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Meprins are oligomeric, glycosylated cell surface or secreted metalloendopeptidases that are composed of multidomain disulfide-linked subunits. To investigate whether subunit oligomerization is critical for intracellular transport or for the enzymatic and/or physical properties of the proteinase, specific cysteine residues were mutated, and the mutants were expressed in 293 cells. Mutation of mouse meprin alpha Cys-320 to Ala in the MAM domain (an extracellular domain found in meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu) resulted in expression of a monomeric form of meprin, as determined by SDS-polyacrylamide gel electrophoresis and nondenaturing gel electrophoresis. The monomeric subunits were considerably more vulnerable to proteolytic degradation and heat inactivation in vitro compared with the oligomeric form of the enzyme. Proteolytic activity of the monomeric meprin using a bradykinin analog or aminobenzoyl-Ala-Ala-Phe-p-nitroanilide as substrate was similar to that of disulfide-linked oligomeric meprin; however, activity against azocasein was markedly decreased. Mutation of another cysteine residue in the MAM domain (C289A), predicted to be involved in intrasubunit disulfide bridging, resulted in disulfide-linked oligomers and monomers. These results indicated that this mutant was capable of forming intersubunit disulfide bonds but less efficiently than wild-type meprin subunits. Mutant C289A also retained activity toward peptides but not the protein substrate and was more vulnerable to proteolytic degradation and heat inactivation compared with the wild-type enzyme. Both Cys mutants were expressed and secreted into the medium at levels comparable with the wild type and had slightly altered glycosylation. This work indicates that 1) Cys-320 of mouse meprin alpha is most likely responsible for the covalent interactions of the subunits; 2) covalent dimerization of subunits is not essential for efficient biosynthesis, trafficking, or posttranslational processing of the secreted protease; and 3) mutations in the MAM domain affect noncovalent interactions of the subunits and the stability and activity of the protease domain, indicating that domain-domain interactions are critical for structure and function of the enzyme.

Meprins, zinc metalloendopeptidases of kidney and intestinal brush border membranes, are composed of differing ratios of alpha and beta subunits. Previous work indicated that the beta subunit was expressed in kidney and intestine of all mouse strains, but that the alpha subunit was only expressed in kidney of random-bred and some inbred strains and not in mouse intestine. The work herein, however, reports that low levels of meprin alpha subunit mRNA and protein are detectable in mouse intestine and are present in increasing concentrations from the duodenum to the ileum. In ICR mice, the duodenum expressed less than 1% of the meprin alpha mRNA (micrograms/g tissue) relative to kidney, the ileum approximately 20%. The large intestine contained approximately 10% of the message found in kidney. An inbred mouse strain, C3H/He, found previously to contain only meprin beta subunits in kidney and intestine, displayed very low levels of meprin alpha mRNA (approximately 1% of that in ICR kidney) in both the kidney and intestine. Intestinal meprin beta mRNA in ICR and C3H/He mice, by contrast, was expressed at similar levels to that found in kidney, and for both strains there was an increase (two- to threefold) in the beta message in the ileum relative to duodenum or jejunum. In general, the pattern of the meprin alpha protein along the intestine was similar to that of alpha mRNA, and activity and response of intestinal meprin A to inhibitors were typical of the enzyme isolated from kidney. These data indicate that meprin alpha can be detected in mouse small and large intestine and that expression is not only tissue- and strain-specific but also longitudinally variable in intestine. The expression pattern for both a and beta subunits indicates an ileal function for the meprins.

Meprin A is a metalloendopeptidase in the proximal tubular epithelium of rodents that is capable of hydrolyzing a great variety of peptides and proteins. The aim of the present investigation was to investigate effects of ureteral ligation on the expression of meprin subunits. Ureteral ligation resulted in marked decreases in the expression of both alpha- and beta-meprin subunits within 12 h of ureteral obstruction. Even greater downregulation of expression of meprin alpha- and beta-mRNA was noted at 24, 48, and 96 h after ureteral ligation. The greatest decrease in meprin mRNA expression in obstructed kidneys over contralateral unobstructed control kidneys (CUK) occurred at 24 h postunilateral ureteral obstruction (post-UUO) for the meprin alpha-subunit (20-fold decrease compared with controls) and at 48 h for the meprin beta-subunit (90-fold decrease). On immunolabeling, the intensity for the two meprin subunits at the corticomedullary junction was dramatically decreased at 24 to 96 h after ureteral ligation in contrast to the CUK specimens. Results of in situ hybridization indicated that the CUK specimens expressed meprin beta-mRNA at the corticomedullary junction, whereas the obstructed kidneys exhibited a decrease in mRNA signal for meprin beta-subunit. There was a steady increase in the interstitial macrophage number in UUO rat kidneys over the 96 h of evaluation post-UUO. ED-1-positive macrophages were observed almost exclusively in the peritubular cortical interstitial space in a ringlike pattern with a preponderance of macrophage clusters around glomeruli. Unexpectedly, after reversal of UUO, the interstitial macrophage number remained higher than controls, despite the demonstrable decompression of the renal pelvis and caliceal system. In summary, this investigation demonstrates downregulation of meprin alpha and beta within hours of UUO and indicates a novel tubular response to ureteral obstruction.

Meprins, metalloendopeptidases of the astacin family, are composed of alpha and/or beta subunits and are expressed at high levels in mammalian renal and intestinal brushborder membranes. Only one mRNA has been identified previously for each of the subunits in adult human and rodent tissues; a 3.6-kilobase message for the alpha subunit and a 2.5-kilobase message for the beta subunit. The present study reports that a larger beta subunit message (2.7 kilobases, referred to as beta'), and no alpha subunit message, is expressed in embryonal carcinoma cell lines, F9 and Nulli-SSC1, and in human colon adenocarcinoma cells, HT-28-18-C1. Furthermore, in Nulli-SSC1 cells, the beta isoform is induced by the morphogen retinoic acid. The beta' isoform differs from beta only in a portion of the 5'-coding (corresponding to the signal and prosequence domains of the protein) and noncoding region. Only one gene was found for the beta subunit in the mouse and human genome. The deduced amino acid sequence of beta' has no homology with beta in the first 35 NH2-terminal residues, but the two sequences are identical after that. In vitro translation experiments indicated that the size of the protein product of beta' cDNA was similar to that of the beta cDNA protein product, and, in the presence of microsomal membranes, both were glycosylated. These studies indicate that the messages for the meprin beta and beta' subunit result from differential promoter usage and alternate splicing. Expression of the two isoforms may be regulated differentially depending on cell type and/or differentiation state of the cell.

Molecular Aspects of Proteinases and Inhibitors: The Metzincinsuperfamily of Zincpeptidases W. Bode, et al. Structure and Biosynthesis of Meprins P. Marchand, J.S. Bond Involvement of Tissue Inhibitor of Metalloproteinases (TIMPs) during Matrix Metalloproteinase Activation H. Nagase, et al. Protein Turnover in Health and Disease: Structural Aspects of Autophagy P.O. Seglen, et al. Mechanism of Autophagy in Permeabilized Hepatocytes M. Kadowaki, et al. Lysosomal Proteinolysis Based on Decreased Degradation of a Specific Protein, Mitochondrial ATP Synthetase Subunit C: Batten Disease J. Ezaki, et al. Functional Aspects of Proteolytic Systems: Endopeptidase 24.11 Neprilysin and Relatives: Twenty Years On A.J. Turner, et al. Function of Calpains: Possible Involvement in Myoblast Fusion M. Hayashi, et al. Ubiquitin and Proteasome Related Proteolysis: Protein and Gene Structures of 20S and 26S Proteasomes K. Tanaka, et al. The Proteasome and Protein Degradation in Yeast W. Hilt, et al. Pathological Aspects of Protein Turnover: Cellular Proteases Involved in the Pathogenicity of Human Immunodeficiency and Influenza Viruses H. Kido, et al. HIV Proteinase Mutations Leading to Reduced Inhibitor Susceptibility B. Korant, et al. 7 additional articles. Index.

Meprins are cell surface and secreted metalloendopeptidases that are expressed in kidney and intestine of mice, rats, and humans (Jiang et al., 1992; Gorbea et al., 1993; Corbeil et al., 1992; Johnson and Hersh, 1992; Sterchi et al., 1988; Yamaguchi et al., 1994). The enzymes are capable of hydrolyzing a variety of proteins and biologically active peptides, such as bradykinin, glucagon, transforming growth factor-α, and parathyroid hormone (Butler et al., 1987; Choudry and Kenny, 1991; Wolz et al., 1991). Meprins are implicated in the degradation of extracellular and membrane-bound proteins and peptides, and it has been suggested that they are involved in the processing and inactivation of peptide hormones in vivo (Kenny et al., 1989; Yamaguchi et al., 1994).

Meprins are cell membrane, oligomeric metalloendopeptidases composed of two distinct but evolutionarily related subunits, alpha and beta. The structural genes for the meprin subunits, Mep-1 alpha and Mep-1 beta, have been previously mapped to chromosomes 17 and 18, respectively, of the mouse genome. We now report the localization of MEP1A and MEP1B in the human genome. MEP1A mapped to the short arm of chromosome 6 by the use of radiation and somatic cell hybrids. More specifically, it is localized between the centromere and GSTA2 in 6p11-p12. MEP1B mapped to chromosome 18, by the use of somatic cell hybrids, in 18q12.2-q12.3, proximal to the TTR/PALB gene. As in the mouse genome, the two homologous human structural genes for alpha and beta (50% identical on the cDNA level) are unlinked. These new markers on human chromosomes 6 and 18 extend the region of known linkage homology with mouse chromosomes 17 and 18, respectively, and provide new molecular access to regions of the human genome.

[Figure][1] CREDIT: GEORGINA PALMER/ISTOCKPHOTO.COM Graduate education in the biomedical sciences has recently been the subject of much discussion ([ 1 ][2]–[ 5 ][3]). In the Policy Forum “Promoting convergence in biomedical science” (29 July, p. [527][4]), P. A. Sharp and R. Langer

Meprins, which are cell surface metalloendopeptidases, consist of two types of subunits, alpha and beta. Genetic factors determine which subunits and oligomeric forms of the enzyme exist in kidney in different strains of mice. In order to further explore factors that determine the concentration and activity of meprins, the rates of biosynthesis and degradation of the meprin subunits were determined in an organ culture system using ICR and C3H/He mouse kidneys. In biosynthesis experiments, the rate of incorporation of radiolabeled amino acids into immunoprecipitable forms of the alpha and beta subunits was determined. The rate of loss of radiolabel from the subunits was measured in pulse-chase experiments to determine the half-lives of the subunits. The rate of synthesis of the alpha subunit was twofold greater than that of the beta subunit: 861 +/- 32 vs 361 +/- 23 dpm/micrograms subunit protein/min for alpha vs beta. The rate of synthesis for total kidney protein in both strains was approximately 700 dpm/micrograms/min. The half-life for alpha was 8.9 +/- 0.24 h compared to 12.1 +/- 0.7 h for beta; the half-life for total protein in kidney was approximately 35 h. Thus, the half-lives of alpha and beta were similar and shorter than the half-lives of the average protein in kidney cells. The higher rate of synthesis of alpha is probably responsible for the greater abundance of this protein compared to beta in microvillus membranes.

Activity of renal meprin, a membrane-bound proteinase in the proximal tubule brush border, was measured in normal rats and in two disease groups: chronic puromycin aminonucleoside nephropathy for 12 weeks and streptozocin-induced diabetes for 6 months. Enzyme activity in kidney homogenates was assayed using azocasein as substrate. The mean activity of mephrin was 3.22 +/- 0.34 U/g kidney weight in normal rats. In diabetic animals, enzyme activity was 8.58 +/- 2.11 U/g kidney weight, P0.01. In contrast, meprin activity was decreased in rats with puromycin-induced glomerulopathy, 2.13 +/- 0.17 U/g kidney weight, P0.01. These findings indicate that meprin activity is elevated in experimental diabetes. Diminished activity of this luminal membrane enzyme in puromycin aminonucleoside nephropathy may contribute to renal injury in this disease model associated with massive urinary protein excretion.

Crystal structures available for four metalloendopeptidases have revealed zinc ligands for these enzymes. New sequence information has made it possible to compare the primary structures of the zinc-binding site in metalloendopeptidases. A scheme based on the zinc-binding site is proposed to classify metalloendopeptidases into five distinct families: thermolysin, astacin, serratia, matrixin, and snake venom metalloproteinases. Two histidines and one glutamate are zinc-ligands in the thermolysin family. Three histidines and one tyrosine are zinc ligands in other four families, which are further distinguished by the identity of the residue following the third histidine and by the environment surrounding the tyrosine.

Abstract 1364 Poster Board I-386 Meprin metalloproteases are abundantly expressed in kidney and intestinal epithelial cells, and have been implicated in inflammatory bowel disease. Recently, we demonstrated that meprin deficiency compromises homeostasis of monocytes and natural killer cells. However, the role(s) of meprins in the hematological system and the pathological consequences of meprin deficiency have not been fully characterized. We report here that mouse and human monocytes expressed meprins, and the expression of meprin α was down-regulated in monocyte-derived mature macrophages (Mφ). In a mouse model of septic shock induced by peritoneally injected bacterial lipopolysaccharide (LPS), mice carrying germline null mutation of both meprin α and βgenes (meprin double knockout, dKO) developed a more severe form of septic shock, characterized by deeper hypothermia, higher levels of blood nitrate/nitrites and suppressed monocytosis, in comparison to the wild-type mice. In addition, LPS-challenged meprin dKO mice exhibited a greater elevation of plasma IL-12, MCP-1, IL-6 and TNF-α, even though these pro-inflammatory cytokines were at lower levels in the dKO mice than the wild-type mice in the steady state. Furthermore, meprin dKO mice harbored a higher number of peritoneal Mφ than the wild-type mice, and thereby produced more soluble cytokines in response to ex vivo LPS stimulation. These results indicate that meprin deficiency is associated with accumulation of peritoneal Mφ, which may lead to hyperactive cytokine responses to inflammatory stimuli and result in increased susceptibility to LPS-induced septic shock. Disclosures No relevant conflicts of interest to declare.

To investigate the expression of meprin-A, a brush-border metalloproteinase in mouse tissues, immunohistochemical studies were conducted using a monoclonal antibody prepared against a purified form of kidney meprin-A form male mice. Kidney slices from female mice displayed markedly less immunoreactivity compared with similar preparations from male mice using this antibody. However, the specific activities of meprin-A in kidney homogenates and purified preparations of meprin-A from male and female mice were not significantly different. Western blots of kidney membrane proteins from several mouse strains indicated that the female form of meprin-A had a decreased mobility relative to the male form when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; this difference could be eliminated by treatment of preparations with endoglycosidase F, which removes some asparagine-linked oligosaccharides. These data and lectin blots of membrane proteins indicate that there are differences in the glycosylation (specifically in the complex type oligosaccharides) of meprin-A in adult (8 wk old) male and female mice. Juvenile (3 wk old) male and female mice displayed similar amounts of immunohistochemical staining in kidney slices, as well as similar meprin-A electrophoretic mobilities and lectin affinities. Administration of 17 beta-estradiol to gonadectomized adult mice decreased the immunoreactivity of meprin-A in kidney slices and the electrophoretic mobility of meprin-A. These studies indicate that estrogens affect posttranslational modifications of meprin-A.

The extended substrate-binding site of meprin-A, a tetrameric metalloendopeptidase from brush border membranes of mouse kidney proximal tubules, was mapped with a series of peptide substrates. Previous studies led to the development of the chromogenic substrate Phe5(4-nitro)bradykinin for meprin-A. With this substrate, several biologically active peptides were screened as alternate substrate inhibitors, and, of these, bradykinin (RPPGFSPFR) was found to be the best substrate with a single cleavage site (Phe5-Ser6). Three types of bradykinin analogues were used for a systematic investigation of substrate specificity: (1) nonchromogenic bradykinin analogues with substitutions in the P3 to P3' subsites were used as alternative substrate inhibitors of nitrobradykinin hydrolysis, (2) analogues of nitrobradykinin with variations in the P1' position were tested as substrates, and (3) intramolecularly quenched fluorogenic bradykinin analogues with substitutions in the P1 to P3 sites were tested as substrates. A wide variety of substitutions in P1' had little effect on KM (174-339 microM) but markedly affected kcat (51.5 s-1 = A greater than S greater than R greater than F greater than K greater than T greater than E = 0). Substitutions in P1 had a greater effect on KM (366 microM-2.46 mM) and also strongly affected kcat (98.5 s-1 = A greater than F much greater than L greater than E greater than K = 2.4 s-1). The variety of allowed cleavages indicates that meprin-A does not have strict requirements for residues adjacent to the cleavage site. Substitutions farther from the scissle bond also affected binding and hydrolysis, demonstrating that multiple subsite interactions are involved in meprin-A action.(ABSTRACT TRUNCATED AT 250 WORDS)

Meprin is a membrane-bound metalloproteinase which is expressed at high levels in the brush border membrane of proximal tubules of kidneys of some mouse strains (referred to as high meprin-activity mice). The mature active proteinase is not present in kidneys of many inbred strains of mice; however, these low meprin-activity mice possess a kidney protein that crossreacts with polyclonal antibodies prepared against meprin. In the present studies, immunohistochemical methods were used to determine the presence of meprin in liver, pancreas, spleen, testis, thymus, kidney, salivary glands, stomach, duodenum, and skin. Meprin crossreactivity was observed only in kidney and salivary glands. In salivary glands, the enzyme was found on the luminal surface of intercalated and striated ducts of submandibular and parotid glands and on interlobular ducts of the latter. In both kidney and salivary glands, the intensity of immunochemical staining was greater in males compared with females. For both sexes, immunoreactivity was markedly greater in the high meprin-activity mice compared to the low meprin-activity mice. These studies indicate that meprin has a limited tissue distribution, and that genetic and hormonal factors that regulate the proteinase are similar in kidney and salivary glands. The localization of the proteinase implies that the enzyme functions in modifying proteins and peptides that are secreted or re-absorbed in the ducts of these tissues.

Phe5(4-nitro)-bradykinin has been identified as a good synthetic substrate to study the kinetics and mechanism of action of the metalloendopeptidase meprin. No convenient substrate for kinetic analysis of the enzyme had been previously described. HPLC analyses indicated that meprin cleaved bradykinin and nitrobradykinin between Phe5 (or Phe5(NO2)) and Ser6. Reaction rates for bradykinin were determined by quantitative HPLC analyses, whereas rates for nitrobradykinin were measured by continuous monitoring of the spectral change that occurs at 310 nm when the Phe(NO2)-Ser bond is hydrolyzed. For nitrobradykinin and unmodified bradykinin, respectively, Km values were 281 and 425 microM, kcat values were 28 and 22 s-1, and kcat/Km values were 9.7 x 10(4) and 5.1 x 10(4)M-1. The two products of bradykinin hydrolysis were not substrates for the enzyme, but they were inhibitors. The initial rates of hydrolysis of nitrobradykinin increased linearly with enzyme concentration (0.09-2.2 micrograms/ml), and increased linearly with temperature in the range from 15 to 55 degrees C. Hydrolysis of the substrate was optimal at alkaline pH values. The cysteine endopeptidases papain and cathepsin L and the metalloproteases thermolysin, angiotensin-converting enzyme, and neutral endopeptidase (EC 3.4.24.11) also cleaved nitrobradykinin, but at different peptide bonds than meprin. The single cleavage of nitrobradykinin at the Phe(NO2)-Ser bond and the concomitant spectral shift that occurs at alkaline pH makes this a particularly suitable substrate for meprin.

Meprin metaloproteases are highly expressed in the apical brush-border of the intestinal and renal proximal tubule epithelia. However, the expression and function of these metaloproteinease in other tissues have not been well established. Using a specific antisera raised against rat but cross-reactive with human Meprin Alfa, we examined the expression of this protein in human peripheral blood cells. Flow cytometry analysis showed that the post-, but not the pre-, immune sera stained strongly for the CD14+ monocytes and moderately for CD56+ NK cells, but not for CD3+ T and CD19+ B cells. To confirm Meprin Alfa expression at the RNA level, peripheral blood cells were fractionated based on the expression of the above surface markers, followed by detection of Meprin Alfa message RNA by semi-quantitative RT-PCR. Consistent with the flow cytometry data, Meprin Alfa messages were detected only in cell fractions containing monocytes and NK cells but not T and B cells. Subsequent sequencing of the PCR amplified fragments confirmed the specificity of the PCR amplification. Interestingly, flow cytometry analysis further revealed that while the expression of Meprin Alfa was maintained in the macrophages differentiated in vitro from isolated monocytes, its expression was reduced in monocytes-derived immature dendritic cells (DC) and diminished in mature DC. The differential expression of Meprin Alfa in the peripheral blood cells suggests important physiological function of this protein in the immune system.

A sensitive colorimetric assay for arginase was developed. Urea produced by arginase was hydrolyzed to ammonia by urease, the ammonia was converted to indophenol, and the absorbance was measured at 570 nm. The assay is useful with low concentrations of arginase (0.5 munit or less than 1 ng rat liver arginase) and with a wide range of arginine concentrations (50 microM to 12.5 mM). Michaelis-Menten kinetics and a Km for arginine of 1.7 mM were obtained for Mn2+-activated rat liver arginase; the unactivated enzyme did not display linear behavior on double-reciprocal plots. The kinetic data for unactivated arginase indicated either negative cooperativity or two types of active sites on the arginase tetramer with different affinities for arginine. The new assay is particularly well suited for kinetic studies of activated and unactivated arginase.

Disulfides (glutathione disulfide, cystine, cystamine) caused a first-order inactivation of rabbit-muscle fructose-1,6-bisphosphate aldolase at pH values of 7.4 and above. Inactivation by glutathione disulfide was partially reversed by reducing agents, but the enzyme became irreversibly inactivated with time. The disulfide-inactivated aldolase had a lower transition temperature and enthalpy of denaturation than the native enzyme. In addition, the disulfide-inactivated enzyme was extensively degraded by proteinases, whereas the native enzyme was resistant. Mixed disulfides were formed; a maximum ratio of 4-5 mol of glutathione/mol of the aldolase tetramer was found. The number of titratable--SH groups on aldolase decreased by 16 (out of 32 total on the control enzyme) after inactivation by glutathione disulfide, indicating that other oxidation reactions in addition to those resulting in mixed disulfides occurred. The substrate, fructose 1,6-bisphosphate, prevented inactivation of aldolase by glutathione disulfide, the formation of glutathione-enzyme mixed disulfides, thermodynamic destabilization of the enzyme, and a decrease of--SH groups on the enzyme. These data indicate that covalent modification of aldolase by biological disulfides is important in modulating enzyme stability and vulnerability to proteinases as well as enzyme activity and that the substrate protects against modification by disulfides.

SUMMARY The effect of streptozotocin-induced diabetes on the turnover of acidic and basic mouse liver cytosol proteins was assessed. Previous work by others had demonstrated that acidic proteins are degraded more rapidly than basic proteins in livers of normal but not in severely diabetic animals. In this study, a milder form of diabetes was investigated to determine whether insulin-dependent diabetes in the absence of starvation, large weight losses, and impending death results in fundamental changes in degradation of the normally labile (acidic) proteins and stable (basic) proteins. The relative rates of degradation of liver proteins were measured by a double-isotope technique and by the loss of protein radiolabeled with [14C]-bi-carbonate. The relative rates of synthesis of proteins were estimated by incorporations of [3H]-leucine. No fundamental change in the relative rates of synthesis or degradation of acidic and basic proteins was observed. There was a general decrease in the incorporation of radiolabeled amino acids in the diabetic state and an increase in the specific activity of some amino acid–metabolizing enzymes, indicating changes in amino acid metabolism in liver. A study of the quantity and subcellular distribution of several liver cell proteinases revealed little if any changes in the proteolytic machinery of liver cells in this form of insulin-dependent diabetes. Thus, the fundamental changes in protein degradation seen in severe diabetes are not observed in a less severe form of the disease.

Meprin, a glycoprotein with potent metalloendopeptidase activity, is an integral component of the brush border membrane of mouse kidney. Previously we reported that genealogically related inbred mouse strains (C3H and CBA) are markedly deficient in the activity of this enzyme. We report here that meprin deficiency is inherited as an autosomal recessive trait and that several other inbred strains also express low levels of meprin activity. All of the inbred strains deficient in meprin activity are of the H-2k haplotype; however, two strains of this haplotype (C58 and C57BR/cd) expressed normal levels of the proteinase. Congeneic and recombinant mouse strains were examined to determine whether the deficiency was linked to the H-2 complex. The gene controlling the activity of meprin (Mep-1) maps on chromosome 17 to the right of the D end of the major histocompatibility complex. The Mep-1 gene is closely linked to a gene that controls isoenzyme patterns of phosphoglycerate kinase (Pgk-2). This work represents the localization of a gene that determines the activity of an integral cellular endopeptidase in mammalian tissues. In addition, the Mep-1 gene is the only identified gene linked to the major histocompatibility complex that regulates a proteinase activity.

Hepatic arginase ( l -arginine amidinohydrolase, EC 3.5.3.1) is an oligomer composed of three or four subunits. The present studies indicate heterogeneity in the size and charge of arginase subunits in mouse liver. Two types of arginase subunits with molecular weights of approximately 35,000 and 38,000 have been found. These two subunits are detected in liver cytosol or in purified preparations of arginase after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Two dimensional SDS-PAGE revealed multiple ionic forms of arginase for both the 35,000 and 38,000 subunits; the subunits contain basic proteins (pI range 7.8–9.1) and acidic proteins (pI range 5.8–6.4). Limited proteolysis by trypsin eliminated the molecular weight differences between the subunits without substantially affecting either their isoelectric points or activity. Comparative peptide maps and amino acid analyses of the 35,000- and 38,000-Da subunits showed that they were very similar. The data indicate that a neutral peptide (approx 3000 Da) is responsible for the differences in subunit molecular weight and that the multiple sized and charged forms are variants of the Same protein.

SummaryCytosol or serum proteins promote the cosedimentation of rat liver lysoso-mal enzymes with the large granular fraction membranes at neutral pH. Cytosol promoted cosedimentation of three lysosomal enzymes but not a mitochondrial or a perox-isomal enzyme. Dialyzed or boiled cytosol are as effective as untreated cytosol whereas γ-globulin is more effective than albumin in promoting cosedimentation.

Inbred mice can be phenotypically divided into two groups: those that contain high levels of a kidney metallo-endopeptidase activity (meprin-a) and those with low meprin-a activity. In studies to investigate the molecular basis for the heterogeneity in the expression of this proteinase activity, we found a latent metallo-proteinase activity associated with kidney membranes of C3H/HeJ mice, a low activity strain. The latent proteinase was activated by treatment of kidney brush border membranes with trypsin and was purified from solubilized C3H kidney membranes. Purified preparations of the C3H latent proteinase (referred to as meprin-b) contained three major proteins of subunit molecular weights 90,000, 140,000, and 160,000. In the absence of reducing agents, four 90,000-Da subunits are covalently linked by S-S bridges. The two higher molecular mass proteins are not covalently linked to each other or to the 90,000-Da subunits. However, cross-linking and affinity chromatography studies indicated that the proteins in the meprin-b preparation were tightly associated. By contrast, purified meprin-a contains only 85,000-Da subunit proteins linked by S-S bridges to form a tetramer. Endoglycosidase F treatment decreased the mass of the 90,000-Da meprin-b subunit and the 85,000-Da meprin-a subunit to polypeptides of 65,000-70,000 Da. The 90,000- and 85,000-Da subunits are immunologically similar, in that polyclonal antibodies prepared against one of the subunits cross-react with the other. The substrate specificities and inhibitor profiles of purified preparations of meprin-a and meprin-b are also similar. These data are consistent with the proposition that meprin-b is a polymorphic form of meprin-a that is incompletely processed in vivo.

An intracellular serine protease from the bacterium Rhodococcus erythropolis was partially purified and characterized. The enzyme, which is present in the 100,000g supernatant fraction of disrupted cells, was purified 160-fold by ammonium sulfate fractionation, and chromatography on DEAE-cellulose, Sephadex G-150, hydroxyapatite, and benzamidine-glycylglycine-Sepharose. The molecular weight of the protease estimated by gel filtration was 82,000. The subunit molecular weight of the protein was estimated to be 90,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, the enzyme appears to be a monomer. The purified enzyme preparation hydrolyzed several arginine-containing synthetic substrates; benzoyl-arginine-4-methyl-7-coumarylamide was routinely used as substrate. Hemoglobin, casein, and azocasein were also hydrolyzed. No carboxypeptidase activity was detected. The pH optimum of the enzyme was 7.0–7.2. The protease was not affected by inhibitors of cysteine proteases (iodoacetate), aspartic proteases (pepstatin), or metallo-proteases (EDTA). Inhibition was observed with the serine protease inhibitor diisopropylfluorophosphate. Several trypsin inhibitors (tosyl-lysine chloromethyl ketone, antipain, leupeptin, 4-aminobenzamidine, ovomucoid, and gramicidin S) inhibited the Rhodococcus protease, but others did not (phenylmethylsulfonylfluoride, lima bean trypsin inhibitor, aprotinin, α-1-antitrypsin). Salts with monovalent cations (e.g., NaCl, KCl) activated the protease two-to threefold at ionic strengths of 0.2 to 0.5. MgCl2 stimulated activity fourfold at ionic strengths of 0.05 to 0.15; CaCl2 stimulated activity (1.5-fold) maximally at an ionic strength of 0.05 and inhibited above 0.15.

The Mep-1 gene on chromosome 17 in mice controls the activity of meprin, a kidney brush border metalloendopeptidase. Most inbred mouse strains of the k haplotype (e.g., CBA, C3H, AKR) are markedly deficient in meprin activity; these mice carry the Mep-1 ballele. Mouse strains in which meprin activity levels are normal are designated Mep-1 a Studies using congenic and recombinant strains mapped the Mep-1 gene telomeric to H-2D near the Tla gene. To further study the relationship between the major histocompatibility complex and Mep-1, a linkage study was conducted. Mep-1 a F1 hybrids [C3H.A (K k D d ) × C3H.OH (K d D k )] were backcrossed with Mep-1 b C3H.OH (K d D k ) parents. The progeny were assayed for H-2D markers, Pgk-2 isozymes, and meprin activity. Recombination between H-2D and Mep-1 occurred in 6 out of 284 mice, a crossover frequency of 2.1%. Mep-1 is therefore 2.1 crossover units telomeric to H-2D and approximately 0.6 crossover units from Tla. The Mep-1 locus provides a new genetic marker for the future mapping of this important area of the mouse genome.

When beef liver extracts were incubated with Mn2+ or Co2+, arginase ( l -arginine amidinohydrolase, EC 3.5.3.1) was activated and its susceptibility to proteolytic inactivation was increased. Mg2+, which did not activate, did not affect proteolytic susceptibility. Increasing concentrations of Mn2+ from 1 to 50 mM increased the extent of both activation and tryptic susceptibility. Trypsin and chymotrypsin were much more effective than pronase or subtilisin in inactivating Mn2+-treated arginase. The chromatographic behavior of arginase on Sephade G-200 before and after activation with Mn2+ was the same. Amino acids protected purified preparations of arginase against tryptic inactivation. The most effective protection was afforded by cysteine, isoleucine, leucine, valine and alanine. Isoleucine protected at concentations as low as 0.38 mM, which is in the physiological range.

The cytosol proteins of mouse or rat liver were separated on the basis of charge and characterized with respect to molecular size, turnover in vivo , and several chemical properties. The basic proteins, which were synthesized and degraded slower than acidic proteins, were generally smaller, as multimers and subunits, than the acidic proteins. Charge and size, therefore, did not appear to be independent characteristics for soluble liver proteins. The amino acid composition of the smaller, basic, stable proteins was very similar to that of the larger, acidic, labile proteins. The charge differences between these two protein fractions could be attributed to the ratio of acidic to basic amino acid residues rather than to carbohydrate, nucleic acid, or phosphate content. The percentage of hydrophobic amino acid residues in the two protein fractions was the same. The acidic labile proteins were more vulnerable to proteolysis and associated with the lysosomal-mitochondrial fractions somewhat more extensively in vitro than the basic, stable proteins. These studies indicate that the information determining the half-lives of soluble enzymes is in the protein moiety of those enzymes and that the factors that correlate with turnover may not be independent variables for soluble mammalian proteins.

All living cells regulate the content and composition of their resident proteins, but the mechanisms by which this is accomplished are not understood. The process of protein degradation has an important role in determining steady state and fluctuations of protein concentrations in mammalian cells. This process may be regulated by innate properties of the protein substrates, by factors that interact or "brand" proteins for degradation or by the degradative machinery of the cell. For a specific protein, there appears to be a committed step, an irreversible event that leads to rapid and extensive degradation. That initial event may or may not involve 1) proteolysis, 2) a nonproteolytic covalent modification or branding event (e.g., oxidation, ubiquitin conjugation), 3) denaturation or unfolding of the protein, or 4) sequestration. The degradative machinery of cells may either recognize proteins committed to degradation or initiate degradation, but the process must be selective because there is great heterogeneity in the rates of degradation for different proteins of one cell. The degradative process certainly requires proteases, and it is probable that lysosomal and extralysosomal proteases are involved in the catabolism of cellular proteins. We review here briefly what is currently known about the factors that may determine the half-life of a protein in a mammalian cell, the role of the protein substrate and sequestration in the process, the proteolytic and nonproteolytic enzymes that may initiate the degradative process, and the regulation of extensive degradation of proteins in cells.

Combinations of pactamycin and Escherichia coli 026: B6 Boivin lipopolysaccharide (LPS) or Salmonella minnesota R595 lipid A, as a bovine serum albumin-lipid A complex, administered simultaneously, killed BALB c mice synergistically. The enhanced lethality of the combination was not the result of a reduced rate of elimination of LPS or pactamycin from the circulation. Pactamycin administered iv, but not ip, accumulated in the lungs immediately after injection; thereafter the drug progressively disappeared from lung tissue. Concurrent administration of LPS did not alter the accumulation of pactamycin in the lungs nor its rate of elimination from blood and lung. The amount of pactamycin recoverable after incubation with liver or lung homogenates was markedly decreased at both 4 and 37°C. No products of the antibiotic were detected chromatographically after incubation with homogenates. Pactamycin and LPS in combination increased the in vitro fragility of hepatic and renal lysosomes, as measured by the release of cathepsin and β-glucuronidase; the effect of the combination was approximately equivalent to the sum of effects of LPS and pactamycin alone. Concanavalin A and cyclic adenosine 3′,5′-monophosphate did not modify the lethality of pactamycin and LPS, singly or in combination, whereas caffeine, ethylenediamine tetraacetate, or 6α-methylprednisolone protected mice from the lethal action of LPS and the synergistic combination but not that of pactamycin. Lethal doses of LPS and the synergistic combination, but not pactamycin, provoked marked hypothermia and elevated concentrations of blood urea nitrogen. These results indicate that pactamycin rendered the mice more susceptible to LPS and that LPS was the causal lethal agent.

This communication reports early events in the most dramatic example of cellular metaplasia: the transformation of iris cells into lens cells. During the early stages of lens regeneration the activities of tyrosine aminotransferase, isocitrate dehydrogenase and creatine phosphokinase remain constant while that of glutamine synthetase increases. Cyclic AMP concentrations decrease prior to the enhancement of RNA and protein synthesis and then appear to increase prior to the initiation of DNA synthesis. This may indicate a negative and a positive control of RNA and DNA synthesis, respectively.

The susceptibility of five enzymes to proteolytic attack has been compared in rat liver extracts. The results show that enzymes with short in vivo half-lives were especially vulnerable to proteolytic attack, as measured by loss of activity in vitro , in contrast to the long-lived enzymes which were resistant to attack. Furthermore, there was a good correlation between the relative rates of inactivation in vivo and in vitro only with specific proteases such as trypsin and chymotrypsin, not with non-specific proteases such as pronase and subtilisin. This suggests that proteolytic enzymes with some degree of specificity are involved in the degradation of intracellular enzymes.

Photooxidation of 3-phosphoglyceraldehyde dehydrogenase in the presence of rose bengal did not effect the rate or extent of acetylation of the enzyme with p-nitrophenyl acetate; however, the rate of deacetylation of the S-acetyl-enzyme was selectively inhibited by 50 to 60%. Amino acid analysis of the photooxidized enzyme showed about 1 histidine residue per monomer was destroyed. No other changes in amino acid content were found. Peptide mapping studies showed that a specific histidine was photooxidized. The sequence of the tetradecapeptide containing the photosensitive histidine is as follows: Val-Asp-Val-Val-Ala-Ile-Asn-Asp(Pro,Phe)Ile-Asp-Leu-His Photooxidation also inhibited oxidation of 3-phosphoglyceraldehyde by 50 to 60% suggesting that the histidine residue is involved in the principal physiological reaction carried out by the enzyme. At high dye to enzyme ratios, rose bengal in the dark also inhibited the esterase and dehydrogenase activities. Rose bengal was a competitive inhibitor with respect to DPN and arsenate, but a noncompetitive inhibitor with respect to 3-phosphoglyceraldehyde or p-nitrophenyl acetate. The effects of photooxidation produced a permanent change in the enzyme whereas the dark inhibitions were completely reversed by removal of the dye from the enzyme surface with charcoal treatment. The role of histidine in the various reactions catalyzed by the enzyme is discussed.

Publisher Summary Meprin, the name is an acronym for metallo-endopeptidase from renal tissue, is an intrinsic plasma membrane proteinase. The enzyme was discovered as the activity in mouse kidney homogenates that was responsible for the high rate of degradation of the general proteinase substrate, azocasein. Meprin was purified, was named, some of its properties were characterized, and its subcellular localization was determined in the laboratories. An inherited deficiency of meprin activity was subsequently discovered in some inbred mouse strains, a rare if not unique example of a heritable deficiency of a cellular proteinase. In addition, the gene that controls meprin activity has been located on chromosome 17 near the major histocompatibility complex. Proteases, endopeptidases and exopeptidases, have been the focus of numerous scientific studies. Indeed purified proteolytic enzymes have provided the material for many investigations of the fine structure of enzymes, the physicochemical interactions between macromolecules or between macromolecules and small ligands, and the chemical kinetics of enzymes.

SummaryThe specific activities of cathepsins in whole homogenates, cell particles, or soluble fractions of muscle or liver were the same in guinea pigs given vitamin E-deficient diets as those on control diets. The proportion of total cathepsin activity in supernatant fractions (70,000g for 45 min) was not affected in animals given the deficient diet for 15 days. The proportion of total cathepsin activity in the supernatant fraction of liver was increased in animals given the deficient diet, compared with the supplemented diet, for 21 days. The sum of the activities found in the soluble plus particulate fractions of muscle from animals maintained on vitamin E-deficient diets for 21 days was greater than activity found in whole homogenates. An inhibitor of cathepsin activity may be present in muscle from the vitamin E-deficient animals.

Meprin is a membrane-bound metallo-proteinase that is present in high concentrations in the kidney brush border of mice and rats (Bond and Beynon, 1986). The enzyme has been purified and found to exist as a tetrameric glycoprotein that contains one mol zinc and three mol calcium per 85,000 molecular weight subunit (Beynon et al., 1981; Butler et al., 1987). Meprin hydrolyzes a variety of peptide and protein substrates (e.g., insulin B chain, glucagon, angiotensins I and II, bradykinin, hemoglobin, casein, aldolase, and phosphorylase kinase); the substrate used most often to assay activity is azocasein, a good general substrate for neutral and alkaline proteinases.

Meprin, an 85,000 molecular weight metalloendopeptidase is a major component of the kidney brush border membrane in mice. Some inbred mouse strains exhibit low levels of meprin activity. These strains were characterized by little, if any, protein in brush border preparations corresponding to the native enzyme. However, material exhibiting partial identity to meprin was identified by Ouchterlony immunodiffusion. Immunoblots of brush border proteins confirmed that this immunoreactive material was present but of higher molecular weight than the native enzyme. The implication of these data is that the structural gene for meprin is expressed, albeit incorrectly, in the low-meprin strains.

Meprin is an intrinsic protein of the brush border, a specialized plasma membrane, of the mouse kidney. It is a metalloendopeptidase that contains 1 mol of zinc and 3 mol of calcium per mol of the 85,000-Mr subunit. The enzyme is isolated, and active, as a tetramer. The behaviour of the enzyme on SDS/polyacrylamide gels in the presence and absence of beta-mercaptoethanol indicates that the subunits are of the same Mr (approx. 85,000) and held together by intersubunit S–S bridges. Eight S-carboxymethyl-L-cysteine residues were detected after reduction of the enzyme with beta-mercaptoethanol and carboxymethylation with iodoacetate. The enzyme is a glycoprotein and contains approx. 18% carbohydrate. Most of the carbohydrate is removed by endoglycosidase F, indicating that the sugar residues are N-linked. The isoelectric point of the enzyme is between pH 4 and 5, and the purified protein yields a pattern of evenly spaced bands in this range on isoelectric focusing. The peptide-bond specificity of the enzyme has been determined by using the oxidized B-chain of insulin as substrate. In all, 15 peptide degradation products were separated by h.p.l.c. and analysed for their amino acid content and N-terminal amino acid residue. The prevalent peptide-bond cleavages were between Gly20 and Glu21, Phe24 and Phe25 and between Phe25 and Tyr26. Other sites of cleavage were Leu6-Cysteic acid7, Ala14-Leu15, His10-Leu11, Leu17-Val18, Gly8-Ser9, Leu15-Tyr16, His5-Leu6. These results indicate that meprin has a preference for peptide bonds that are flanked by hydrophobic or neutral amino acid residues, but hydrolysis is not limited to these bonds. The ability of meprin to hydrolyse peptide bonds between small neutral and negatively charged amino acid residues distinguishes it from several other metalloendopeptidases.

An inherited deficiency of a metalloendopeptidase (meprin) activity occurs in kidneys of many inbred mouse strains. To clarify whether meprin protein is present in low-activity strains and determine the distribution of meprin in kidneys of mice with high- and low-meprin activities, kidney slices were stained through the use of the indirect immunoperoxidase technique and examined by light and electron microscopy. Light microscopy at high dilutions of anti-meprin IgG confirmed the brush border localization of meprin in high-meprin activity strains and revealed no detectable cross-reactive material in low-meprin activity strains. However, light and electron microscopy studies that use lower dilutions of anti-meprin immunoglobulin G (IgG) revealed cross-reactivity in low-activity strains, also at the luminal surface of the proximal tubules. Studies at lower magnifications indicated that meprin is primarily associated with the juxtamedullary region of the kidney in both high- and low-activity strains. Western blots of urinary proteins showed significant amounts of meprin-like proteins, but only in the urine of mice with high-meprin activity. The low activity of meprin in some inbred mouse strains is not associated with the presence of the protein in compartments of kidney cells other than the brush border or with secretion of the protein into the urine.

A cysteine metalloproteinase that degrades 125I-insulin B chain at neutral pH values was isolated from C3H mouse liver. The enzyme was partially purified from the 100,000g supernatant fraction by ammonium sulfate precipitation, DEAE-cellulose chromatography, and fast protein liquid chromatography. The molecular weight of the proteinase was estimated to be 190,000 by gel filtration on Sephadex G-200. Degradation of 125I-insulin B chain by the proteinase was inhibited by p-hydroxymercuribenzoate (PHMB) and iodoacetate (cysteine proteinase inhibitors) and by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (metalloproteinase inhibitors). The proteinase also degraded 125I-glucagon but did not hydrolyze 125I-insulin, leucine-2-naphthylamide, or several large proteins. Equivalent levels of EDTA- and PHMB-inhibitable 125I-insulin B chain-degrading activity were observed in the 100,000g supernatant fractions of brain, liver, lung, kidney, heart, and spleen from four mouse strains (C3H/HeN, CBA/J, ICR, and C57BL/6). High levels of 125I-insulin B chain-degrading activity were found in the particulate fraction of kidneys and lungs from these four mouse strains; these activities were inhibited by EDTA but not by PHMB. The activity of the soluble liver cysteine metalloproteinase was not altered in C3H mice treated ip with metal chelators, bacterial endotoxin, phenobarbital, dexamethasone, or insulin. Starvation for 24 or 48 hr and alloxan-induced diabetes diminished total activity of this enzyme in liver by about 50 and 30%, respectively. This soluble polypeptide-degrading enzyme appears to be ubiquitous in mice and to be regulated by nutritional conditions.

Cathepsin L was capable of destroying rabbit muscle aldolase ( d -fructose-1,6-bisphosphate d -glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) activity towards the substrate fructase 1,6-bisphosphate. The rate of loss of activity towards this substrate was stimulated (approx. 2-fold) by physiological concentrations of ATP and to a lesser degree by GTP, CTP, UTP, ADP and cyclic AMP, while PP i and P i decreased the rate of inactivation. Other proteinases (cathepsin B, cathepsin D, trypsin and chymotrypsin) also decreased aldolase activity toward fructose 1,6-bisphosphate more rapidly in the presence of ATP and more slowly in the presence of P i . Cathepsin L, at higher concentrations, was capable of inactivating aldolase activity towards fructose 1-phosphate and extensively degrading the enzyme; these reactions were not affected by ATP and P i . The thermostability of aldolase was also unaffected by these ligands. ATP and P i had no effect on the rates of hydrolysis of other proteins (hemoglobin, bovine serum albumin, casein and azocasein) by cathepsin L. These data indicate that the effects of ATP and P i was due to interactions of these ligands with aldolase that make the enzyme more vulnerable to limited but not extensive proteolysis; these ligands do not directly affect cathepsin L activity.

Arginase activity is elevated in livers of diabetic animals compared to controls and there is evidence that this is due in part to increased specific activity (activity/mg arginase protein). To investigate the molecular basis of this increased activity, the physicochemical and kinetic properties of hepatic arginase from diabetic and control mice were compared. Two types of arginase subunits with molecular weights of 35,000 and 38,000 were found in both the diabetic and control animals and the subunits in these animals had similar, multiple ionic forms. Kinetic parameters of purified preparations of arginase for arginine (apparent K m and V max values) and the thermal stability of these preparations from diabetics and controls were also similar. Furthermore, no difference was found in the distribution of arginase activity among different subcellular liver fractions. Separation of basic and acidic oligomeric forms of arginase by fast-protein liquid chromatography resulted in a slightly different distribution of activity among the forms in the normal and diabetic group. The apparent K m values for Mn 2+ of the basic form of the enzyme were 25 and 33 μ m for the enzyme from normal and diabetic animals, respectively; for acidic forms, for which two apparent K m values were measured, the values were 8 and 197 μ m for arginase from controls and 35 and 537 μ m from diabetics. These results indicate that in diabetes, while no marked changes in the physicochemical characteristics of arginase are obvious, some changes are found in the interaction of arginase with its cofactor Mn.

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.