Dietary regulation of levels of active mRNA coding for amylase and serine protease zymogens in the rat pancreas

Translation products of a reticulocyte lysate reaction, programmed with poly(A)-rich RNAs from the male mouse submaxillary gland, were subjected to affinity chromatography on a tubulin-Sepharose column. Analysis of the bound proteins in sodium dodecylsulfate/polyacrylamide gels revealed two polypeptides of Mr 27 000 and 45 000, that were shown to bind to tubulin in a specific manner. These polypeptides were absent from the translation products coded by poly(A)-rich RNAs from the female mouse. They were eluted from the tubulin-Sepharose resin under conditions similar to those employed for the dissociation of immune complexes. The Mr-27 000 and Mr-45 000 proteins were identified by immunoprecipitation with specific antisera as the precursors of the gamma subunit of the nerve growth factor (NGF) and renin respectively. These two precursors as well as a third, unidentified polypeptide of Mr 38 000, probably unrelated to the beta subunit of NGF, bound also to microtubules. The mature form of renin, purified from the submaximillary gland, also displayed an affinity for the microtubules. In contrast, the mature form of the gamma subunit of NGF did not bind to the microtubules. The possible involvement of the microtubules (tubulin) in the biosynthesis of these two secretory proteins is discussed.     

Growth hormone induces two mRNA species of the serine protease inhibitor gene family in rat liver

In order to study the molecular actions of growth hormone on gene expression, we have cloned and characterized two unique, but related, cDNA sequences from rat liver, lambda Spi-1 and lambda Spi-2. These two cDNA sequences are complementary to rat hepatic mRNA species previously designated as Spots 3 and 20 when assayed by in vitro translation and two-dimensional gel electrophoresis. By Northern blot, the two mRNAs are both 1900 bases in length and growth hormone administered to hypophysectomized rats increases the levels of both of these mRNAs. In contrast, the combined administration of thyroxine, corticosterone, and dihydrotestosterone to hypophysectomized rats did not augment these mRNAs. The simultaneous administration of all four hormones resulted in a level greater than that observed for animals treated with growth hormone alone. Analysis of genomic DNA suggests the presence of two similar, but not identical, genes. DNA sequencing of lambda Spi-1 and lambda Spi-2 revealed that they were 90% homologous at the nucleotide level and 87% homologous at the amino acid sequence level. lambda Spi-2 has 78% homology with mouse contrapsin, 60% with human alpha 1-antichymotrypsin, and 51-55% with alpha 1-antitrypsins, all members of the serine protease inhibitor gene family. The nucleotide and deduced amino acid sequences of lambda Spi-1 and lambda Spi-2 which align with the reactive centers of known members of this family differ substantially from each other and from other members of the family. The difference in the reactive center suggests that the specificity or function of these proteins may differ from other members of serine protease inhibitor gene family.     

Ontogenesis of TRH mRNA in the rat pancreas

The rapid changes in TRH levels in the rat pancreas during the neonatal period make this organ an interesting model for the study of the regulation of TRH biosynthesis. Pancreatic RNAs were isolated by the guanidinium thiocyanate method and layered onto CsCl cushion. Northern blot preparations were hybridized with 32P labeled TRH cDNA probe. Pancreatic TRH mRNA was first detected in 19-day old fetuses and reached the highest level on day 0, then decreased, being barely detectable 14 days after birth. The neonatal injection of streptozotocin induced a dramatic drop of TRH mRNA levels 24 hours later. This result suggests that the peculiar evolution of TRH level in pancreas is partly due to the evolution of the expression of the TRH gene.     

Characterization of a membrane-bound arginine-specific serine protease from rat intestinal mucosa

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.     

Insights into the serine protease mechanism based on structural observations of the conversion of a peptidyl serine protease inhibitor to a substrate

BackgroundSerine proteases are one of the most studied group of enzymes. Despite the extensive mechanistic studies, some crucial details remain controversial, for example, how the cleaved product is released in the catalysis reaction. A cyclic peptidyl inhibitor (CSWRGLENHRMC, upain-1) of a serine protease, urokinase-type plasminogen activator (uPA), was found to become a slow substrate and cleaved slowly upon the replacement of single residue (W3A).MethodsBy taking advantage of the unique property of this peptide, we report the high-resolution structures of uPA in complex with upain-1-W3A peptide at four different pH values by X-ray crystallography.ResultsIn the structures obtained at low pH (pH 4.6 and 5.5), the cyclic peptide upain-1-W3A was found to be intact and remained in the active site of uPA. At 7.4, the scissile bond of the peptide was found cleaved, showing that the peptide became a uPA substrate. At pH 9.0, the C-terminal part of the substrate was no longer visible, and only the P1 residue occupying the S1 pocket was identified.ConclusionsThe analysis of these structures provides explanations why the upain-1-W3A is a slow substrate. In addition, we clearly identified the cleaved fragments of the peptide at both sides of the scissile bond in the active site of the enzyme, showing a slow release of the cleaved peptide.General significanceThis work indicates that the quick release of the cleaved P′ fragment after the first step of hydrolysis may not always be needed for the second hydrolysis.     

Evidence of a new serine protease in the rat pure pancreatic juice that degrades somatostatin

Somatostatin (SS) is found in the endocrine pancreas and has been reported in the pure pancreatic juice (PPJ) of different species. Characterization by gel filtration of immunoreactive SS (irSS) in the rat PPJ (rPPJ) results in a single peak corresponding to 23kDa molecular weight. Incubation of the 23kDa fraction with labeled or synthetic SS results in time dependent degradation of both peptides. This degradation is inhibitable by PMSF, calcium and by heat, whereas specific inhibitors of trypsin and chymotrypsin are without effect. These data suggest that irSS previously measured in rPPJ samples by RIA without confirmation of radioactive tracer stability may lead to false positive results. Indeed, our study indicates the presence of a 23kDa enzyme in the rPPJ degrading radiolabeled somatostatin during the RIA procedure. This putative new enzyme found into the rPPJ may thus be partially responsible for the apparent irSS presence.     

Fat-rich diet induces inflammatory changes in the intact rat pancreas

The effects of chronic ethanol ingestion combined with fat-rich, protein-rich or carbohydrate-rich diets on the histology of the intact rat pancreas were studied. 192 male Wistar rats were randomly divided into four different dietary groups. Half of each group received 15% (v/v) ethanol in their drinking solution and the rest were used as controls and given tap water. After a 12-week diet period the pancreas were removed and histological specimens were stained with hematoxylin and eosin. No significant difference was observed between the groups in occurrence of edema, but inflammatory cells were found in (9/24) rats in the fat-rich group receiving water (p less than 0.01. In the fat-rich diet group receiving ethanol this finding was observed in 5 of 24 rats. In these groups slight parenchymal cell necrosis was also observed in conjunction with the inflammatory cells. All specimens in the other groups were normal. It is suggested that inflammatory changes caused by a fat-rich diet may be due to unknown toxic effects of this diet.     

Involvement of a serine protease in tumour-necrosis-factor-mediated cytotoxicity

We investigated the effect of various protease inhibitors on the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells. 1. The following serine-type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethane, N alpha-p-tosyl-L-phenylalanyl chloromethane, N alpha-p-tosyl-L-arginine methyl ester, L-leucine methyl ester, DL-phenylalanine methyl ester, N-acetyl-DL-phenylalanine-beta-naphthyl ester, p-nitrophenyl p'-guanidino-benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF. 2. Inhibition of TNF-mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide. 3. TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1,3-3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with N alpha-p-tosyl-L-lysine chloromethane did not influence its biological activity. 4. The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage. 5. Protease inhibitors did not influence TNF binding, internalization or metabolization. 6. No increase in supernatant protease activity or in cell-associated protease activity could be detected after treatment of L929 cells with TNF. Our results document the involvement of protease activity, acting quite late during the cytolytic and growth inhibiting processes induced by TNF.     

A novel serine protease highly expressed in the pancreas is expressed in various kinds of cancer cells

We have isolated a cDNA that encodes a novel serine protease, prosemin, from human brain. The cDNA of human prosemin is 1306 bp, encoding 317 amino acids. It showed significant homology with the sequence of a chromosome 16 cosmid clone (accession no. NT_037887.4). The prosemin gene contains six exons and five introns. The amino acid sequence of prosemin shows significant homology to prostasin, gamma-tryptase, and testisin (43%, 41%, and 38% identity, respectively), the genes of which are also located on chromosome 16. Northern hybridization showed that prosemin is expressed predominantly in the pancreas and weakly in the prostate and cerebellum. However, western blot and RT-PCR analyses showed that prosemin is expressed and secreted from various kinds of cancer cells, such as glioma, pancreas, prostate, and ovarian cell lines. Prosemin is secreted in the cystic fluid of clinical ovarian cancers. Furthermore, immunohistochemistry showed prosemin protein localized in the apical parts of ovarian carcinomas. Recombinant prosemin was expressed in COS cells and was purified by immunoaffinity chromatography. Recombinant prosemin preferentially cleaved benzyloxycarbonyl (Z)-His-Glu-Lys-methylcoumaryl amidide (MCA) and t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA. Our results suggest that prosemin is a novel serine protease of the chromosome 16 cluster that is highly expressed in the pancreas. The usefulness of this serine protease as a candidate tumor marker should be further examined.     

Crystallization and amino acid composition of a serine protease from rat skeletal muscle

A serine protease from rat skeletal muscle was crystallized in good yield, and the homogeneity of the preparation was proved by ultracentrifugical analysis and polyacrylamide disc electrophoresis. The S_{20, w} value of the enzyme was 2.2 S and the molecular weight was calculated to be 22,000–24,000 from the results of sedimentation equilibrium analysis. Analysis showed 87% coincidence in the amino acid composition with that of a serine protease from the small intestine. The apparent Km and k_cat(sec^?1) values for N-acetyl-L-tyrosine ethyl ester were 1.1 × 10^?3 M and 9.0, respectively.     

Nitrogen excretion in rats on a protein-free diet and during starvation

Nitrogen balances (six days) were determined in male Wistar rats during feeding a diet with sufficient protein or a nearly protein-free diet (n = 2 x 24), and then during three days of starvation (n = 2 x 12). The objective was to evaluate the effect of protein withdrawal on minimum nitrogen excretion in urine (UN), corresponding to endogenous UN, during feeding and subsequent starvation periods. The rats fed the protein free-diet had almost the same excretion of urinary N during feeding and starvation (165 and 157 mg/kg W(0.75)), while it was 444 mg/kg W(0.75) in rats previously fed with protein, demonstrating a major influence of protein content in a diet on N excretion during starvation. Consequently, the impact of former protein supply on N losses during starvation ought to be considered when evaluating minimum N requirement necessary to sustain life.     

Effects of protein-free diet in amino acid homeostasis of rat blood plasma and gut contents

Amino acids in rat systemic and portal vein plasma and jejunal and ileal gut contents after 7 days of feeding normoprotein (NP) and protein-free diet (PF) are investigated. Amino acid analyses revealed that ingestion of PF diet resulted in unusual amino acid patterns in both plasmas. Thus, while the levels and/or molar ratios of all indispensable amino acids were significantly decreased, those of several gluconeogenetic amino acids, especially of glycine and alanine, were increased in both plasmas, but particularly in portal. By contrast, the molar ratios of the majority of amino acids in jejunal and ileal contents were not changed by PF diet.