The heterogeneity of prorennin was studied by chromatography on DEAE-cellulose and microgranular DEAE-cellulose columns, as well as by polyacrylamide-gel electrophoresis. Prorennin prepared by alum treatment, salting-out and chromatography was resolved into three components by a compound gradient of sodium phosphate on microgranular DEAE-cellulose. Polyacrylamide-gel electrophoresis confirmed the chromatographic results, but crystalline rennin was shown to consist of four bands. When prorennin was isolated directly by chromatography, four zymogen components were resolved on microgranular DEAE-cellulose with a modified compound gradient of sodium phosphate. Polyacrylamide-gel electrophoresis confirmed the existence of four multiple forms of prorennin as well as homogeneity of the chromatographic fractions.
Activation of the four separate components of prochymosin (prorennin) at pH 5.0 demonstrated that each zymogen was the precursor to an electrophoretically distinct chymosin (rennin). When the increase in milk-clotting activity with time was analysed, the mechanism of activation of unfractionated prochymosin, individual prochymosin components, and a mixture of the prochymosin fractions at pH 5.0 was shown to follow essentially autocatalytic kinetics. The activation of prochymosin C was completed in 70 h, whereas the other three fractions each required more than 110 h for complete activation under the same conditions. Intact prochymosin, the mixture of four components and prochymosin C were activated at similar rates. Interaction of the individual fractions during activation is suggested to explain the increased rate of the activation for the mixture. Comparison of autocatalytic activation of unfractionated prochymosin purified chromatographically at pH 6.7 and 5.7 demonstrated an increased rate of reaction of the zymogen prepared at the lower pH value. The possibility that prochymosin became susceptible to activation during preparation at pH values slightly below 6.0, as a result of changes in the proportion of the components or a conformational change and exposure of the active site, is discussed.
Binding of 63Ni(Il) to ultrafiltrable constituents of rabbit serum was studied (a) after in vitro incubation (2 h, 37 °C) of rabbit serum with 63NiCl2 (10-100 µmol/liter), and (b) at intervals (0.25-2 h) after in vivo administration of 63NiCl2 (40-160 µmol/kg body wt, i.v.). Serum ultrafiltrates were fractionated by thin-layer chromatography, and the separated compounds made visible by autoradiography and by ninhydrin staining. Several (≃5) ultrafiltrable 63Ni-complexes were demonstrable as distinct radiodense 63Ni-bands with chromatographic mobilities corresponding to those of ninhydrin-positive bands. Unbound 63Ni(II) was not detected in serum ultrafiltrates in either the in vitro or in vivo experiments. In sera (n = 10) incubated in vitro with 63Ni(II) (10 µmol/ liter), the mean percentage of ultrafiltrable 63Ni was 36% (range = 33-38) of total serum 63Ni. In contrast, in sera (n = 10) obtained 2 h after i.v. injection of 63Ni(II) (40 µmol/kg), the mean concentration of total serum 63Ni was 10.8 µmol/liter (range = 6-14), and the mean percentage of ultrafiltrable 63Ni was 15% (range = 9-21) of total serum 63Ni. The disparity between the percentages of ultrafiltrable 63Ni obtained in vitro and in vivo was obviated when the in vivo experiments were performed in rabbits bilaterally nephrectomized, with ligated common bile ducts. This investigation confirms the existence of several nickel receptors in serum ultrafiltrates and substantiates the role of ultrafiltrable complexes in the excretion of nickel.
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