Rat seminal vesicle FAD-dependent sulfhydryl oxidase. Biochemical characterization and molecular cloning of a member of the new sulfhydryl oxidase/quiescin Q6 gene family.

Rat FAD-dependent sulfhydryl oxidase was purified; partial sequencing indicated that it was homologous to human quiescin Q6. A cDNA (GenBank accession no. AF285078) was cloned from rat seminal vesicles, and active recombinant sulfhydryl oxidase was expressed in Chinese hamster ovary epithelial cells. This 2472-nucleotide cDNA has an open reading frame of 1710 base pairs, encoding a protein of 570 amino acids including a 32-amino acid leader sequence and two potential sites for N-glycosylation. One of them is used and the 64,000 M(r) purified protein was transformed to 61,000 by the action of endoglycosidase F. Northern blotting and reverse transcription-polymerase chain reaction analyses showed that there were small amounts of sulfhydryl oxidase in the rat testis, prostate, lung, heart, kidney, spleen, and liver, and that the gene was highly expressed in seminal vesicles and epididymis. Rat sulfhydryl oxidase cDNA corresponds to the human cell growth inhibiting factor cDNA, which could be a differently spliced form of quiescin Q6. Comparing sulfhydryl oxidase sequences with those of human quiescin Q6 and mammalian and Caenorhabditis elegans quiescin Q6-related genes established the existence of a new family of FAD-dependent sulfhydryl oxidase/quiescin Q6-related genes containing protein-disulfide isomerase-type thioredoxin and yeast ERV1 domains.

Interactions between ovine cathepsin L, cystatin C and alpha 2-macroglobulin. Potential role in the genital tract.

The specific inhibitor of cysteine proteinases, cystatin C, was purified from ram rete testis fluid and the conditioned medium of Sertoli cells. This molecule associated with sheep liver cathepsin L at one of the fastest rates ever described for a proteinase/inhibitor interaction (1.75 +/- 0.20 x 10(8) M-1.s-1). But the association rate constant for the interaction of cathepsin L with alpha 2-macroglobulin, a non-specific inhibitor of proteinases, was also extremely high (8.8 +/- 0.75 x 10(6) M-1.s-1). Cathepsin L complexed with alpha 2-macroglobulin was protected from inhibition by type 2 and type 3 cystatins. The data indicate that cystatin C is the most potent inhibitor of cathepsin L in mammalian male genital tract fluids, whereas alpha 2-macroglobulin may act as a terminal acceptor of this enzyme. These inhibitors could therefore inhibit the activated form of procathepsin L which may appear during the complex process of spermatozoa production and maturation in the testis and epididymis.

Evolution of grass-cutter (Thryonomys swinderianus) inflammation markers: comparison with rabbit (Oryctolagus cuniculus) and rat (Rattus norvegicus).

An inflammatory reaction was induced in grass-cutters (Thryonomys swinderianus) by injecting turpentine. The changes in the plasma haptoglobin, fibrinogen, alpha 2 macroglobulin and immunoglobulin G was followed for 23 days by immunonephelometry. The results were compared to rat and rabbit. They showed that (a) the inflammatory reaction is delayed in the grass-cutter compared to rats and rabbits; (b) the concentration of haptoglobin increases less than in rat and rabbit; (c) the fibrinogen concentration is very low in the grass-cutter, despite hypercoagulability of blood; (d) the changes in the plasma alpha 2-macroglobulin in the grass-cutter seems to be comparable to that of rabbit alpha 1 macroglobulin in amplitude and in its slow return to the initial concentration; and (e) fibrinogen and haptoglobin are suitable markers for grass-cutter inflammation monitoring.

Posttranslational folding of alpha 1-inhibitor 3. Evidence for a compaction process.

alpha 1-inhibitor 3 (alpha 1 I3) is a rodent-specific proteinase inhibitor of about 190 kDa belonging to the alpha 2-macroglobulin family. It consists of five globular domains, three of which are connected by disulfide bridges, and contains an intramolecular thiol ester which can react with attacking proteinases. To explore the folding of newly synthesized alpha 1 I3, we have used rat hepatocytes and pulsechase experiments. In one of the analyses, the radiolabeled protein was isolated from cell lysates by immunoprecipitation and its Asp-Pro bonds cleaved by treatment with formic acid. The size of the major fragment, as assessed by electrophoresis under nonreducing conditions, was found to increase from 100 to 150 kDa upon the chasing. This result, together with knowledge of the positions of the cleavage sites and the disulfide arrangement, indicates that one of the interdomain disulfide bonds is formed after the synthesis of the polypeptide. Analysis of the same material by limited proteolysis and by velocity centrifugation showed that the folded regions became larger and that the protein became more compact; the thiol ester was found to be formed after these conformational changes. These results suggest that the domains of alpha 1 I3 are only partially developed directly after the synthesis of the polypeptide and that they acquire their final structure as the protein condenses and the domains interact with one another.

A simple method for calibrating collagenase/pronase E ratio to optimize heart cell isolation.

A mixture of crude collagenase and non-specific proteases has been used to isolate guinea pig ventricular heart cells. Measurements of collagenase activity with Wünsch's substrate and protein content with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggest that collagenase enzymes do not play a major role in heart cell isolation. On the other hand, an important factor in heart digestion seems to consist of some fractions of the proteases present in crude collagenase. It is also noted that crude collagenases do not present any sensitivity to added calcium but because this ion is important to obtain isolated cells its role is discussed. According to our results, the SDS-PAGE method can be used to determine the appropriate enzyme concentrations to obtain calcium-tolerant myocytes. These myocytes have electrophysiological properties as reported in the literature.

Production of the cysteine proteinase inhibitor cystatin C by rat Sertoli cells.

The Sertoli cells of the rat testis produce cystatin C, a cysteine proteinase inhibitor. Primary culture of Sertoli cells secreted both unglycosylated and glycosylated forms of rat cystatin C. Despite the low concentration of cystatin C in rete testis fluid, equilibrium dissociation constants (Ki) for the interaction between cystatin C and lysosomal cathepsins indicate that this molecule could be involved in the local regulation of testicular cysteine proteinase activity which may be necessary for spermatogenesis and spermiogenesis.

Intracellular modifications of rat alpha 1 inhibitor3. Formation of disulphides, internal thiolester and sulphation.

alpha 1 Inhibitor3 (alpha 1I3) is a monomeric protease inhibitor of about 190 kDa which is secreted by rodent hepatocytes. We have studied intracellular modifications of this protein in [35]methionine-labelled rat hepatocytes by pulse/chase experiments followed by immunoprecipitation and gel electrophoresis under reducing and nonreducing conditions. Directly after the pulse, most of the unreduced alpha 1I3 migrated faster than the reduced form, indicating that disulphide bridges are formed during or shortly after synthesis yielding a compact structure. With increasing chase time however, an increasing portion of the unreduced alpha 1I3 migrated with a mobility lower than that of the reduced protein, half-maximal conversion occurring after about 10 min. This finding suggests that alpha 1I3 undergoes a conformational change in the endoplasmic reticulum, possibly becoming more elongated. During 10-30 min of chase, the protein acquired the capacity to undergo autolytic cleavage upon heating, a property due to the existence of an internal thiolester bond [Howard, J. B., Vermeulen, M. & Swenson, R. P. (1980) J. Biol. Chem. 255, 3820-3823; Esnard, F., Gutman, N., El Moujahed, A. & Gauthier, F. (1985) FEBS Lett. 182, 125-129]. Analysis by subcellular fractionation indicated that this bond is formed in the endoplasmic reticulum. Finally, we show that secreted alpha 1I3 is sulphated, presumably at Tyr618.

The cDNA structure and expression analysis of the genes for the cysteine proteinase inhibitor cystatin C and for beta 2-microglobulin in rat brain.

Tissue patterns of gene expression were analyzed by measuring mRNA levels and incorporation of radioactive amino acids for cystatin C and beta 2-microglobulin, the two extracellular proteins in the brain with the highest ratio of concentration in cerebrospinal fluid over that in blood plasma. The primary structure of rat cystatin C mRNA from choroid plexus was determined by nucleotide sequencing of cloned cDNA and the tissue patterns of gene expression were analysed by RNA blot analysis and in situ hybridization. Cystatin C was found to be composed of 120 amino acids and to contain a potential site for N-linked glycosylation. The tissue with the highest cystatin C mRNA level was the choroid plexus of the brain. Cystatin C mRNA was also detected in lower levels in other areas of the brain, testis, epididymis, seminal vesicles, prostate, ovary, submandibular gland, and, in trace amounts, in liver. Choroid plexus pieces in culture secreted radioactive cystatin C when incubated with radioactive leucine. Rat beta 2-microglobulin cDNA was cloned and identified by nucleotide sequencing and comparison of the obtained sequence with that of mouse and human beta 2-microglobulin cDNA. Tissue levels of beta 2-microglobulin mRNA in the rat were measured by hybridization to rat beta 2-microglobulin cDNA. The highest levels of beta 2-microglobulin mRNA were observed in liver and choroid plexus. Other parts of the brain and testis contained lower levels of beta 2-microglobulin mRNA.

Two rat homologues of human cystatin C.

Two immunochemically related forms of cystatin C-like inhibitors which differ in their Mr app and isoelectric point have been found both in urine and seminal vesicles of rats. Amino-terminal sequences of these two cystatins are identical within the same fluid and exhibit a high degree of homology with that of human cystatin C. However, cystatins C purified from urine lack eight residues at their amino-terminal end when compared to those of seminal vesicles. The occurrence of two cystatin C-like components in rat fluids has been found to be due to the presence of a glycosylated form reported here as cystatin Cg which specifically binds concanavalin A and is susceptible to endo-beta-N-acetylglucosaminidase treatment.

Purification of the cystatin C-like inhibitors from urine of nephropathic rats.

Two cysteine proteinase inhibitors of the cystatin C type have been purified from urine of sodium chromate-treated rats. Both strongly inhibit papain as well as rat liver cathepsin L (Ki less than 10(-11) M) whereas rat liver cathepsins B and H are inhibited to a lesser extent. They differ by their apparent molecular mass of 17 kDa and 22 kDa and by their isoelectric point greater than or equal to 9.5 and 7.7 respectively. These two molecules share complete immunochemical identity and are precipitated by antibodies directed against human cystatin C but not by anti rat thiostatin and anti rat H-kininogen antibodies. They are also found in large amounts in seminal vesicles where they represent most of the cysteine proteinase inhibitory capacity.

Cysteine-proteinase-inhibiting function of T kininogen and of its proteolytic fragments.

Previous attempts to liberate T kinin from T kininogen [Moreau et al. (1986) Eur. J. Biochem. 159, 341-346; Gutman et al. (1988) Eur. J. Biochem. 171, 577-582] have shown that complete fragmentation of the precursor molecule into inhibitory peptides was achieved before any vasoactive peptide was released, suggesting a possible physiological significance for this phenomenon. In this study, cysteine-proteinase-inhibiting properties of rat T kininogen and of its proteolytic fragments issuing from trypsin and submaxillary gland endopeptidase k hydrolysis, have been investigated using rat lysosomal cathepsins B, H and L, papain and bovine calpains I and II. All three lysosomal cathepsins were inhibited by T kininogen but tighter interactions were observed with cathepsin L and papain. Though higher Ki values were obtained for cathepsins B and H, rate constants for association were found to have high and almost similar values (in the 10(6) M-1 s-1 range) whatever the enzyme used. Proteolytic fragments also inhibited cathepsin L and papain very strongly and even better than the entire molecule for some of them, but no significant inhibition of cathepsins B and H was observed. Bovine calpains were not inhibited by T kininogen nor by its proteolytic fragments. From the results of this kinetic analysis, which indicates that both the association and the dissociation of lysosomal cysteine proteinases with T kininogen may occur rapidly, an hypothesis has been put forward on the possible in vivo functioning of T kininogen as a proteinase inhibitor.

Relationship between the cysteine-proteinase-inhibitory function of rat T kininogen and the release of immunoreactive kinin upon trypsin treatment.

The potential kininogenic function of rat T kininogen has been studied in parallel with the cysteine-proteinase-inhibitory function also carried by this molecule. Proteolytic cleavage of the molecule was observed upon incubation with catalytic amounts of trypsin. These conditions do not permit any significant release of immunoreactive kinin and do not modify the total papain-inhibiting capacity of T kininogen. As trypsin concentration increases in the reaction mixture, immunoreactive kinin is liberated and the total papain-inhibiting capacity decreases accordingly, as indicated by titration studies. This decrease, however, does not exceed 50% of the initial value even at a trypsin concentration as high as 75 microM, indicating that only one of the two inhibitory sites has been inactivated. The remaining inhibitory fragment corresponds to a peptide of apparent Mr 24 000, which binds papain at least as well as native T kininogen. T kininogen, therefore, appears as a potent proteinase inhibitor and/or a proteinase inhibitor precursor, whereas its kininogenic function remains questionable since no specific kininogenase able to release T kinin or another kinin under physiologically compatible conditions has been found so far.

[Nuclear proteins preferentially bound to the beta-globin gene: cellular specificity and variation during terminal differentiation of mouse erythroblasts]. / Protéines nucléaires se liant préférentiellement au gène beta-globine: spécificité cellulaire et variations au cours de la différenciation terminale des érythroblastes de souris.

Restriction fragments of the mouse beta major globin gene and of the long terminal repeat (LTR) DNA fragment of the mouse mammary tumor provirus as a control, were used to analyze the specificity of DNA-protein interactions in nuclear extracts of mouse erythroleukemia (MEL) cells and of other differentiated mouse cultured cell lines. After gel electrophoresis and transfer to nitrocellulose, DNA-binding proteins with a preferential affinity for the cloned beta-globin genomic sequence were characterized and related to the level of globin gene expression during induction of differentiating mouse erythroblasts. Two proteins (110 K and 75 K) appear in differentiated MEL cells while another one (100 K), for which we have localized the binding site on the beta-globin gene, is present only in immature MEL cells.

Rat plasma alpha 1-inhibitor3: a member of the alpha-macroglobulin family.

The overall mechanism of interaction with proteinases of alpha 1-inhibitor3, a plasma proteinase inhibitor so far specific to the rat, has been shown to be closely similar to that described for alpha-macroglobulins. This mechanism includes: (i) the cleavage of at least one susceptible peptidic bond which leads to structural changes in the molecule. (ii) The cleavage of a putative thiol ester bond in another site of the molecule which permits the covalent linkage of the enzyme. Moreover, fragmentation of alpha 1-inhibitor3 upon heating as observed for alpha-macroglobulin quarter subunits has been demonstrated. The question is raised of the presence of such a molecule in rat plasma in addition to two alpha-macroglobulin species, all of these proteinase inhibitors being antigenically unrelated.

An abnormal plasma antithrombin with no apparent affinity for heparin.

Congenital antithrombin abnormality was found in several members of a French family. No history of thrombotic episodes was associated with this abnormality. Plasma antithrombin concentration as well as the rate of thrombin inactivation by defibrinated plasma in the absence of heparin were normal. However, the heparin cofactor activity was decreased by about 50% in plasma of affected patients. Accordingly, about half the amount of plasma antithrombin did not bind to gel bound heparin. Moreover the crossed immunoelectrophoretic pattern in the presence of heparin demonstrated two peaks of antithrombin, the slower one migrating as normal antithrombin when heparin was omitted from the first agarose gel. It was concluded that molecular alteration of the antithrombin molecule seemed to affect only the heparin binding site thus preventing from any rate enhancement of thrombin inactivation.

Inhibition of human liver cathepsin L by alpha 2 cysteine-proteinase inhibitor and the low-Mr cysteine proteinase inhibitor from human serum.

The inhibition of human liver cathepsin L by two specific proteinase inhibitors present in human serum, namely alpha 2 cysteine-proteinase inhibitor and the low-Mr cysteine-proteinase inhibitor, was studied. Kinetic parameters, including inhibition constants (Ki) and rate constants for association and dissociation (k+1 and K-1), were determined. The values found are consistent with a possible physiological function of these inhibitors to control cathepsin L activity. Furthermore, a transfer of active proteinase from the complex with either cysteine-proteinase inhibitor species to alpha 2-macroglobulin was demonstrated in vitro. Given the rate of dissociation of both cathepsin-L-cysteine-proteinase inhibitor complexes, a function of transitory inhibitor can therefore be hypothesized for these proteins and might then provide an explanation of the clearance of lysosomal proteinases.

Protease inhibitor localization in control and streptozotocin-diabetic skeletal muscles.

alpha 1-Antitrypsin and alpha 1-inhibitor-3 were localized for the first time inside skeletal muscle cells. Their content, especially that of alpha 1-inhibitor-3, was greatly reduced following streptozotocin-induced diabetes. alpha 1-Antitrypsin and alpha 1-inhibitor-3 were also observed in the vascular components and interstitial space surrounding both control and diabetic soleus muscles as revealed by immunofluorescence. In diabetic muscles, the non-myofibre locale of alpha 1-inhibitor-3 was reduced, and to a lesser extent, alpha 1-antitrypsin. Both myofibre and extracellular patterns were reversed to control levels by insulin replacement.

Rat alpha 1-cysteine proteinase inhibitor. An acute phase reactant identical with alpha 1 acute phase globulin.

alpha 1-Cysteine proteinase inhibitor was isolated from normal and acute phase rat serum. The procedure, which includes successive fractionations on AcA 34, Cibacron blue Sepharose, DEAE-Sephacel, and hydroxyapatite, but avoids the use of an affinity chromatography step on a cysteine proteinase gel, led to the preparation of two electrophoretically distinct components. These resolved to a single band of apparent Mr = 68,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A slightly higher Mr was estimated from gel filtration studies, possibly related to the rather high carbohydrate content (15.25% of the dry weight). The purified protein exhibited strong inhibiting capacity towards papain with a Ki of 5 X 10(-11) M. As shown by immunochemical quantitation as well as functional activity, a 6-10-fold increase in the alpha 1-cysteine cysteine proteinase inhibitor content was recorded in inflammatory serum, thus demonstrating the protein to be a typical acute phase reactant. Its partial physicochemical characterization (Mr, isoelectric point, extinction coefficient, amino acid and carbohydrate compositions) shows large similarities with alpha 1 acute phase globulin whose biological function remains unknown, and which is present at the same concentration (about 0.5 g/liter) in normal serum. Identity between the two molecules has been further demonstrated by double immunodiffusion analysis since a continuous precipitating line was observed when purified alpha 1-cysteine proteinase inhibitor was reacted against anti-alpha 1 acute phase globulin and anti-alpha 1-cysteine cysteine proteinase inhibitor antibodies.

Comparison of immunochemical and biological properties of human anti thrombin during blood clotting and upon interaction with exogenous thrombin.

Modification of immunological and biological properties of human antithrombin were studied in plasma-serum pairs and in defibrinated plasma supplemented with human thrombin. Modified antithrombin obtained through whole-blood clotting or upon addition of exogenous thrombin appeared the same with regards to its electrophoretic or biological properties. However, amounts of thrombin higher than that physiologically available, had to be used to obtain a "serum-like" antithrombin in thrombin supplemented plasma suggesting different pathways for this transformation. This was in agreement with the observation in plasma of a modification of antithrombin antigenic properties upon thrombin addition whereas no difference was demonstrated when comparing serum to normal plasma. It may be concluded that the inactivation of antithrombin and the appearance of electrophoretically modified forms in normal serum is not mainly due to the formation of enzyme-inhibitor complexes and therefore that proteolytically modified, enzyme-free forms of antithrombin demonstrated in purified systems (Fish et al. 1979) could be of physiological relevance.