCA-hydrolyzing activity to an extent that correlates with their ability to inhibit malignant transformation in vitro, this endopeptidase activity may be a cellular target of the anticarcinogenic protease inhibitors.

Animals↗
PubMed1995

Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.

Animals↗
PubMed1998

Protease activity in gingival crevicular fluid: presence of free protease.

Our aim was to study protease activity in GCF from inflamed sites with or without tissue destruction. 19 patients with both periodontitis and gingivitis sites and 12 patients having gingivitis alone participated in the study. GCF samples were collected by an intracrevicular washing method. The protease activity was measured as degradation of FITC-conjugated casein. To obtain a semiquantitative estimate of the harvested GCF volume, we measured the transferrin concentration in the wash-fluid. The protease activity was significantly higher in the deep pockets in periodontitis patients than in shallow pockets in the same patients. This difference was still higher when the ratio of protease activity to the amount of transferrin in the sample was plotted. Although protease activity was lower in samples from gingivitis patients than in the deep pockets in periodontitis patients, the difference was not significant. About 90% of the activity could be inhibited by the addition of an excess amount of alpha-1-antitrypsin (A1AT). This study shows that protease activity is higher in inflamed sites with tissue destruction than in inflamed sites without. Most of this activity could be inhibited by A1AT, which suggests that the activity is due to an imbalance between protease and antiprotease rather than to proteases insensitive to A1AT.

Adult↗
PubMed1987

Characterization of Erwinia chrysanthemi extracellular proteases: cloning and expression of the protease genes in Escherichia coli.

Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three antigenically and structurally distinct proteases, A, B, and C and produces a protease inhibitor, a low-molecular-weight, heat-stable protein which remains mostly intracellular and which binds specifically to the A, B, and C proteases. The structural genes for proteases A, B, and C and for the inhibitor are clustered on a ca. 40-kilobase DNA fragment present in cosmid pEW4. Escherichia coli strains harboring pEW4 secrete the three proteases into the medium during the exponential phase of growth, without intracellular accumulation and in the absence of detectable cell lysis. An 8.5-kilobase EcoRI fragment derived from the cosmid encodes proteases B and C and the inhibitor as well as functions involved in the synthesis or secretion (or both) of the proteases. The inhibitor is not required for protease synthesis or secretion.

Cloning, Molecular↗
PubMed1996

Conditional human immunodeficiency virus type 1 protease mutants show no role for the viral protease early in virus replication.

The human immunodeficiency virus type 1 protease plays a critical role in the proteolytic processing of precursor polyproteins during virion maturation. Contradictory evidence has been obtained for a possible role for the protease early after infection, i.e., during DNA synthesis and/or integration. We have reexamined this question by using conditional mutants of the protease. In one set of experiments, protease mutants that confer a temperature-sensitive phenotype for processing were used to assess the need for protease activity early after infection. No significant difference from results with wild-type virus was seen when infections were carried out at either 35 or 40 degrees C. In a separate set of experiments, infections were carried out in the presence of a protease inhibitor. In this case, both wild-type virus and a drug-resistant variant were used, the latter as a control to ensure a specific effect of the inhibitor. Infection with either virus was not inhibited at drug concentrations that were up to 10-fold higher than those needed to inhibit intracellular processing by the viral protease. The results obtained by both of these experimental protocols provide evidence that the human immunodeficiency virus type 1 protease does not play a role early after infection.

Cell Line↗
PubMed1997

Upregulation of signalase processing and induction of prM-E secretion by the flavivirus NS2B-NS3 protease: roles of protease components.

Recently, we have shown that the ability of the flavivirus NS2B-NS3 protease complex to promote efficient signalase processing of the C-prM precursor, as well as secretion of prM and E, does not appear to depend strictly on cleavage of the precursor at its Lys-Arg-Gly dibasic site by the protease. We suggested that the association of the protease with the precursor via NS2B may be sufficient by itself for the above effects. To study the proposed association in more detail, we have developed an assay in which processing at the C-prM dibasic cleavage site is abolished by Lys-->Gly conversion. We constructed deletion mutants and chimeras of the West Nile (WN) flavivirus NS2B protein and expressed them in the context of [5'-C-->NS3(243)] containing either wild-type C-prM or its cleavage site mutant. All NS2B variants were able to form active protease complexes. Deletion of the carboxy-terminal cluster of hydrophobic amino acids in NS2B had no apparent effect on the formation of prM and prM-E secretion for the cassettes containing either wild-type or mutated C-prM precursor. Deletion of the amino-terminal hydrophobic cluster in NS2B did not affect prM-E secretion for the cassettes with wild-type C-prM but abrogated prM-E secretion for the cassettes with the mutated dibasic cleavage site in C-prM. Similarly, the NS2B-NS3(178) protease of Japanese encephalitis (JE) virus, when substituted for the WN virus NS2B-NS3(243) protease, was able to promote prM-E secretion for the cassette with the wild-type C-prM precursor but not with the mutated one. Replacement of the deleted amino-terminal hydrophobic cluster in the WN virus NS2B protein with an analogous JE virus sequence restored the ability of the protease to promote prM-E secretion. On the basis of these observations, roles of individual protease components in upregulation of C-prM signalase processing are discussed.

Cloning, Molecular↗
PubMed1999

Proteases and protease inhibitors in taurocholate-induced acute pancreatitis in rats.

CONCLUSION: Taurocholate-induced acute pancreatitis (AP) in the rat mimics early necrotizing human pancreatitis. Protease activation and protease inhibitor consumption occur consistent with a two-stage development, and contact-phase activation is a possible primary event in this model. BACKGROUND: Proteases and protease inhibitors have been indicated to play an important role in both human and experimental acute pancreatitis, although little is known about them in rats. METHODS: Three percent sodium taurocholate was infused into the bilio-pancreatic duct to induce AP, and over 0-72 h we measured lipase, amylase, albumin, prekallikrein, factor X, alpha-1-macroglobulin, alpha-2-antiplasmin, antithrombin III, alpha-1-protease inhibitor, and C1-esterase inhibitor (all in plasma) and histologic and macroscopic findings. RESULTS: A severe necrotizing, nonlethal, AP was induced with an early increase in plasma lipase and alpha-amylase activity levels and peritoneal exudate followed by a return to near control levels after 72 h. Histologic score and pancreatic wet weight ratio increased initially and remained high during the observation period. The protease inhibitors C1-esterase inhibitor, alpha-2-antiplasmin, and antithrombin III decreased early, within 0-6 h, whereafter levels normalized. The protease inhibitors alpha-1-macroglobulin and alpha-1-protease inhibitor later gradually decreased over the 72 h.

Animals↗
PubMed1999

Defining protease specificity with proteomics: a protease with a dibasic amino acid recognition motif is regulated by a two-component signal transduction system in Salmonella.

Microbial proteases play diverse and important roles in bacterial virulence but their detection and characterisation is often hampered by their limited abundance or lack of expression in the absence of suitable environmental signals. We describe here a sensitive proteomic approach to detect proteases that are under the control of a virulence regulator and to characterise their recognition motifs. Using MG++-depleted growth media or a mutant strain of Salmonella in which the PhoP-PhoQ virulence regulatory system is constitutively active, truncated forms of DnaK, elongation factor G, elongation factor Tu and ribosomal protein S1 proteins were detected. Two other global regulatory mutants and cells exposed to acid or to oxidative stress failed to produce the truncated proteins, indicating specific control of the protease activity by the PhoP-PhoQ system. Our results suggest that at least two proteases are induced. To define the proteolytic cleavage sites of one of the proteases, peptides from each of the truncated proteins were identified by tryptic mass fingerprinting/nanoelectrospray mass spectrometry and mapped onto the sequence of the intact protein. Alignment of the regions around the cut site indicates that the protease recognises a dibasic amino acid motif characteristic of the omptin protease family. The induction of such proteases in bacteria depleted of Mg++ ions may contribute to the PhoPQ-mediated resistance of Salmonella to cationic antimicrobial peptides. Additionally, our results suggest it would be prudent to keep the concentration of this ion above micromolar levels during bacterial sample preparation for proteomic analyses.

Acids↗
PubMed2001

Cysteine protease activity is up-regulated in inflamed ankle joints of rats with adjuvant-induced arthritis and decreases with in vivo administration of a vinyl sulfone cysteine protease inhibitor.

OBJECTIVE: Cysteine proteases are postulated to play a role in tissue destruction in the joints of animals with arthritis. The purpose of the present study was to confirm the concept that cysteine proteases are enzymes involved in the pathology of rheumatoid arthritis (RA). METHODS: Arthritis was induced in Lewis rats by adjuvant injection (adjuvant-induced arthritis [AIA] model) and scored for inflammation. At necropsy, the rear paws were either fixed in formalin and assigned a histologic score (based on synovial cell proliferation, cartilage erosion, bone erosion, and fibroproliferative pannus) or frozen, cryosectioned, and assayed for enzyme activity either by in situ cytochemical staining with a post-azo-coupling method using a chromogenic substrate (Z-arg-arg-MNA) or by a novel assay placing the tissue section directly in a cuvette using the fluorogenic substrate Z-arg-arg-AMC. RESULTS: Enzymatic activity, measured either in frozen sections in situ or in the cuvette assay, was positively correlated with joint destruction (r = 0.7) and inflammation (r = 0.8). Activity was not inhibited significantly by Pefabloc (a serine protease inhibitor), EDTA (a metalloprotease inhibitor), or pepstatin A (an aspartyl protease inhibitor) but was inhibited by E-64 and vinyl sulfone irreversible inhibitors of cysteine proteases. The effect of one of the vinyl sulfone cysteine protease inhibitors, Mu-Leu-HomoPhe-vinylsulfone, was tested in vivo by dietary administration at 2.2 mg/kg/day in the AIA model; this resulted in a significant decrease in inflammation and in the amount of cysteine protease activity measured in the joint tissue. CONCLUSION: Cysteine protease activity levels increase in the diseased state and may be an important target for designing small molecule inhibitors to reduce the inflammation and tissue destruction associated with RA.

Animals↗
PubMed1996

The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70-kDa aspartic proteinase show preferences among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates.

Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate prohormone processing enzymes. The chromaffin granule proenkephalin processing proteases have been characterized as the novel cysteine protease prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for prohormone substrates. The recombinant prohormones proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular prohormones. PTP preferred proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the prohormones tested. Importantly, these results indicate that prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular prohormone.

Animals↗
PubMed2006

Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe.

The creation of protease-deficient mutants to avoid product degradation is one of the current strategies employed to improve productivity and secretion efficiency of heterologous protein expression. We previously constructed a set of single protease-deficient mutants of the fission yeast Schizosaccharomyces pombe by respective disruption of 52 protease genes, and we succeeded in confirming useful disruptants (Idiris et al., Yeast 23:83-99, 2006). In the present study, we attempted multiple deletions of 13 protease genes, single deletions of which were previously confirmed as being beneficial for reducing extracellular product degradation. Using PCR-based gene replacement, a series of multiple deletion strains was constructed by multiple disruption of a maximum of seven protease genes. Effects of the resultant multiple deletion strains on heterologous expression were then measured by practical expression of a proteolytically sensitive model protein, the human growth hormone (hGH). Time profiles of hGH secretion from each resultant mutant demonstrated significantly enhanced hGH productivity with processing of the multiple protease deletions. The data clearly indicated that disruption of multiple protease genes in the fission yeast is an effective method for controlling proteolytic degradation of heterologous proteins particularly susceptible to proteases.

Biotechnology↗
PubMed1981

Purification and characterization of alkaline protease and neutral protease from chromatin of rats.

It was previously reported that, in addition to a known chymotrypsin-like protease capable of hydrolyzing histones with an optimum pH of 8 (neutral protease), another protease is bound to the chromatin of various rat tissues and in situ hydrolyzes casein more quickly than histones with an optimum pH of 10 (alkaline protease). In the present study, the alkaline protease was purified 14 000-fold to approx. 75% purity from the chromatin of Rhodamine sarcoma. This tumor contains both proteases at higher levels than normal tissues. For purification, affinity columns of Sepharose with bound soybean trypsin inhibitor, casein and histones were successively used. Also, the neutral protease was purified 920-fold to an apparently homogeneous state by affinity chromatography on a Sepharose column with bound soybean trypsin inhibitor under conditions, in which an excess amount of the enzyme was applied on the column so that part of the enzyme would pass through the column without adsorption and the enzyme thus adsorbed was then eluted. The purified alkaline and neutral proteases had molecular weights of approx. 18 000 and 27 000, respectively, and isoelectric points of approx. 11. The former enzyme hydrolyzed casein (100) in preference to histones (18) with an optimum pH of 9.5, whereas the latter enzyme preferred histones (100) to casein (32) with an optimum pH of 8. Their actions against other proteins and synthetic substrates were also studied.

Animals↗