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Background Hip fractures in older people result in increased mortality. Objective We developed and validated an accurate and simple prognostic scoring system for hip fractures that can be used preoperatively. Design Retrospective study. Setting Multicenter. Participants Patients aged ≥65 years with hip fractures who underwent surgery between 2011 and 2021 were enrolled. Methods The significant factors were determined with logistic regression analysis, and a scoring system was developed. The patients were classified into three groups, and a log-rank test was performed to evaluate 1-year survival rates. The model was internally and externally validated using the 5-fold cross-validation and data from another hospital, respectively. Results We included 1026 patients. The analysis revealed eight significant prognostic factors: sex, body mass index, history of chronic heart failure and malignancy, activities of daily living (ADLs) before injury, hemoglobin and the prognostic nutritional index (PNI) at injury, and the American Society of Anesthesiologists Physical Status. The area under the receiver operating characteristic curve (AUC) after internal validation was 0.853. The external validation data consisted of 110 patients. The AUC of the model for the validation data was 0.905, showing outstanding discrimination. Sensitivity and specificity were 88.7% vs. 100% and 93.3% vs. 95.2% for the development and validation data, respectively. Conclusions We developed and validated an accurate and simple prognostic scoring system for hip fractures using only preoperative factors. Our findings highlight PNI as an important predictor of prognosis in hip fracture patients. [ABSTRACT FROM AUTHOR]

A 65-year-old woman presented with right elbow pain after a fall. Imaging showed an avulsion fracture of the olecranon. The patient subsequently underwent surgery using the suture bridge technique with anchors. However, loosening was observed intraoperatively. Therefore, a McLaughlin cerclage with an artificial ligament was added, resulting in a rigid fixation. Bone union was achieved at 4 months postoperatively. At 18 months postoperatively, no limitation was observed in the range of motion of the elbow joint; the disabilities of the arm, shoulder and hand score was 0. McLaughlin cerclage with an artificial ligament provided additional fixation, demonstrating greater strength compared with suture anchors and minimizing the risk of cut-through in the osteoporotic bone. This approach offers a promising alternative for such cases by combining firm fixation with a reduced risk of complications, particularly in older patients with osteoporosis. [ABSTRACT FROM AUTHOR]

Strobilurins A and X, isolated from Mucidula venosolamellata culture extracts, demonstrated potent inhibition of human melanoma G-361 cell proliferation. Strobilurin X exhibited milder inhibitory effects on human fibroblast cells (NB1RGB) compared to strobilurin A. Additional strobilurin-related compounds were isolated from the other mushroom species. Oudemansins A and B displayed weaker activities on G-361 cells than strobilurins A and B, respectively, emphasizing the importance of a conjugated double-bond structure. Among isolated compounds, strobilurin G showed the lowest IC50 value for G-361 cells. Additional strobilurins bearing various substituents on the benzene ring were synthesized. Synthetic intermediates lacking the methyl β-methoxyacrylate group and a strobilurin analogue bearing modified β-methoxyacrylate moiety showed almost no inhibitory activity against G-361 cells. The introduction of long or bulky substituents at the 4′ position of the benzene ring of strobilurins enhanced the activity and selectivity, suggesting differential recognition of the benzene ring by G-361 and NB1RGB cells. [ABSTRACT FROM AUTHOR]

Microbial production of cis,cis-muconate (ccMA) from phenolic compounds obtained by chemical depolymerization of lignin is a promising approach to valorize lignin. Because microbial production requires a large amount of carbon and energy source, it is desirable to establish a ccMA-producing strain that utilizes lignin-derived phenols instead of general sources like glucose. We isolated Pseudomonas sp. strain NGC7 that grows well on various phenolic compounds derived from p-hydroxyphenyl, guaiacyl, and syringyl units of lignin. An NGC7 mutant of protocatechuate (PCA) 3,4-dioxygenase and ccMA cycloisomerase genes (NGC703) lost the ability to grow on vanillate and p-hydroxybenzoate but grew normally on syringate. Introduction of a plasmid carrying genes encoding PCA decarboxylase, flavin prenyltransferase, vanillate O-demethylase, and catechol 1,2-dioxygenase into NGC703 enabled production of 3.2 g/L ccMA from vanillate with a yield of 75% while growing on syringate. This strain also produced ccMA from birch lignin-derived phenols. All these results indicate the utility of NGC7 in glucose-free ccMA production. [ABSTRACT FROM AUTHOR]

Nociceptive receptors enable animals to sense tissue-damaging stimuli, thus playing crucial roles in survival. Due to evolutionary diversification, responses of nociceptive receptors to specific stimuli can vary among species. Multispecies functional comparisons of nociceptive receptors help elucidate their evolutionary process and molecular basis for activation. The transient receptor potential ankyrin 1 (TRPA1) ion channel serves as a nociceptive receptor for chemical and thermal stimuli that is heat-activated in reptiles and frogs while potentially cold-activated in rodents. Here, we characterized channel properties of avian TRPA1 in chicken. Chicken TRPA1 was activated by noxious chemicals that also activate TRPA1 in other vertebrates. Regarding thermal sensitivity, chicken TRPA1 was activated by heat stimulation, but not cold, thus thermal sensitivity of avian TRPA1 does not coincide with rodent TRPA1, although both are homeotherms. Furthermore, in chicken sensory neurons, TRPA1 was highly coexpressed with TRPV1, another nociceptive heat and chemical receptor, similar to mammals and frogs. These results suggest that TRPA1 acted as a noxious chemical and heat receptor, and was coexpressed with TRPV1 in the ancestral terrestrial vertebrate. The acquisition of TRPV1 as a novel heat receptor in the ancestral terrestrial vertebrate is likely to have affected the functional evolution of TRPA1 regarding thermal sensitivity and led to the diversification among diverse vertebrate species. Additionally, we found for the first time that chicken TRPA1 is activated by methyl anthranilate (MA) and its structurally related chemicals used as nonlethal bird repellents. MA-induced responses were abolished by a TRPA1 antagonist in somatosensory neurons, indicating that TRPA1 acts as a MA receptor in chicken. Furthermore, TRPA1 responses to MA varied among five diverse vertebrate species. Utilizing species diversity and mutagenesis experiments, three amino acids were identified as critical residues for MA-induced activation of chicken TRPA1. [ABSTRACT FROM PUBLISHER]

Aspergilloglutamic peptidase from Aspergillus niger var. macrosporus (AGP) is one of the so-called pepstatin-insensitive acid endopeptidases, which are distinct from the well-studied aspartic peptidases. Among the known homologues of the glutamic peptidases, AGP is a unique two-chain enzyme with a light chain and a heavy chain bound non-covalently with each other, and thus is an interesting target for protein structure–function relationship studies. In this article, we report the crystal structure of a dimeric form of the enzyme at a resolution of 1.6 Å. This form has a unique structure in which the C-terminal region of the light chain of one of the molecules binds to the active site cleft of the other molecule like a part of a substrate. This form mimics the enzyme-activation product complex produced upon autoproteolysis, and provides a structural clue that could help to clarify the activation mechanism. This type of dimeric structure of a peptidase is here reported for the first time. [ABSTRACT FROM PUBLISHER]

The aim of this report was to review the use of orthopedic surgeries performed to manage rheumatoid arthritis (RA). Our review of studies published in English indicates that there has been a decrease in RA-associated surgeries in Western countries. Improvements in medical treatment may partly explain the reduction in numbers of orthopedic joint surgeries, which also suggests a worldwide trend toward improved long-term outcomes. However, the results of our multicenter study in Japan indicate that the number of RA-associated operations has not decreased, and that the numbers of operations performed annually have been relatively stable from 1998 to 2008. Although there definitely has been a decline in the numbers of synovectomy surgeries, the numbers of operations on the upper limbs and foot arthroplasties have increased. With the trend toward milder disease because of improved medical treatment, we speculate that RA patients may want and need better function for the activities of daily living. The combination of medical treatment and surgical intervention is thought to improve outcomes in RA patients who will develop joint destruction. Additional studies, including analyses of RA databases containing long-term data on a variety of surgical interventions, are needed. [ABSTRACT FROM AUTHOR]

The article presents a study regarding the stability of nepenthesin when treated with urea or guanidine hydrochloride compared with porcine pepsin A. It says that the enzyme assay was conducted at pH 2.0 with hemoglobin as substrate and that Nepenthes alata was incubated at different temperatures. Results show that marked pH/temperature stability was exhibited by nepenthesins compared with porcine pepsin.

The article presents a study which investigates the P1 and P1' residue specificities of the serine-carboxyl peptidase Physarolisin I produced by the true slime mold Physarum polycephalum. The specificities were examined by the use of combinatorial peptide substrates. Results show that the P1 position prefers certain hydrophobic and acidic residues and the P1' position prefers some hydrophobic residues. It notes that the specificity is partially explained by the nature of the S1 subsite residues.

RNA interference (RNAi) provides a powerful means of sequence-specific gene silencing. Several studies show that RNAi may provide promising strategies to treat human diseases by suppressing disease responsible genes in vivo. In locomotor diseases, the progression of collagen-induced arthritis (CIA) is suppressed by tumor necrosis factor-α (TNF-α)-specific small interfering RNA (siRNA) delivered into the joint. The aim of this study, is to compare the effects of intraarticularly administered siRNAs targeting TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and receptor activator of NF-κB ligand (RANKL) on CIA in rats. We confirmed that the silencing effects of siRNA duplexes specific for rat TNF-α, IL-1β, IL-6 and RANKL in vitro. Each siRNA was also delivered into the knee joint of CIA rats by the in vivo electroporation method 7, 10, 13 and 16 days after immunization with collagen. Local delivery of TNF-α or IL-1β-specific siRNA ameliorated CIA in rats effectively at the gross morphological, radiographical and histological evaluations. Our results suggested that TNF-α and IL-1β were the cytokines to be targeted in the joint for the treatment of rheumatoid arthritis. The in vivo siRNA transfection method may be useful for selection of target molecules to be silenced for treatment of joint diseases. [ABSTRACT FROM AUTHOR]

The article presents the study which focuses on the purification and characterization of a major collagenase. It states that the purification of the collagenase was taken from the crude enzyme sample of Streptomyces parvulus through successive phases of chromatography. Accordingly, the purified enzyme revealed a relative molecular mass and it was strongly inhibited by metal-chelating agents. Moreover, purified collegenases from Streptomyces species have been characterized partially only.

The article presents a study on the structural evidence for establishing the relationship of scytalidolisin with the sedolisin family of serine-carbonyl peptidases. It details the elution as well as the effuluent monitoring of the enzyme by measuring their absorbance. Peptide peak fractions were collected and lyophilized while peptide residues are identified. The amino acid sequences alignment of major peptides and sequences of typical serine carboxyl peptidases are also presented.

In this study, we investigated the usefulness of contrast-enhanced MRI with maximum intensity projection (MIP) as a convenient tool for detecting early rheumatoid arthritis (RA). A total of 21 patients with undiagnosed arthritis of the hands at the initial visit were enrolled in a prospective study over a 1-year period. The number of swollen joints found during physical examination at this first visit, the results of serological tests and the number of synovitis joints diagnosed on MIP images were compared between the RA group and non-RA group. Of the 21 patients, 17 (81%) from the initial study who were followed up for an additional 1 year entered this study. Of these, 5 met the conditions for diagnosis of RA during follow-up, and 12 did not. MIP images were used to review the arthritis of RA patients, and a significant difference was found in the number of synovitis inflammations detected with MIP images when compared with findings after physical examinations. The two criteria of positive CARF and/or anti-CCP antibody and symmetrical synovitis in bilateral hands on MIP images allowed the prediction of RA with 100% sensitivity and 75% specificity. Thus, MIP is a useful tool for making early diagnosis of RA because it yields clear visualization even with just one image. [ABSTRACT FROM AUTHOR]

The article reports on results of a study of the substrate specificities of porcine and bovine enteropeptidases toward the peptide Val-(Asp)4-Lys-Ile-Val-Gly and its analogs. A description of the study design and method is presented. The study concluded that in addition to P1 Lys, P2 Asp in the substrates is important, that P3 Asp is additionally important and that P5 Asp contributes to the susceptibility.

Escherichia coli signal peptidase I (SPase I) is a membrane-bound serine endopeptidase that catalyses the cleavage of signal peptides from the pre-forms of membrane or secretory proteins. Our previous studies using chemical modification and site-directed mutagenesis suggested that Trp300 and Arg77, Arg222, Arg315 and Arg318 are important for the proper and stable conformation of the active site of SPase I. Interestingly, many of these residues reside in the C-terminal region of the enzyme. As a continuation of these studies, we investigated in the present study the effects of mutations in the C-terminal region including amino acid residues at positions from 319 to 323 by deletions and site-directed mutagenesis. As a result, the deletion of the C-terminal His323 was shown to scarcely affect the enzyme activity of SPase I, whereas the deletion of Gly321-His323 or Ile319-His323 as well as the point mutation of Ile322 to alanine was shown to decrease significantly both the activity in vitro and in vivo without a big gross conformational change in the enzyme. These results suggest a significant contribution of Ile322 to the construction and maintenance of the proper and critical local conformation backing up the active site of SPase I. [ABSTRACT FROM PUBLISHER]

Previously, we characterized glutathione S-transferase (GST) B1-1 from Escherichia coli enzymologically and structurally. Besides GST B1-1, E. coli has seven genes that encode GST-like proteins, for which, except SspA, neither biological roles nor biochemical properties are known. Here we show that the GST-like YfcF and YfcG proteins have low but significant GSH-conjugating activity toward 1-chloro-2,4-dinitorobenzene and GSH-dependent peroxidase activity toward cumene hydroperoxide. Analysis involving site-directed mutagenesis suggested that Ser16 and Asn11 were important for the activities of YfcF and YfcG, respectively. On the contrary, no residue around the catalytic site of GST B1-1 has been demonstrated to be essential for catalytic activity. Deletions of the gst, yfcF, and yfcG genes each decreased the resistibility of the bacteria to hydrogen peroxide, which was recovered by transformation with the expression plasmid for the deleted enzyme. The inactive YfcF(S16G) and YfcG(N11A) mutants, however, could not rescue the knockout bacteria. Thus, E. coli has at least three GSTs of distinct classes, all of which are important for defense against oxidative stress in spite of the structural diversity. This seems consistent with the hypothesis that GSTs constitute a protein superfamily that has evolved from a thioredoxin-like ancestor in response to the development of oxidative stress. [ABSTRACT FROM PUBLISHER]

The article discusses the examination on the function of n-linked oligosaccharides of Aspergillus awamori feruloyl esterase for thermostability and catalysis. The study found out that when A or Q N-glycosylation-free N79A replaced N79 and mutant enzymes had lower activity compared to the glycosylated recombinant AwFAEA wil-type enzyme toward α-naphthylbutyrate, α-naphthylcaprylate and phenolic acid methyl esters.

Mitochondria could be a good target for anti-parasitic drugs. The α and β subunits of mitochondrial processing peptidase (MPP) and the core subunits of the cytochrome bc1 complex, UCR-1 and UCR-2, are homologous to one another and are important for mitochondrial functions. However, our knowledge of these proteins in nematodes is very limited. Caenorhabditis elegans, a free-living nematode, has six genes coding for proteins homologous to these subunits. On primary structure comparison, and immunochemical and enzymological analyses, the gene products were assigned as follows: Y71G12B.24, α-MPP; ZC410.2, β-MPP; F56D2.1, UCR-1; VW06B3R.1, T10B10.2; and T24C4.1, UCR-2. The primary structures of β-MPP and UCR-1 from Brugia malayi, a parasitic nematode causing human filariasis, were deduced from their cDNA structures. Phylogenetic analysis showed that the UCR-1s from both C. elegans and B. malayi were less related to mammalian UCR-1s than to MPPs from various organisms. MPP and the bc1 complex are essential for the life cycle of C. elegans, because their reverse genetic inhibition is lethal. This suggests the possibility that these proteins are also essential for the viability of B. malayi and other parasitic nematodes, and are potential targets for anti-parasitic agents. [ABSTRACT FROM PUBLISHER]

Previously we isolated and characterized a membrane-bound, arginine-specific serine protease from pig intestinal mucosa [J. Biol Chem 269, 32985–32991 (1994)]. For further characterization of this type of enzyme, we cloned a cDNA from rat intestinal mucosa encoding the precursor of a similar protease. The partial amino acid sequences determined for the pig enzyme were found to be shared almost completely by the rat enzyme. The serine protease domain of the rat enzyme, heterologously expressed in Escherichia coli, specifically cleaved Arg (or Lys)-X bonds with a marked preference for Arg-Arg or Arg-Lys, similar to the pig enzyme. The mRNA for the rat enzyme was shown to be distributed mainly in intestine, and the enzyme was detected in the duodenal mucosa as a 70 kDa protein. Immunohistochemical analysis of the small intestinal tissue showed that the enzyme is localized mainly on brushborder membranes. [ABSTRACT FROM AUTHOR]

Amino acid sequences in H2O2-oxidized bovine serum albumin (BSA) that are susceptible to proteolytic cleavage by oxidized protein hydrolase (OPH) were investigated. When oxidized BSA was treated with OPH, low-molecular-weight fragments (54, 46, 24, 22, 20, and 8 kDa) were produced as analyzed by SDS-PAGE. N-Terminal amino acid sequence analysis of these fragments indicated that oxidized BSA -was cleaved by OPH at three major sites, Leu218-Ser219, Tyr410-Thr411, and Phe506-Thr507, at an early stage of the proteolytic degradation. In the three-dimensional structure of BSA deduced by computer modeling, these cleavage sites were found to be located slightly inside the BSA molecule, in positions not easily accessible by OPH. The influence of oxidation on the tertiary structure of BSA was then investigated by hypothetically replacing all the four methionine and two tryptophan residues with their oxidized forms, methionine sulfoxide and N′-formyl-kynurenine, respectively. The three-dimensional structure of the hypothetically oxidized BSA indicated that all the three cleavage sites in the protein could become more exposed to the solvent than in unoxidized BSA. These results suggest that, upon oxidation of BSA, the amino acid sequences that are potentially cleav-able by OPH but present inside the molecule become exposed on the surface and susceptible to proteolysis by OPH. This is the first report demonstrating the cleavage sites of oxidized protein by oxidized protein-selective protease, suggesting the possible mechanism of oxidized protein-selective degradation by the enzyme. [ABSTRACT FROM AUTHOR]

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. BioL Chem. 269, 19976–19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/ or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and n, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates. [ABSTRACT FROM AUTHOR]

Eight bromelain inhibitor (BI) isoform fractions were isolated from pineapple stem, and then submitted to conventional amino acid sequencing after performic acid oxidation and subsequent separation of the resulting light and heavy chains. The results revealed that all fractions exhibited microheterogeneity, containing at least two major components, but that all the isoinhibitors have a common double-chain structure (Mr =ca. 5, 700-5, 900) with five disulfide bonds and similar amino acid sequences. Notably, Fraction BI-VIII exhibited less than 40% of the specific inhibitory activity toward stem bromelain as compared with the other inhibitor fractions. This fraction was a mixture of two isoforms, BI-VIII(1) and BI-VIII(2), the latter lacking the arginine or glutamine residue at the COOH-terminus of the light chain. Furthermore, the oxidized light chain of BI-III, used as a representative normal isoinhibitor, was found to exhibit significant inhibitory activity, whereas the oxidized light chain of BI-VIII(2) lacking the COOH-terminal Arg or Gin showed only very low inhibitory activity. Therefore, the major bromelain-inhibitory site was indicated to be the COOH-terminal residue, Arg or Gin, of the light chain. This is consistent with the three dimensional structure model constructed by computer modeling for the hypothetical complex between BI-VI and papain, a close homolog of bromelain. [ABSTRACT FROM AUTHOR]

Proteolytic trimming of isoprenylated proteins, including Ras, at the C-terminal CAAX motifs is a key event in their activation. However, the protease responsible for the proteolysis has not been well characterized yet. In this study, we established a novel assay method for the enzyme using a fluorescent substrate, dansyl(Dns)-KSKTKC(S-farnesyl)-VIM, with which we can assess the proteolytic activity with high sensitivity and more easily than by the former assay methods using radio-labeled substrates. Using this assay method, we purified the protease 104-fold from bovine brain microsomal membranes by Sepharose CL-6B gel filtration and DE-52 chromatography. The partially purified enzyme was shown to be an endoprotease specific to the farnesylated peptide and to have a Km value of 1.0 μM for Dns-KSKTKC(S-farnesyl)VIM. o-Phenanthroline and zinc chloride strongly inhibited the activity. Interestingly, however, m- and p-phenanthrolines were as effective as o-phenanthroline, indicating that the inhibition by o-phenanthroline is not simply due to its chelating action. The molecular mass of the protease was deduced to be 480 kDa by gel filtration. The enzymatic activity was lost during further attempts at chromatographic purification, but was partially recovered by mixing the chromatographic fractions which had apparently lost the activity. These results suggest that this protease consists of multiple subunits. [ABSTRACT FROM AUTHOR]

An extracellular metalloprotease was purified from the culture supernatant of Pseudomonas fluorescens strain KT1 to apparent homogeneity and shown to consist of a single polypeptide chain (Mr 46,000–47,000). The enzyme was strongly inhibited by chelating agents such as EDTA and o-phenanthroline, and activated by certain detergents. Among the peptidyl 4-methylcoumaryl-7-amide (MCA) substrates examined, t-butyloxycarbonyl-Arg-Val-Arg-Arg-MCA was the best one. With this substrate, the enzyme exhibited a pH optimum of around pH 5.5 in the absence of Co2+ ions, whereas it showed two different pH optima (at pHs around 5.5 and 8–9) in the presence of Co2+ ions due to remarkable activation by Co2+ ions in the alkaline pH range. On the other hand, a single broad pH optimum of around 6 to 8 was obtained with some peptides in both the presence and absence of Co2+ ions, and no activation by Co2+ was observed. The enzyme showed trypsin-like specificity, preferentially cleaving certain arginyl peptide bonds, and hydrolyzed the basic protein, histone, most rapidly among various proteins examined. Partial amino acid sequence analysis revealed that the enzyme is highly homologous with proteases of the serralysin family, a group of zinc metalloproteases. [ABSTRACT FROM AUTHOR]

The complete primary structure of the major component of tuna pepsinogens was determined by conventional protein chemistry methods. It was composed of a prosegment of 37 residues and a pepsin moiety of 323 residues, having a relative molecular mass of 39,364. The essential aspartyl residues in the active site and the three disulnde bonds common to other pepsinogens were conserved; however, several unique substitutions and/or deletions characteristic of tuna pepsinogen were found at various positions, especially in the prosegment and subsite regions, as compared with the sequences of other pepsinogens, which may affect the rate of activation of the zymogen, and/or the catalytic function and substrate specificity of the enzyme. Tuna pepsinogen is the least acidic among pepsinogens. The sequence identity between tuna pepsinogen and other pepsinogens ranged from 45 to 52%. A phylogenetic tree based on the primary structures suggested that tuna pepsinogen diverged from the pepsinogen A and prochymosin groups in an early period of pepsinogen evolution. [ABSTRACT FROM AUTHOR]

Pepsinogen was purified from the gastric mucosa of soft-shelled turtle (Trwnyx sinensis) by a series of chromatographies on DEAE-cellulose, Sephadex G-100, and Q-Sepharose. Upon chromatography on Q-Sepharose, it was separated into nine isoforms. These isoforms showed a relative molecular mass of approximately 43,000 Da on sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and isoforms 4 through 9 contained carbohydrate (approx. 2% each). Insofar as they were examined, their NH2-terminal sequences differed only in showing substitution at a few positions. At pH 2.0, they were rapidly activated to the corresponding isoforms of pepsin in a stepwise manner. The nine isoforms showed similar specific activity toward hemoglobin and hydrolyzed N-acetyl-L-phenylalanyl- L-diiodotyrosine, a good substrate for pepsin A, at somewhat different rates. They were inhibited by pepstatin to various extents, more strongly than human pepsin C but less strongly than human pepsin A. All isoforms appeared to have similar cleavage specificity toward oxidized insulin B chain, which resembled those of both human pepsins A and C. A cDNA clone for one of the zymogen isoforms was isolated and sequenced. The amino acid sequence thus deduced was more homologous with those of mammalian pepsinogens A than those of mammalian pepsinogens C or prochymoain. [ABSTRACT FROM AUTHOR]