Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on human alpha-fetoprotein. Isolation and characterization of monomeric and polymeric forms and amino-terminal sequence analysis.

Human alpha-fetoprotein has been isolated from the serum and ascitic fluid of a patient with hepatoma by a combination of immunoadsorbent column chromatography and Sephadex G-150 gel filtration. Human alpha-fetoprotein is a sialylated glycoprotein with an estimated molecular weight of 67 500, composed of a single-chain polypeptide of approximately 580 amino acid residues and 3.6% carbohydrate. It is a negatively charged protein with an acid isoelectric point (pH 4.57). In addition to the monomeric form of alpha-fetoprotein, we have identified human alpha-fetoprotein polymers, including dimeric and trimeric forms, which dissociate to the monomer only upon exposure to disulfide-reducing reagents, implying that their formation is dependent upon intermolecular disulfide bonds. These polymers are found in human alpha-fetoprotein isolated by isoelectric focusing in both the major (pI 4.57) and minor (pI 5.2) alpha-fetoprotein fractions. The first 17 residues of the NH2-terminal amino acid sequence of the hepatoma-derived human alpha-fetoprotein have been identified. Fetal alpha-fetoprotein is indistinguishable from hepatoma alpha-fetoprotein by several criteria, including immunoelectrophoresis, acryalmide gel electrophoresis, and proclivity for dimerization.     

Identity of urinary trypsin inhibitor-binding protein to link protein

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, directly binds to some types of cells via cell-associated UTI-binding proteins (UTI-BPs). Here we report that the 40-kDa protein (UTI-BP(40)) was purified from the cultured human chondrosarcoma cell line HCS-2/8 by UTI affinity chromatography. Purified UTI-BP(40) was digested with trypsin, and the amino acid sequences of the peptide fragments were determined. The sequences of six tryptic fragments of UTI-BP(40) were identical to subsequences present in human link protein (LP). Authentic bovine LP and UTI-BP(40) displayed identical electrophoretic and chromatographic behavior. The UTI-binding properties of UTI-BP(40) and LP were indistinguishable. Direct binding and competition studies strongly demonstrated that the NH(2)-terminal fragment is the UTI-binding part of the LP molecule, that the COOH-terminal UTI fragment (HI-8) failed to bind the NH(2)-terminal subdomain of the LP molecule, and that LP and UTI-BP(40) exhibited significant hyaluronic acid binding. These results demonstrate that UTI-BP(40) is identical to LP and that the NH(2)-terminal domain of UTI is involved in the interaction with the NH(2)-terminal fragment of LP, which is bound to hyaluronic acid in the extracellular matrix.     

Demonstration of pancreatic secretory trypsin inhibitor in serum-free culture medium conditioned by the human pancreatic carcinoma cell line CAPAN-1

Serum-free culture medium conditioned by an established human pancreatic adenocarcinoma cell line, CAPAN-1, contains copious amounts of immunoreactivity due to pancreatic secretory trypsin inhibitor (PSTI) as demonstrated by radioimmunoassay. The immunoreactive substance was purified from the conditioned medium to apparent homogeneity by trypsin affinity and gel filtration chromatography with an overall recovery of 40%, and its primary structure was determined by Edman degradation. The immunoreactive substance is a peptide of 56 amino acid residues with a calculated molecular weight of 6,241. Its amino acid composition, primary structure, and inhibitory effect against trypsin are indistinguishable from those of human pancreatic juice PSTI, indicating that this substance is PSTI itself. This is the first direct demonstration that tumor cells secrete PSTI in vitro. When CAPAN-1 was inoculated into a nude mouse, it produced a tumor and the tumor synthesized human PSTI in vivo, as demonstrated by the fact that the tumor extract contained 99.0 +/- 26.2 ng of human PSTI/mg of protein, while PSTI was not detected in extracts from other tissues examined. Furthermore, high levels of human PSTI (14.3 +/- 2.6 ng/ml) were detected in the serum of tumor-bearing mice but not in that of nontumor-bearing mice, suggesting that PSTI secreted from the tumor appears in the blood circulation. Taken together, these results strongly support the view that the serum levels of PSTI are elevated in cancer-bearing patients due to secretion of this peptide from tumor cells per se.     

Purification of alpha 1-microglobulin produced by human hepatoma cell lines. Biochemical characterization and comparison with alpha 1-microglobulin synthesized by human hepatocytes

alpha 1-Microglobulin (alpha 1m) was determined by radio-immunoassay in the supernatants of five human hepatoma cell lines. High amounts of alpha 1m were produced by PLC/PRF/5, intermediate ones by Hep G2 and Hep 3B and very low ones by Malhavu and SK Hepl. alpha 1m isolated from hepatoma cell lines PLC/PRF/5 or Hep G2 supernatants displayed the same physicochemical properties as that purified from human urines: the apparent molecular mass was 26 kDa and the pI from 5.6 to 6.4 as measured after two-dimensional polyacrylamide gel electrophoresis in denaturating conditions; for the native molecule the pI was estimated to be 4.0-4.9. Both urinary and hepatoma alpha 1m migrate as a diffuse band in the alpha zone in agarose gel at pH 8.6 in non-denaturing conditions and present a brown chromophore covalently associated with the molecule. After biosynthetic labelling with [35S]methionine, proteins extracted from hepatoma cell line PLC/PRF/5 and from isolated hepatocytes of human liver were separated by two-dimensional PAGE and transferred to a nitrocellulose membrane. alpha 1m was identified and found to be identical in both cases. However, when compared with the alpha 1m isolated from cell supernatants, less charge heterogeneity but also minor additional spots of higher molecular mass were observed.     

Omentin, a novel adipocytokine inhibits TNF-induced vascular inflammation in human endothelial cells

Omentin is a recently identified adipocytokine with insulin-sensitizing effect. While lack of omentin may be related to the pathogenesis of obesity-related cardiovascular diseases, its effect in vasculature is largely unknown. We examined effects of omentin on vascular endothelial inflammatory states. Western blotting was performed to analyze inflammatory signal transduction in cultured vascular endothelia cells. The cyclic guanosine monophosphate (cGMP) content was measured using enzyme immunoassay. Treatment of human umbilical vein endothelial cells with omentin (300 ng/ml, 20 min) induced phosphorylation of 5′-AMP-activated protein kinase (AMPK) (Thr 172) and endothelial nitric oxide (NO) synthase (eNOS) (Ser 1177). Consistently, omentin increased the cGMP level. Pretreatment with omentin (300 ng/ml, 30 min) significantly inhibited the phosphorylation of JNK as well as expression of cyclooxygenase (COX)-2 by TNF-α (5 ng/ml, 20 min–24 h). An inhibitor of JNK significantly inhibited the TNF-α-induced COX-2 expression. Inhibitory effect of omentin on TNF-α-induced COX-2 was reversed by a NOS inhibitor. The present results demonstrate for the first time that omentin plays an anti-inflammatory role by preventing the TNF-α-induced COX-2 expression in vascular endothelial cells. Omentin inhibits COX-2 induction via preventing the JNK activation presumably through activation of AMPK/eNOS/NO pathways.     

Isolation and characterization of epidermal growth factor from human milk

Epidermal growth factor (EGF) has been purified from human milk. The purification was monitored with a human placental membrane radioreceptor assay using murine salivary epidermal growth factor I (mEGF I) as a competitive ligand and was achieved exclusively by the use of reverse-phase liquid chromatography (RPLC). The sequential use of preparative, semipreparative and analytical RPLC on an octylsilica support with solvent systems of different solute selectivity such as pyridine formate, triethylammonnium phosphate or perfluorocarbonic acids in the presence of n-propanol or acetonitrile allowed purification to homogeneity with 5 consecutive runs. The molecular mass, amino acid composition and NH2-terminal sequence of human EGF were determined. Gas-phase microsequencing of residues 1-17 revealed the following sequence: Asn-Ser-Asp-Ser-Glu-X-Pro-Leu-Ser-His-Asp-Gly-Tyr-X-Leu-X-Asp which is identical with the NH2-terminof urogastrone from human urine. The purified polypeptide competes with mEGF for the placental membrane receptor with a ki of 1 ng. Furthermore, it stimulates the anchorage-dependent as well as -independent proliferation of human and rat indicator cells with half-maximal stimulation at 1 and 2.5 ng/ml, respectively. Although human epidermal growth factor has been unequivocally identified in human milk and -for the first time-shown to be identical with urogastrone from human urine, the high-resolution techniques employed have also revealed the presence of EGF-related molecules which await further characterization. It is possible that EGF and the EGF-related growth factors possess important regulatory functions in normal growth of the human breast during pregnancy and lactation as well as in abnormal growth during mammary tumor formation and progression.     

Elastase inhibition by the inter-alpha-trypsin inhibitor and derived inhibitors of man and cattle

The inhibitory properties of HI-14 and BI-14, the active 14-kDa parts released from the corresponding human and bovine inter-alpha-trypsin inhibitors, are compared. The structurally homologous inhibitors composed of two tandem Kunitz-type domains differ in their inhibitory specificity, although the reactive site residue in position P1 is occupied by identical (arginine in the C-terminal domain II) or similar (methionine and leucine in the N-terminal domain I of HI-14 and BI-14, respectively) amino-acid residues. The N-terminal domain I of HI-14 is completely inactive against chymotrypsin and pancreatic elastase, whereas BI-14 is a strong inhibitor of these enzymes. Elastase from polymorphonuclear granulocytes interacts with both inhibitors but with different affinities. Compared with the bovine inhibitor, the human inhibitor shows a much lower affinity from this enzyme. Human ITI and its physiological 30-kDa derivative (HI-30) show the same inhibitory properties as HI-14. The differences between human and bovine inhibitors might be explained by a preceding oxidation of Met in vivo of the reactive site residue in position P1 and/or by the influence of the environmental parts connected with this antielastase reactive site region in human ITI or in the active domains thereof.     

Molecular cloning and sequencing of the cDNA for human membrane-bound carboxypeptidase M. Comparison with carboxypeptidases A, B, H, and N

Carboxypeptidase M, a widely distributed membrane-bound carboxypeptidase that can regulate peptide hormone activity, was purified to homogeneity from human placenta (Skidgel, R. A., Davis, R. M., and Tan, F. (1989) J. Biol. Chem. 264, 2236-2241). The NH2-terminal 31 amino acids were sequenced, and two complementary oligonucleotide probes were synthesized and used to isolate a carboxypeptidase M clone from a human placental cDNA library. Sequencing of the cDNA insert (2009 base pairs) revealed an open reading frame of 1317 base pairs coding for a protein of 439 residues. The NH2-terminal protein sequence matched the deduced amino acid sequence starting with residue 14. Hydropathic analysis revealed hydrophobic regions at the NH2 and COOH termini. The NH2-terminal 13 amino acids probably represent part of the signal peptide, and the COOH-terminal hydrophobic region may act either as a transmembrane anchor or as a signal for attachment to a phosphatidylinositol glycan moiety. The carboxypeptidase M sequence contains six potential Asn-linked glycosylation sites, consistent with its glycoprotein nature. The sequence of carboxypeptidase M was 41% identical with that of the active subunit of human plasma carboxypeptidase N, 41% identical with bovine carboxypeptidase H (carboxypeptidase E, enkephalin convertase), and 15% with either bovine pancreatic carboxypeptidase A or B. Many of the active site residues identified in carboxypeptidases A and B, including all of the zinc-binding residues (2 histidines and a glutamic acid), are conserved in carboxypeptidase M. These data indicate that all of the metallocarboxypeptidases are related, but the nondigestive carboxypeptidases with more specialized functions, present in cell membranes, blood plasma, or secretory granules (i.e., carboxypeptidase M, carboxypeptidase N and carboxypeptidase H), are more closely related to each other (41-49% identity) than they are to carboxypeptidase A or B (15-20% identity).     

cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta

Two nearly full-length cDNAs for placental plasminogen activator inhibitor (PAI) have been isolated from a human placenta lambda gt11 cDNA library. One positive (lambda PAI-75.1) expressed a protein that could adsorb and purify anti-PAI antibodies. The expressed protein inhibited the activity of human urokinase in a fibrin autography assay, and formed a 79-kDa (reduced) covalent complex with 125I-urokinase that could be immunoprecipitated with anti-PAI. The cDNA insert of the longer isolate (lambda PAI-75.15) consisted of 1909 base pairs, including a 5'-noncoding region of 55 base pairs, an open reading frame of 1245 base pairs, a stop codon, a 3'-noncoding region of 581 base pairs, and a poly(A) tail. The size of the mRNA was estimated to be 2.0 kilobases by Northern blot analysis. The translated amino acid sequence consisted of 415 amino acids, corresponding to a 46.6-kDa protein. The sequence was related to members of the serpin gene family, particularly ovalbumin and the chicken gene Y protein. Like these avian proteins, placental PAI appears to lack a cleavable NH2-terminal signal peptide. Residues 347-376 were identical to the partial sequence reported recently for a PAI isolated from the human monocytic U-937 cell line. Placental PAI mRNA was apparently expressed at low levels in human umbilical vein endothelial cells, but was not detectable in HepG2 hepatoma cells. It was present in U-937 cells and was inducible at least 10-fold by phorbol 12-myristate 13-acetate. Thus placental PAI is a unique member of the serpin gene family, distinct from endothelial-type PAI. It is probably identical to monocyte-macrophage PAI.     

Growth modulatory effects of a liver-derived growth inhibitor, transforming growth factor beta 1, and recombinant tumor necrosis factor alpha, in normal and neoplastic cells

The growth modulatory effects of a rat liver-derived growth inhibitor (LDGI), transforming growth factor beta 1 (TGF-beta 1), and recombinant tumor necrosis factor (rTNF-alpha) were examined in a variety of liver-derived and nonliver-derived normal and neoplastic cell culture systems. Normal rat liver epithelial (RLE) cells were highly sensitive to the growth inhibitory effects of LDGI (ID50 = 0.2 ng/ml) and TGF-beta 1 (ID50 = 0.25 ng/ml) but were less sensitive to rTNF-alpha (ID40 = 5000 Units/ml). Aflatoxin B1-transformed RLE cells showed sensitivity to the cytostatic effects of LDGI (ID50 = 1.5 ng/ml); however, these cells were completely resistant to the antiproliferative effects of TGF-beta 1 and rTNF-alpha. Clones isolated from these transformed cells, exhibited a wide range of sensitivities to LDGI but all of the clones were resistant to the growth inhibitory effects of both TGF-beta 1 and rTNF-alpha. Rat hepatoma Reuber cells were extremely sensitive to the antiproliferative effects of rTNF-alpha (ID50 = 10 Units/ml) but exhibited sensitivity to LDGI only at concentrations above 1.5 ng/ml and were resistant to the antiproliferative effects of TGF-beta 1. Rat hepatoma UVM 7777 cells and human hepatoma HepG2 cells, however, were insensitive to the growth inhibitory effects of all three factors. Among the nonliver-derived cells, human breast carcinoma (MCF-7) cells were extremely sensitive to rTNF-alpha (ID50 = 20 Units/ml, exhibited some sensitivity to LDGI (ID50 = 1 ng/ml), and were resistant to the antiproliferative effects of TGF-beta 1. In contrast, the rate of DNA synthesis is rat kidney fibroblasts and human foreskin fibroblasts was significantly stimulated in response to TGF-beta 1, LDGI, and rTNF-alpha. These data demonstrate that LDGI, TGF-beta 1, and rTNF-alpha exert positive and negative modulations of growth in different cell systems and that the growth regulatory effects of LDGI differ from those of TGF-beta 1 and rTNF-alpha in some cell types.     

Human endothelial cells selectively express large amounts of pancreatic-type ribonuclease (RNase 1)

Pyrimidine-specific ribonucleases are a superfamily of structurally related enzymes with distinct catalytic and biological properties. We used a combination of enzymatic and non-enzymatic assays to investigate the release of such enzymes by isolated cells in serum-free and serum-containing media. We found that human endothelial cells typically expressed large amounts of a pancreatic-type RNase that is related to, if not identical to, human pancreatic RNase. This enzyme exhibits pyrimidine-specific catalytic activity, with a marked preference for poly(C) substrate over poly(U) substrate. It was potently inhibited by placental RNase inhibitor, the selective pancreatic-type RNase inhibitor Inhibit-Ace, and a polyclonal antibody against human pancreatic RNase. The enzyme isolated from medium conditioned by immortalized umbilical vein endothelial cells (EA.hy926) possesses an amino-terminal sequence identical to that of pancreatic RNase, and shows molecular heterogeneity (molecular weights 18,000-26,000) due to different degrees of N-glycosylation. Endothelial cells from arteries, veins, and capillaries secreted up to 100 ng of this RNase daily per million cells, whereas levels were low or undetectable in media conditioned by other cell types examined. The corresponding messenger RNA was detected by RT-PCR in most cell types tested so far, and level of its expression was in keeping with the amounts of protein. The selective strong release of pancreatic-type RNase by endothelial cells suggests that it is endowed with non-digestive functions and involved in vascular homeostasis.     

Homologues of the human C and A apolipoproteins in the Macaca fascicularis (cynomolgus) monkey

We used antisera to human A and C apolipoproteins to identify homologues of these proteins among the high-density lipoprotein apoproteins of Macaca fascicularis (cynomolgus) monkeys, and NH2-terminal analysis was used to verify the homology. The NH2-terminal sequence of the M. fascicularis apoA-I is identical with that of another Old World species, Erythrocebus patas, and differs from human apoA-I at only 4 of the first 24 residues. M. fascicularis apoA-II contains a serine for cysteine replacement at position 6 and is therefore monomeric like the apoA-II from all species below apes. Human and monkey apoA-II are not otherwise different through their first 25 residues. About 20% of M. fascicularis apoC-I aligns with human apoC-I through residue 22, and 80% lacks an NH2-terminal dipeptide. Otherwise, the monkey apoC-I differs from the human protein at only 2 of 25 positions. Two forms of M. fascicularis apoC-II were identified. ApoC-II1 is highly homologous with human apoC-II, whereas an NH2-terminal hexapeptide is absent from apoC-II2. ApoC-II2 was the predominant species, and apoC-II1 appears to represent a propeptide from which a hexapeptide prosegment is cleaved at a Gln-Asp bond. Both forms of monkey apoC-II are potent activators of lipoprotein lipase. There are two polymorphic forms of M. fascicularis apoC-III, and their electrophoretic mobilities become identical after treatment with neuraminidase. Except for a glycine for serine substitution at position 10, the first 15 NH2-terminal residues of M. fascicularis and human apoC-III are the same.     

Demonstration of a new acrosin inhibitor in human seminal plasma

We have recently described the purification and characterization of a tumor-associated trypsin inhibitor (TATI). Studies on its N-terminal sequence suggested identity with the pancreatic secretory trypsin inhibitor (PSTI) (Huhtala, M.-L., Pesonen, K., Kalkkinen, N. & Stenman, U.-H. (1982) J. Biol. Chem. 257, 13713-13716). I report here the occurrence of a TATI-like activity in human seminal plasma. Concentrations of this inhibitor in seminal plasma varied considerably (4-500 ng/ml, n = 50). In radioimmunoassay the dose-response curves of the new seminal plasma inhibitor and purified TATI were parallel. The similarity between these two inhibitors was demonstrated by gel filtration, reverse phase liquid chromatography and ion-exchange chromatography. By ion exchange chromatography the new inhibitor could be separated from the main seminal plasma trypsin inhibitors. Purified TATI was shown to inhibit human acrosin effectively.     

Expression and excretion of human pancreatic secretory trypsin inhibitor in lipoprotein-deletion mutant of Escherichia coli

We constructed a gene coding for the 56-amino acid human pancreatic secretory trypsin inhibitor (PSTI), and ligated it on a plasmid downstream from the trp promoter and the signal peptide sequence of alkaline phosphatase. The resulting plasmid was transfected into a lipoprotein deletion mutant (Escherichia coli JE5505) and the plasmid-carrying cells were induced with 3-indoleacrylic acid. A considerable amount (50 micrograms/ml culture) of the mature PSTI protein was detected in the culture supernatant. The excreted PSTI was identical to the natural PSTI protein with respect to the trypsin-inhibiting activity, the N-terminal and the C-terminal amino acid sequences and the amino acid composition.     

The primary structure of the human pancreatic secretory trypsin inhibitor: Amino acid sequence of the reduced S-aminoethylated protein

The amino acid sequence of human pancreatic secretory trypsin inhibitor [Pubols, M. H., Bartelt, D. C., and Greene, L. J. (1974) J. Biol. Chem., 249, 2235–2242] was determined by a combination of selective trypsin and chymotrypsin hydrolysis reactions on the S-2-aminoethylcysteinyl inhibitor and conventional methods (subtractive Edman degradation and exopeptidase hydrolysis) for sequence determination of small peptides. The peptides were ordered on the basis of the identification of the amino- and carboxyterminal residues of the products at each stage of the degradation procedure. The sequence determination was carried out on a mixture of chromatographic forms present in both tissue and pancreatic juice which are identical in amino acid composition, aminoterminal residues, molecular weight, and specific activity, but differ only in asparagine content and susceptibility to enzymatic hydrolysis. The amino acid sequence of the human inhibitor corresponding to chromatographic form A₃ has been shown to be NH₂

.     

Characterization of human C4a anaphylatoxin

Human C4a anaphylatoxin was isolated from a Cls digest of the fourth component of complement. Isolation required a two-step procedure involving ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on Sephadex G-50. Characterization of C4a indicated it is a highly cationic polypeptide (pI = 9.0-9.5) containing 77 residues with Mr = 8,759. C4a is devoid of tryptophan, histidine, and carbohydrate. Judged by the shape and magnitude of its circular dichroism spectrum, 54% of the polypeptide backbone of C4a assumes an alpha-helical conformation. Partial NH2-terminal sequence determination of C4a revealed a sequence identical with that published by Bolotin et al. (Bolotin, C., Morris, S., Tack, B., and Prahl, J. (1977) Biochemistry 16, 2008-2015) for the NH2 terminus of the alpha-subunit of human C4. Comparison of the NH2-terminal sequence of C4a with the sequences of complement activation fragments C3a (Hugli, T.E. (1975) J. Biol. Chem. 250, 8293-8301) and C5a (Fernandez, H.N., and Hugli, T.E. (1978) J. Biol. Chem, 253-6955-6962) showed that of the first 24 NH2-terminal residues of C4a, 6 were identical with those of C3a (25% homology) and 8 were identical with those of C5a (33% homology). These data represent the first chemical evidence for the existence of an evolutionary relationship among anaphylatoxins C3a, C4a, and C5a, and imply that a similar relationship exists among their precursor proteins.     

Production of recombinant human pancreatic secretory trypsin inhibitor by Escherichia coli

A synthetic gene for human pancreatic secretory trypsin inhibitor (PSTI) was fused to the coding sequence for the amino-terminal 135 amino acid residues of human interferon-gamma (IFN-gamma) by interposing a methionine codon sequence, and the resulting hybrid gene was efficiently expressed in Escherichia coli cells. Recombinant human PSTI (rHu-PSTI) was separated from the IFN-gamma/PSTI fused protein by cleavage at the methionine residue with cyanogen bromide. Finally, rHu-PSTI was purified by affinity chromatography on a bovine trypsin-CH-Sepharose 4B column. The amino acid composition, partial amino-terminal sequence, disulfide formation, human trypsin inhibitory activity, and immunoreactivity against rabbit anti-human PSTI serum of rHu-PSTI corresponded to those of the natural form.     

Absence of trypsinogen autoactivation and immunolocalization of pancreatic secretory trypsin inhibitor in acinar cells in vitro

Summary To establish the significance of the addition of trypsin inhibitors to pancreatic acinar cells maintained in vitro, cells were cultured in the presence or absence of soybean trypsin inhibitor. Both cultures exhibited similar growth pattern, ultrastructural appearance, as well as secretory properties. Moreover, there was no evidence of trypsinogen activation in the culture medium. Using the immunocytochemical approach, pancreatic secretory trypsin inhibitor antigenic sites were revealed with specific polyclonal and monoclonal antibodies. The results obtained demonstrated that this trypsin inhibitor is in fact a typical pancreatic secretory protein being processed through the endoplasmic reticulum-Golgi-granule secretory pathway of the acinar cells in rat and human tissues. While the polyclonal antibody yield labelings of increasing intensities along the secretory pathway, the monoclonal one probably due to the molecular nature of its specific antigenic determinant, gave higher labelings in the endoplasmic reticulum. In conclusion the present study has shown that pancreatic acinar cells secrete a specific pancreatic trypsin inhibitor which most probably is involved in the mechanism to prevent trypsinogen activation.