We investigated the effect of the extracellular protease of Serratia marcescens on human serum constituents such as immunoglobulins, fibronectin, alpha 1-protease inhibitor, alpha 2-macroglobulin, lysozyme, and transferrin. At a very low concentration of Serratia 56-kilodalton protease (56K protease), purified human plasma fibronectin was degraded rapidly into three structural domains or small fragments. Immunoglobulin G3 (IgG3) and IgA1 were also degraded within 30 min with 1 microgram of this protease per ml, more rapidly than their other subclass of IgG or IgA. alpha 1-Protease inhibitor, which did not inhibit the 56K protease, was degraded similarly by the protease. These events were demonstrated by fluorescence polarization and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protease was considerably inhibited by human alpha 2-macroglobulin and chicken ovomacroglobulin. However, when there was a 2 M excess of ovomacroglobulin or a 4 M excess of alpha 2-macroglobulin over the 56K protease, about 25 or 40% proteolytic activity remained, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protease degraded the alpha 2-macroglobulin extensively during prolonged incubation, which paralleled with regeneration of the protease activity. The protease also cleaved human lysozyme, although moderately. Human serum transferrin was degraded slightly, and human serum albumin was almost resistant to the 56K protease. The enzyme seemed to have no effect on reconstituted collagen, but it degraded rat tropocollagen and yielded fragments of beta and gamma chains by cleaving the intramolecular cross-links. Most of the above proteolysis by the 56K protease appears to result in a limited type of substrate specificity. Thus, the present study demonstrates that the protease is capable of degrading defense-oriented humoral proteins and tissue constituents. Furthermore, it is toxic to fibroblasts. These findings also clarified the possible role of Serratia protease as a virulence factor in the pathogenesis of serratial infections. We recently demonstrated this notion in vivo with rabbit cornea (R. Kamata et al., Ophthalmology 92:1452-1459, 1985).

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We previously proposed that the decreased rates of synthesis of collagen and proteoglycans in vitamin C-deficient guinea pigs were unrelated to the role of ascorbate in proline hydroxylation but might result from modulation of hormones known to change during fasting. In the present studies, we found that sera from guinea pigs on an ascorbate-free diet for 24-28 days or from those fasted for 4 days, with vitamin C supplementation, showed similar changes in the concentrations of several hormones. EGF and IGF-II concentrations were unchanged, but cortisol was increased 3-5 times and growth hormone was increased to approximately twice normal levels. Thyroxine and IGF-I concentrations were decreased to 40% and 25-33% of normal levels, respectively. The decrease in serum IGF-I must occur by a growth hormone-independent pathway. The extent of changes in hormone concentrations in sera from ascorbate-deficient guinea pigs was correlated with the extent of weight loss. Sera from scorbutic and fasted guinea pigs failed to stimulate DNA synthesis in quiescent BALB 3T3 cells in the presence of saturating concentrations of EGF and PDGF. Addition of experimental sera to normal serum showed that lack of mitogenic activity was due to the presence of an inhibitor. Inhibition was not related to IGF-I concentrations in the sera, although it was reversed by the addition of IGF-I to sera from scorbutic or fasted animals. These results support our proposed model and suggest that IGF-I, as well as an inhibitor of its activity, plays a role in the regulation of growth by vitamin C and other nutrients.

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Methods were developed for the radioisotopic assay of argininosuccinate synthetase [L-citrulline: L-aspartate ligase (AMP-forming), EC 6.3.4.5] and argininosuccinase [L-argininosuccinate arginine-lyase, EC 4.3.2.1]. The assay of argininosuccinate synthetase was based on the separation of [14C]argininosuccinate formed from aspartate and [carbamoyl-14C]citrulline in the presence of ATP from the substrate citrulline. For this, the product was converted to its anhydride form by boiling for 30 min at pH 2.0 followed by application on a column of Dowex 50W (pyridine form). Argininosuccinic anhydride was eluted with 0.3 M pyridine acetate buffer, pH 4.25, while citrulline was eluted with 0.1 M pyridine acetate buffer, pH 3.80. The assay of argininosuccinase was based on the separation of [14C]argininosuccinic acid formed from arginine and [U-14C]fumaric acid from the substrate fumarate on a column of Dowex 50W(H+ form). The argininosuccinic acid was adsorbed on the column and eluted with 1 M pyridine solution, while fumarate was not adsorbed. The distributions of these two enzymes in various organs and cell fractions were reinvestigated using these methods.

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During metamorphosis, the collagen layer (basement lamella) of the resorbing tail fin of bullfrog tadpole was markedly disintegrated, and was invaded by mesenchymal cells from below, which contained cytoplasmic vacuoles enclosing collagen fibrils. Participation of the locally produced collagenase in the process was discussed.By use of the tissue incubation method, collagenolytic activity was found in the wound tissues of the rabbit dorsal skin, and its significance in the wound healing process was discussed. Some of the repairing tissues after incubation were histologically examined.In the human stomach, high degree of collagenolytic activity was observed in the mucosa affected by gastritis after incubation, and there were several evidences that some kinds of cancerous tissues possessed a certain degree of the activity. The collagenolytic enzyme from the mucosa of gastritis was proved to have the characteristics of animal collagenase, previously obtained from tadpoles or other mammalian tissues. ACTA PATH. JAP. 24: 1-19, 1974. Characterization of the collagenolytic enzyme in the human stomachSmall pieces of the mucosa adjacent to ulcer of the stomach were cultivated a t 37°C for 0. USUKU r; .1.

Abstract. Changes in interstitial collagenase activity in the rat uterine cervix during ripening were clarified in a time-dependent manner. Premature delivery was induced by an antiprogesterone agent, RU486, for rats in late pregnancy. The presence of interstitial collagenase in the extract from the rat cervical tissue was demonstrated, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using the natural and unaffected collagen as a substrate. The collagenase activity was determined as the release of digested peptides from the radio-labeled collagen. Our experiments with RU486 were performed in rats on the 18th day of pregnancy. A single administration of RU486 (15 mg/kg) resulted in the premature delivery of all treated rats within 30 h after the injection (average time was 23.9 h). The marked increase in cervical wet weight was observed up to the time to premature delivery along with a significant acceleration from 18 h after the adminsitration of RU486. In this state, the cervical collagenase activity was enhanced, the highest levels being recorded at 21 h after the administration. The interstitial collagenase in the uterine cervix appears to play a significant role in the regulation mechanisms of cervical ripening in late pregnant rats.