Katsunori Shibata

Purification and immunological determination of α2-macroglobulin in serum from injured rats

Abstract Rat α 2 -macroglobulin was isolated and purified from the pooled sera of turpentine-injected rats by sequential use of dextran sulphate, DEAE-cellulose, and gel filtration chromatography. The final protein product obtained by this procedure proved to be α 2 -macroglobulin of a high degree of purity based on electrophoretic, immunologic and centrifugal analysis. The α 2 -macroglobulin preparation also binds stoichiometrically to trypsin preventing subsequent inhibition by protein trypsin inhibitors. SDS-polyacrylamide gel electrophoresis of rat α 2 -macroglobulin after incubation with trypsin suggested that there are at least two susceptible peptide bonds in the 170 000-dalton α 2 -macroglobulin subunit. The concentration of α 2 -macroglobulin in the sera of rats was measured by electroimmuno assay using a monospecific antiserum against α 2 -macroglobulin. Purified α 2 -macroglobulin was used as a standard. Sera from normal male rats contained 32 ± 4 μ g of α 2 -macroglobulin per ml. To determine the time course of response of α 2 -macroglobulin to inflammation, rats were subjected to either laparotomy or subcutaneous injection of turpentine. After either type of injury, the concentration of α 2 -macroglobulin increased rapidly, reaching a maximum value of 110–140 times that of the control value by 24 h. Little difference was noted in responsiveness between the two sexes.

Rat α1-acid glycoprotein Purification and immunological estimation of its serum concentration

A rapid method is described for the preparation of serum alpha1-acid glycoprotein from rats with inflammation induced with turpentine oil injection. The protein obtained by two purification steps, batchwise adsorption with DEAE-cellulose followed by chromatography on CM-cellulose, was proved to be native alpha1-acid glycoprotein in a high degree of purity by electrophoretical, immunological, ultracentrifugal and carbohydrate analysis. The monospecific and potent antiserum to this protein was prepared by immunizing rabbits with the desialyzed material emulsified with Freunds incomplete adjuvant. Using purified alpha1-acid glycoprotein and its specific antiserum, the concentration of alpha1-acid glycoprotein in rat serum was determined by single radial immunodiffusion. Abnormally high levels of its concentration (5-6 times higher than the control) were observed in inflammatory and tumor bearing rats.

Photochromism of single crystals composed of dioxazolylethene and dithiazolylethene

Although dioxazolylethene 1a did not show any photocoloration in the single crystalline phase, 1a displayed photochromism in the mixed crystal containing both 1a and dithiazolylethene 2a ; the mixed crystal changed color from colorless to red, orange, and yellow upon irradiation with light of appropriate wavelengths.

Distribution of α2-macroglobulin in normal, inflammatory, and tumor tissues in rats

Distribution of uptake and catabolism of intravenously administered125l-labeled rat α2-macroglobulin(125I-α2MG) were examined in normal, inflammatory, and tumor tissues of rats. Clearance of intravenously administered [125l]α2MG from the circulation was rapid. Accumulation of this compound into inflammatory tissue was 2–3 times more extensive than in normal tissues. The accumulation into sarcoma tissue was much less. Radioactivity in TCA-PTA precipitates remained fairly constant for the first 12 h in inflammatory tissue and for the first 24 h in sarcoma. These patterns of accumulation were never observed in the normal tissues. As the kidney preferentially accumulated large amounts of [125l]α2MG in the nondegraded form and its degradation products, the tissue may play a special role in the metabolism of α2MG. Rapid clearance from the circulation and relatively small amounts of accumulation in tissues suggest that α2MG may function as a protease inhibitor, mainly in the circulation rather than in the tissues.

Developmental Studies on Glucosamine Metabolism

Summary Changes in hepatic hexosamine metabolism and serum seromucoid concentrations during postnatal development were investigated in the rat. A relatively low activity of hepatic L-glutamine: D-fructose-6-phosphate aminotransferase (AT) was observed within 24 hr of birth. This rapidly increased to a maximum at about 2 weeks of age, followed by a decline to adult levels after another 2 weeks. The developmental pattern of hepatic UDP-N-acetylglucosamine 2′-epimerase (EP) closely resembled that of AT. It was relatively low in the newborn, increased to a maximum at about 2 weeks and then declined to adult values after another 2 weeks. The serum seromucoid concentration was low in new born rats and then increased gradually with development to adult levels at about 50 days. The hepatic concentrations of UDP-GlcNAc and CMP-NANA both were relatively high at 24 hr after birth compared to values observed at 50 days. The developmental pattern observed for both over this time period was quite similar and was almost the reciprocal to that for the seromucoid fraction. After 50 days the concentration of UDP-GlcNAc remained constant at adult levels as did that of the seromucoid fraction, but the concentration of CMP-NANA showed a marked increase. It is suggested that the hepatic concentrations of these amino-sugar nucleotides during development primarily reflect the demand for the hepatic synthesis and secretion of plasma glycoproteins as reflected in the seromucoid fraction. The authors wish to thank Dr. A. M. Chandler, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma, for his assistance in preparation of this manuscript. The authors also express their thanks to Dr. Kazunori Kawamura, The Second Department of Medicine, Miyazaki Medical College, for his advice.

Type-Dependent Difference in the Metabolism of Human Haptoglobins

SummaryMetabolic properties of haptoglobin 1-1 and 2-2 proteins were investigated by performing turnover studies in normal rabbits in a steady state concerning haptoglobin metabolism. Studies in six rabbits showed that the average plasma half-lives of iodinated haptoglobin 1-1 and 2-2 were similar with values of 2.3 and 2.4 days, respectively. The distribution in the body compartments, however, was significantly different at the equilibrium time and was such that the extravascular compartments contained 57.0% of the total body pool of haptoglobin 1-1, compared only 36.9% in the case of haptoglobin 2-2. These findings suggest that the former protein is higher than the latter in capillary permeability.These phenomena may be attributable to the difference in the molecular size and/or polymeric structure rather than that in the primary structure between two haptoglobins. The present results would reflex the survival of human haptoglobins in normal man, although the results were obtained by tracing human haptoglobins injected in rabbits. Finally, the biological significance of the type-dependent metabolism of haptoglobins was discussed.