Extracellular Protease Inhibitors Research Articles

The cerebral hemorrhage-producing cystatin C variant (L68Q) in extracellular fluids

A variant of the normal extracellular cysteine protease inhibitor cystatin C (L68Q-cystatin C), is the amyloid precursor in hereditary cystatin C amyloid angiopathy (HCCAA). It has been suggested that the mutation causes cellular entrapment ofL68Q-cystatin C in vivo and that the variant protein is not secreted to extracellular fluids. In order to test this hypothesis, we used matrix-assisted laser desorption ionization time-of-flight mass spectrometry in an effort to demonstrate the presence ofL68Q- along with wildtype cystatin C in plasma and cerebrospinal fluid (CSF) of HCCAA-patients. Plasma from all five investigated HCCAA-patients contained both L68Q- and wildtype cystatin C. The presence of approximately equal amounts of cystatin C dimers and monomers was demonstrated in plasma from HCCAA-patients, whereas only monomers could be found in normal plasma. L68Q-wildtype-cystatin C heterodimers seem to be present in the dimeric cystatin C population. CSF from six HCCAA-patients also contained cystatin C-dimers and monomers, but the dimeric fraction was minute. CSF from control patients did not contain dimeric cystatin C These results suggest that the milieu of L68Q-cystatin C is important for its stability and dimerization status and that certain milieus might hinder its further development into oligomers, amyloid fibrils and other precipiting aggregates.

Purification and Characterization of Extracellular Cysteine Protease Inhibitor from Chlorella sp.

An extracellular cysteine protease inhibitor (ECPI) from Chlorella sp. was purified to homogeneity by DEAE-Cellulose column chromatography and Sephacryl S-300 column chromatography. The molecular mass of the inhibitor was estimated to be 284 kDa by SDS-PAGE and 285 kDa by gel filtration on Sephacryl S-300 column chromatography. ECPI retained 100 % of its original activity even after heating at 100°C for 20 min. It showed an inhibitory activity against the proteolytic activity of papain, ficin, chymopapain, calcium activated protease (calpain) and bromelain, but not against trypsin or α-chymotrypsin. ECPI contained 83.4% carbohydrate residues by weight and inhibited papain at a ratio of 1 : 1. The inactivated inhibitor, treated by sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME), was reactivated by keeping it at 10°C and pH 8.5 after elimination of SDS and 2-ME.

Kinetics of equine neutrophil elastase release and superoxide anion generation following secretagogue activation: a potential mechanism for antiproteinase inactivation

Man and horses both suffer from neutrophil mediated pulmonary diseases however there are striking species differences in the underlying pathology. In particular while pulmonary emphysema is a common pathological sequel to human respiratory disease it is not a major feature of the common equine neutrophil mediated condition, chronic obstructive pulmonary disease (COPD). The proposed reason for this difference is that equine neutrophils contain less elastase than equivalent human cells and therefore there is a reduced risk of excess and/or uninhibited elastase activity, which is considered the major cause of pulmonary emphysema in man, in the horse lung. In previous studies equine neutrophil elastase (ENE) has been assayed by measuring elastinolytic activity whereas human neutrophil elastase content has been determined using immunological techniques. Neutrophils contain several intracellular protease inhibitors therefore measurement of elastase activity may underestimate the total NE content. The aim of the current study was to develop immunological techniques to allow investigation of the cellular content, distribution and release of ENE from purified equine neutrophils. Equine neutrophil elastase 2A (ENE 2A), the most abundant elastase in equine neutrophils, and equine alpha-1-proteinase inhibitor (API), the main inhibitor of elastase were found to be present at 0.813 pg ± 0.179 and 0.021 pg ± 0.003 (mean ± SEM, n = 11 individual horses) per neutrophil, respectively. This represents twice as much elastase as previously found in the equine neutrophil and a comparable amount to that reported in human neutrophils. Immunolocalisation demonstrated that ENE 2A has a granular distribution within the cytosol of neutrophils, whereas API exhibits a uniform non-granular cytoplasmic appearance. In addition the kinetics of simultaneous generation and release of superoxide anions (SOA) and release of ENE 2A from equine neutrophils, stimulated in vitro by zymosan-activated serum (ZAS) in the presence and absence of the cation chelator ethylene glycol- N, N, N′, N′-tetraacetic acid (EGTA), showed a close relationship between total SOA generation and total ENE 2A release during the initial 90 min post-ZAS stimulation and the dependence of both events on extracellular cations. In conclusion these studies have shown that horse and human neutrophil elastase content and mediator release functions are more closely matched than was previously thought. This suggests that the species differences in pathology resulting from neutrophil-mediated respiratory disease are determined by other factors such as differences in the abundance and function of intra- and extra-cellular protease inhibitors.

Enzymatic action of human glandular kallikrein 2 (hK2). Substrate specificity and regulation by Zn2+ and extracellular protease inhibitors.

Human glandular kallikrein 2 (hK2) is a serine protease expressed by the prostate gland with 80% identity in primary structure to prostate-specific antigen (PSA). Recently, hK2 was shown to activate the zymogen form of PSA (proPSA) in vitro and is likely to be the physiological activator of PSA in the prostate. hK2 is also able to activate urokinase and effectively cleave fibronectin. We studied the substrate specificity of hK2 and regulation of its activity by zinc and extracellular protease inhibitors present in the prostate and seminal plasma. The enzymatic activity and substrate specificity was studied by determining hK2 cleavage sites in the major gel proteins in semen, semenogelin I and II, and by measuring hydrolysis of various tripeptide aminomethylcoumarin substrates. HK2 cleaves substrates C-terminal of single or double arginines. Basic amino acids were also occasionally found at several other positions N-terminal of the cleavage site. Therefore, the substrate specificity of hK2 fits in well with that of a processor of protein precursors. Possible regulation mechanisms were studied by testing the ability of Zn2+ and different protease inhibitors to inhibit hK2 by kinetic measurements. Inhibitory constants were determined for the most effective inhibitors PCI and Zn2+. The high affinity of PCI for hK2 (kass = 2.0 x 10(5) M-1 x s-1) and the high concentrations of PCI (4 microM) and hK2 (0.2 microM) in seminal plasma make hK2 a very likely physiological target protease for PCI. hK2 is inhibited by Zn2+ at micromolar concentrations well below the 9 mM zinc concentration found in the prostate. The enzymatic activity of hK2 is likely to be reversibly regulated by Zn2+ in prostatic fluid. This regulation may be impaired in CAP and advanced metastatic cancer resulting in lack of control of the hK2 activity and a need for other means of control.

Expression and characterization of an epididymis-specific gene.

A truncated cDNA coding a rabbit epididymal protein (BE-20) was identified in a previous study. In the present study the full-length cDNA was isolated by the method of rapid amplification of cDNA ends. The BE-20 cDNA consisted of 585 bp with a poly(A) tail of 26 residues and an open reading frame composed of 369 bp encoding a deduced polypeptide containing 123 amino acid residues with a calculated molecular mass of 13 kDa. The N-terminus-contained a leucine-rich segment. BE-20 cDNA has about 76.8% homology with the HE4 gene of human epididymis. Northern blot analysis of mRNAs prepared from 17 different human tissues was performed using as probe a 0.5-kb DNA fragment corresponding to a segment of BE-20 cDNA. Positive reaction was elicited only with epididymal mRNA. A DNA fragment corresponding to a section of the open reading frame of BE-20 cDNA was cloned in Escherichia coli under the control of the T7 promoter. The cellular content of the expressed recombinant protein comprised about 55% of the total protein. The chromatographically purified bacterial product migrated as a single band with an estimated M(r) of 15 kDa on analysis by SDS-PAGE. In conclusion, BE-20 cDNA is expressed only in the epididymis. It is structurally related to the four-disulfide core family of extracellular proteinase inhibitors and may be involved in sperm maturation.

Characterization of a 14 kDa plant-related serine protease inhibitor and regulation of cytotoxic activity in earthworm coelomic fluid

We have purified and characterized the serine protease inhibitor activity contained in the coelomic fluid of the earthworms, Eisenia. Serine protease inhibitor activity was stable between pH 3 and 9.5, not flocculable by pH 3.0 and resistant to 100°C for 15 min. or to 4°C for 24h. Ten μL of coelomic fluid was sufficient to inhibit in vitro the protease activity of 0.12 μg of trypsin. Injection of living bacteria into earthworms resulted in increased serine protease activity 1–2 days post-injection, and increased serine protease inhibitor activity on day 4, suggesting that serine protease inhibitor is responsible for serine protease neutralization. Purified to homogeneity by affinity chromatography on trypsin, the serine protease inhibitor of Eisenia is a monomer of 14 kDa. Its partial NH 2 amino acid sequence revealed a basic hydrophobic fragment which shared 68–75% homologies and 47–60% identities with several plant serine protease inhibitors. Eisenia cytotoxic activity due to the two fetidins of 40 and 45 kDa was stimulable in vitro by several serine proteases. Incubation with soybean trypsin inhibitor variant a (STIa) resulted in less cytotoxicity. The inhibitory effect occurred only when STIa was added before cell disruption. Interpretative cytotoxic scheme involving the release of intracellular cytotoxic proteins, intracellular trypsin-like activator and extracellular serine protease inhibitor suggests regulatory mechanisms for cellular/humoral immune system of earthworms.

Influence of extracellular proteins, proteases and protease inhibitors on grapevine somatic embryogenesis

An embryogenic grapevine rootstock cell suspension, continuously grown in the presence of auxin, was predominantly composed of proembryogenic masses. When transferred to an auxin-free medium, grapevine somatic embryos developed but were rapidly blocked at the heart stage. This inhibition has been related to the presence of extracellular macromolecules (Coutos-Thevenot et al., 1992a). In this study, the initial cell population density has been found to influence markedly embryo development. Inoculations below 5·103 cells per ml were required to obtain fully grown cotyledonary embryos. Interestingly, extracellular proteins of molecular weights of 32, 34, 48 and 52 kDa accumulated in cultures grown at high population cell densities and disappeared in cultures inoculated at densities below 5·103 cells per ml. Protein fractions partially purified by ion exchange chromatography caused both an early inhibition of embryogenesis and a stimulation of secondary embryogenesis. Moreover, to test for the possibility of modulating embryo development through alterations of extracellular proteins, cultures were supplemented with proteases and protease inhibitors. The addition of trypsin increased the rate of embryo development only in cultures inoculated at a low cell population density. Conversely, the protease inhibitor aprotinin inhibited development, arresting embryos at globular and heart stages. Together, these results provide evidence that extracellular proteins modulate somatic embryogenesis and suggest that an extracellular proteolitic mechanism could be implicated in development.

Effects of Transforming Growth Factor-βl and Phorbol Ester on PALI-1 and PA Genes in Human Lung Cells

Transforming growth factor-β (TGF-β) mediates the production of extracellular matrix proteins, proteases and protease inhibitors in epithelial cells. Both TGF-β and phorbol-12-myristate-13-acetate (PMA) exert both positive and negative effects on mitogenesis in these as well as other cell types. Phorbol esters act through stimulation of protein kinase C (PKC) and are among the most potent tumor promoters known. The present study was conducted to determine whether the effect of TGF-β in human non-small cell lung cancer (NSCLC) and normal human bronchial epithelial (NHBE) cells parallels that of the phorbol esters and whether this effect of TGF-β involves PKC. TGF-β1 and PMA increased expression of TGF-βl mRNA 24 hr after their addition to both NSCLC and NHBE cells. The effects of these agents on expression of the mRNAs for TGF-β2 and TGF-β3 were more complex; while TGF-β2 and TGF-p3β mRNAs increased transiently in response to TGF-p1 in NHBE cells and TGF-β3 mRNA increased transiently in some NSCLC cells, expression of these mRNAs decreased in most of these cells in response to PMA with the exception of the carcinoid NCLH727 where TGF-pZ mRNA increased dramatically. TGF-β1 and PMA both caused a persistent increase in expression of the mRNAs for both plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator (PA) up to 24 hr in most NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. In contmst, while TGF-βl also increased expression of PAI-1 mRNA in NHBE cells, the expression of PA mRNA decreased simultaneously. The effect of PMA on PAI-1 and PA mRNAs was opposite of TGF-β1 in these cells, with expression of PAI-1 mRNA decreasing and PA mRNA increasing after addition of PMA. These data show that there is parallel regulation of the genes for TGF-βl, PAI-1 and PA by TGF-β1 and PMA in NSCLC, but differential regulation of the genes for PAL1 and PA by these agents in NHBE cells. The responses of the mRNAs and proteins of TGF-β1, PAI-1 and PA to TGF-βl and PMA were inhibited by the serind threonine kinase inhibitor H7 in NSCLC cells. Treatment of NSCLC cells with TGF-β1 and PMA resulted in a persistent increase in the expression of fibronectin mRNA and protein. This response was blocked by the addition of H7. Inhibition of these effects by H7 in NSCLC cells suggests that H7 blocks TGF-p responses by inhibiting a protein serindthmnine kinase(s). Because the effects of TGF-p and PMA on the different TGF-p isoforms, PA, PA1 and fibronectin in NHBE and NSCLC cells are complex, our data suggest that there are distinct mechanisms for controlling the different TGF-p isoforms, PA, PA1 and extracellular matrix proteins in normal lung and lung cancer cells.

Individual prostate-specific antigen (PSA) forms as prostate tumor markers

Prostate-specific antigen (PSA) is a kallikrein-like serine protease mainly expressed in the human prostate. It is responsible for the proteolysis of the gel-forming proteins in human semen. Two major extracellular protease inhibitors, alpha-1-antichymotrypsin (ACT) and alpha-2-macroglobulin (AMG) may inactivate PSA escaping from the prostate. The predominant immunodetected form of PSA in serum is complexed to ACT but PSA exists also in a free non-complexed form despite the large excess of inhibitors. The concentrations of PSA in serum are normally less than 4 μg/l, but elevated concentrations are found in a majority of patients with prostate cancer (CAP) and the analysis of PSA in serum has become invaluable in the detection and monitoring of patients with CAP. However, it is not an ideal tumor marker in the sense that there are CAP patients with normal PSA concentrations in serum and patients with benign hyperplasia of the prostate (BPH) with elevated PSA concentrations. Analysis of the various PSA forms in serum attracts much interest as there is a higher proportion of PSA in complex with ACT in patients with CAP than in those with BPH. Optimal combinations of monoclonal antibodies have been used to design sensitive noncross-reacting immunoassays for the detection of free PSA, PSA-ACT complexes and the detection of both free PSA and PSA complexes in an equimolar fashion (i.e. total PSA). Several studies have demonstrated that the analysis of the proportions of the free-to-total PSA in serum may increase the diagnostic specificity by 15–20% without significant loss in the sensitivity for detection of CAP.

Cloning, sequencing, and expression of a Thermomonospora fusca protease gene in Streptomyces lividans.

The major Thermomonospora fusca YX extracellular protease gene (tfpA) was cloned into Escherichia coli and Streptomyces lividans and was sequenced. The open reading frame encoded 375 residues, including a 31-residue potential signal sequence, an N-terminal prosequence containing 150 residues, and the 194-residue mature protease that belongs to the chymotrypsin family. The protease was secreted by S. lividans, but evidence suggested that it was bound to an extracellular protease inhibitor. An inhibitor-deficient mutant was selected to produce protease for purification.

Identification of a rabbit epididymal protein gene.

A protein designated as BE-20 was purified from cauda epididymal fluid of the rabbit by preparative polyacrylamide gel electrophoresis and HPLC on a mono Q HR5/5 anion exchange column. The purified protein migrated with an estimated Mt of 20,000 when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequence of the N-terminus of the BE-20 protein was determined. The initial eight amino acid residues were His-Gly-Ala-Asp-Lys-Pro-Gly-Val. The corresponding 23 mer oligonucleotide (5'-CATGGCGCTGACAAGCCTGGGGT-3') was synthesized and used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The purified BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. The BE-20 cDNA had 78.5% identity in 479 bp overlap with human epididymis-specific HE4 cDNA. The amino acid sequences of the initial 30 amino acid residues of the N-terminus of the purified protein and the deduced polypeptides were as follows: N-His-Gly-Ala-Asp-Lys-Pro-Gly-Val-Cys-Pro-Gln-Leu-Ser-Ala-Asp-Leu-Asn-Cy s- Thr-Gln-Asp-Cys-Arg-Ala-Asp-Gln-Asp-Cys-Ala-Glu. The deduced polypeptide contained 16 cysteine residues and had partial sequence homology with proteins belonging to the four-disulfide core family of extracellular proteinase inhibitors. The BE-20 protein may play a role in sperm maturation and/or capacitation.

Biologic Function of Prostate-Specific Antigen: Enzyme Action and Reactions with Extracellular Protease Inhibitors

Biologic Function of Prostate-Specific Antigen: Enzyme Action and Reactions with Extracellular Protease Inhibitors

Biological activity of prostate-specific antigen isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroelution.

Human prostate-specific antigen (PSA), a 33 kDa kallikrein-like serine protease, occurring in the prostate, in seminal plasma and in blood, was prepared under nonreducing conditions in an enzymatically active form from seminal plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by fast copper staining, electroelution from gel slices and dialysis against isotonic phosphate-buffered saline (PBS). Enzymatic activity was demonstrated for the first time directly by cleavage of semenogelin, one of the biological substrates of PSA, isolated by the same procedure, i.e. SDS-PAGE and electroelution, but from seminal vesicle fluid. The purified PSA formed SDS-stable complexes with the two major extracellular protease inhibitors in blood, alpha 1-antichymotrypsin (alpha 1-ACH) and alpha 2-macroglobulin (alpha 2-M). PSA isolated under reducing conditions was enzymatically inactive and could not bind to the protease inhibitors alpha 1-ACH and alpha 2-M.

Cell surface syndecan-1 on distinct cell types differs in fine structure and ligand binding of its heparan sulfate chains.

Heparan sulfate (HS) can bind a large variety of biological effectors, including extracellular matrix components, growth factors, chemokines, degradative enzymes, and protease inhibitors. Where studied, HS is known to be structurally heterogeneous and to vary in sulfation pattern between cells and tissues. Because heparan sulfate can represent several distinct proteoglycans, we asked whether the structural variation in the heparan sulfate chains of a single species of cell surface proteoglycan is a reproducible, differentiated characteristic and whether the variation can result in distinct biological functions. We studied the molecular structure and binding affinity for type I collagen and fibroblast growth factor-2 of syndecan-1 purified from the surfaces of NMuMG normal murine mammary gland epithelia, NIH/3T3 fibroblasts, and BALB/3T3 endothelioid cells. Syndecan-1 from these cell types varied in molecular mass largely due to variation in the length of the HS chains. Although the highly sulfated and N-acetylated domains in these HS chains were organized similarly, the number of highly sulfated domains differed. The disaccharide compositions were also similar except for reproducible and consistent differences in the amount of hexuronic acid-N-sulfated-6-O-sulfated glucosamine and 2-O-sulfated hexuronic acid-N-sulfated glucosamine. These differences were confirmed by oligosaccharide mapping, which showed cell type-specific variations in the composition of the highly sulfated domains. These structural variations correlated with cell type-specific differences in the affinity of syndecan-1 and its isolated HS chains for type I collagen. However, no differences in affinity for fibroblast growth factor-2 were detected. The results indicate that the size, fine structure, and ligand affinity of the HS chains on a single proteoglycan species differ in a consistent and reproducible manner between cell types. Thus, the variation in structure and binding ability of HS on syndecan-1 is a differentiated characteristic of the cell type that can enable cells to respond distinctly to the HS-binding effectors in the cellular microenvironment.

Growth factors in retinal diseases: Proliferative vitreoretinopathy, proliferative diabetic retinopathy, and retinal degeneration

The goal of this review is to present the current knowledge on specific growth factor involvement in posterior segment eye disease. Growth factors can be defined as multifunctional signals which modify cell growth or proliferation, alone or in concert, by binding to specific cell surface receptors. Their biological effects on cells include cell adhesion, migration, survival, differentiation, extracellular matrix secretion, protease and protease inhibitor release, production of other growth activities, and angiogenesis. Growth factors couple the cell to the microenvironment. As some growth factors are soluble mediators of wound repair and angiogenesis, it seems possible that proliferative vitreoretinopathy and proliferative diabetic retinopathy are caused or aggravated by these factors. Other factors act as survival factors and can possibly prevent retinal degeneration. The multifunctional nature of growth factors makes it probable that practical uses will be found for these agents in the future.

Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene.

A novel proteinaceous protease inhibitor was isolated from the culture supernatant of Bacillus brevis HPD31. The protease inhibitor of B. brevis (designated BbrPI) was produced extracellularly in multiple forms having at least three different molecular weights. One of them, BbrPI-a, was purified to near homogeneity and only showed inhibitory activity toward serine proteases, such as trypsin, chymotrypsin, and subtilisin. BbrPI was presumed to form a trypsin-inhibitor complex in a molar ratio of 1:1. The inhibitor was found to be heat resistant at neutral and acidic pHs. The gene coding for BbrPI was cloned into Escherichia coli, and its nucleotide sequence was determined. The sequence suggested that BbrPI is produced with a signal peptide of 24 amino acid residues. The amino acid sequence of the protein deduced from the DNA sequence contained the amino acid sequences of amino termini of the inhibitors, a, b, and c, and their putative precursor determined chemically. The molecular weight of the precursor was about 33,000, and the molecular weights of inhibitors a, b, and c were about 22,000, 23,500, and 24,000, respectively. It is presumed that the secreted precursor protein, which is probably inactive, is cleaved by protease into several active protease inhibitor molecules. BbrPI shows no significant homology to the protease inhibitors described previously and is unique in not having any cysteine residues in its molecule.

Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin

Immunologic measurements of the serum concentration of prostate-specific antigen (PSA), an abundant prostatic-secreted serine proteinase, are frequently used to monitor patients with prostate cancer, though it has not been ascertained whether this immunoreactivity represents a PSA zymogen, the active proteinase, or PSA complexed to extracellular proteinase inhibitors. To characterize the PSA immunoreactivity in serum, we used monoclonal antibodies produced against PSA and a polyclonal rabbit IgG against alpha 1-antichymotrypsin in the design of three noncompetitive PSA assays: assay T, which detected PSA both when present as the active proteinase and when complexed to alpha 1-antichymotrypsin; assay F, which recognized the active proteinase but most poorly detected PSA complexed to alpha 1-antichymotrypsin; and assay C, which was specific for PSA complexed to alpha 1-antichymotrypsin. We used the three assays to measure PSA immunoreactivity in 64 patients' sera and in the effluent after gel chromatography of sera from four patients. This identified an 80- to 90-kDa complex between PSA and alpha 1-antichymotrypsin as the predominant fraction of the PSA immunoreactivity in blood plasma; an immunoreactive 25- to 40-kDa compound was the minor fraction.

A Major Human Epididymis-Specific cDNA Encodes a Protein with Sequence Homology to Extracellular Proteinase Inhibitors1

The amino acid sequence of a major human epididymis-specific protein was deduced from the nucleotide sequence of its cloned cDNA. The encoded product showed characteristics of a secretory protein, with a signal peptide followed by a small (approximately 10-kDa), acidic (pI 4.3), and cysteine-rich polypeptide. The positions of half-cysteines suggested that it was a two-domain member of the family of 'four-disulfide core' proteins to which a number of proteinase inhibitors belong. Southern blot analyses of human genomic DNA showed that the transcripts originated from a single copy gene. Northern blot and in situ transcript hybridization specifically localized the HE4 (human epididymis gene product) mRNA to the epithelial cells of the epididymal duct, predominantly within the distal sections. A possible function in sperm maturation as indicated by amino acid similarities to extracellular proteinase inhibitors of genital tract mucous secretions is discussed in the context of its tissue-specific transcription.

Characterization of an extracellular glycoprotein inhibitor of trypsin (TI-23) produced by Streptomyces sp. 23

The production of an extracellular trypsin inhibitor, TI-23, was found to parallel the growth of Streptomyces sp. 23 at different cultivation temperatures, reaching a maximum level at late exponential phase. Although the different temperatures (18°, 28° and 37°C) did not greatly affect the growth of the microorganism, they proved to be an important factor for extracellular inhibitory activity. Maximum specific rates of both cell growth and production of the inhibitor were recorded during the cultivation of Streptomyces sp. 23 at 37°C. TI-23 proved to be a monomeric glycoprotein containing 17% carbohydrate and differing in amino acid composition from the known extracellular proteinase inhibitors of streptomycetes. The molecular mass of the inhibitor was estimated to be about 13 kDa and the isoelectric point 4.3. The inhibition spectrum of TI-23 included trypsin as well as some microbial alkaline proteinases.

Isolation and characterization of marine bacteria producing alkaline protease inhibitor.

Marine bacteria which produced extracellular alkaline protease inhibitor were isolated and identified. A new and simple casein double layer agar method was employed for a quick screening of the inhibitor producers. Out of the 878 isolates from Aburatsubo Inlet, Kanagawa, Japan, only three strains produced the inhibitor whereas no inhibitor producer was found out of the 1, 961isolates from other sea areas. Among the three inhibitor producers, strain B-10-31 showed the largest inhibitory zone, suggesting that this strain had the strongest inhibitory activity.All the inhibitor producing strains were aerobic, polarly flagellated, Gram-negative, rodshaped organisms with low G+C contents in their DNAs (39.9 to 42.0mol%). They required NaCI for growth, which indicates that they are of marine origin. All the strains hydrolyzed complex polymers like casein, DNA, gelatin and starch. None of them utilized organic acids. Based on these taxonomical characteristics, they were identified as Alteromonas spp. Although the three strains belonged to the same genus, strain B-10-31 differed considerably in its characteristics from the other two (B-3-62 and B-3-87).This is the first report of a protease inhibitor producing bacteria isolated from seawater.