Post-translational heterogeneity of the human vitamin D-binding protein (group-specific component)

The vitamin D-binding protein in human serum (the group-specific component) is an alpha 2-globulin which is genetically polymorphic in all populations studied. Previous work (J. Svasti and B. H. Bowman (1978) J. Biol. Chem. 253, 5188-5194, and J. Svasti, A. Kurosky, A. Bennett, and B. H. Bowman (1979) Biochemistry 18, 1611-1617) has shown that the electrophoretic variations of the proteins controlled by two allelic genes, Gc1 and Gc2, are due to at least three amino acid substitutions between Gc1 and Gc2 (Svasti et al. (1979] and to heterogeneity in the Gc1 phenotype arising from carbohydrate dissimilarities. Gc1 migrates electrophoretically as two protein bands, while Gc2 migrates cathodally as a single band. This study demonstrates a post-translational glycosylation difference occurring in a single area of the Gc1 sequence which accounts for the heterogeneity observed previously. The glycosylation site, a threonine residue, appears to be in a sequence which differs between Gc1 and Gc2. The O-glycosidic bond, which is typical of mucins, is rare in plasma proteins. The cyanogen bromide fragment containing the galactosamine-containing carbohydrate in Gc1 was partially sequenced through 20 residues from the amino terminus. No detectable galactosamine could be found in the homologous cyanogen bromide fragment in Gc2. A new purification procedure for the vitamin D-binding protein in human plasma has been developed. Three chromatographic steps provide purified protein.     

Kinetics of histone H10 accumulation and commitment to differentiation in murine erythroleukemia cells

The accumulation of histone H10 (also denoted IP 25) in murine erythroleukemia cells, induced to differentiate with hexamethylene bis-acetamide, was shown to precede by 15-20 h the appearance in the culture of cells irreversibly committed to differentiate. In addition the rates of accumulation of H10 and of committed cells vary in a similar manner with the HMBA concentration. Flow microfluorimetric analysis demonstrated that the accumulation of H10 did not occur simultaneously in all the cells. This accumulation of histone H10 was initiated first in cell in the G2 phase of the cell cycle and subsequently in the cells situated in all the phases of the cell cycle.     

Cyclic AMP, the hyperresponsiveness factor from hog kidney

The isolation and identification of a material present in the plasma of hypertensive dogs and hypertensive human patients has been under study since 1972. The earliest experiments in relation to this work, noted that plasma from hypertensive dogs cause a hyperresponse to norepinephrine when both were administered by way of the vein. Employing a rat assay system that consisted of an anesthetized rat with polyethylene catheters in the vein for giving norepinephrine and the test fractions and a catheter in the artery for blood pressure monitoring, fractions from hog kidney were tested for hyperresponsiveness activity. The active material is very comparable to cyclic AMP in molecular weight, ultraviolet spectrum, paper chromatography, Enzyme hydrolysis and activity in the anesthetized rat system. This evidence indicates that the hyperresponsiveness factor of renal origin is cyclic AMP.     

Determination of the heat resistance of spores using a solid heating block system

A simple and accurate technique for the determination of the heat resistance of spores is described. The technique combines a modified capillary tube method with a solid heating block. The come-up time of spore suspensions was found to be short and simple and accurate technique is suggested for the correction of the come-up times. Experimental results are presented for the destruction of spores of Bacillus stearothermophilus at 120 which indicates the accuracy and reproducibility of the new method.     

Endocytosis of aggregated immunoglobulin G by rat basophilic leukemia cells; rate, extent, and effects on the endocytosis of immunoglobulin E

Rat basophilic leukemia (RBL) cells have distinct receptors for IgE and IgG. We assessed the endocytosis of chemically and immunochemically cross-linked mouse-IgG and its influence on the simultaneous endocytosis of IgE. We found that at 37 degrees C, aggregates of IgG and IgE were endocytosed at about the same rate with one-half of the maximal endocytosis occurring in 5 to 13 min, and the efficiency of endocytosis for both ligands ranging from 40 to 70%. We also found that endocytosis of cross-linked IgE and IgG occurred simultaneously and neither ligand significantly affected the rate or extent of endocytosis of the other. The cells accumulated the cross-linked IgG, and then released it to the extracellular environment, at a rate (less than 3%/hr) slower than the released endocytosed IgE (greater than 10%/hr). Using an assay that discriminates between unbound and receptor-bound oligomeric IgG, we found that oligomeric IgG is endocytosed with its receptor, and that the bulk of the ligand remains bound to its receptor for greater than 120 min after endocytosis. The differences in the rate of release of endocytosed IgG vs IgE suggests that the intracellular fate or pathway of these two oligomeric ligands may differ.     

Acid proteinase activity in fish--I. Comparative study of extraction of cathepsins B and D from Mujil auratus

Acid catheptic activity was measured in crude extracts of muscle, liver, heart, spleen and gonads from the fishes Mujil auratus, Sparus aurata and Lightonatus mormyrus. The spleen was the organ which showed the highest activity. A comparative study of the seven most commonly used extraction methods was made. Some were modified to account for the characteristics of the fish organs and the activity extracted from them. The Siebert method resulted as the best extraction method only if 1 mM EDTA was present in the medium. The activity from Mujil auratus muscle was strongly inhibited by iodoacetate, N-ethylmaleimide, p-hydroxy mercuribenzoate, and diazo-acetyl-DL-norleucine methyl ester. The results indicated the presence of a carboxyl-proteinase and a thiol-proteinase. According to inhibition studies, the levels of proteinase and amidase activities shown by different organs of Mujil auratus were re-examined. The spleen extract showed the maximum activity for both cathepsins, but muscle extract accounted for more than 95% of total catheptic activity.