Purification and characterization of protease from a newly isolated Rhizopus oryzae

A newly isolated Rhizopus oryzae was found to exhibit some unusual phenomenon of secreting alkaline protease which was purified and characterized. The molecular weight was determined to be 28,600 dalton in gel electrophoresis. The enzyme is stable in the pH range from 3 to 11 and most active at pH 8. The temperature optimum of this thermostable biocatalyst is at 60 °C. The enzyme is sensitive to metal chelators, most of the metal ions (excepting a few monovalent cations) and inhibitor like PMSF. This indicates that the protease of isolated Rhizopus oryzae falls under alkaline serine group.     

Effect of retinoic acid on adenosine diphosphate and collagen-induced alterations in enzymes of GSH-linked antioxidant defence system of human blood platelets in vitro

Collagen stimulation of blood platelets resulted in significant increases in malondialdehyde (MDA) formation and activity of glucose-6-phosphate dehydrogenase (G6PDH) and a decrease in catalase and glutathione peroxidase (GPx). Retinoic acid (RA) pretreatment did not show any appreciable changes except for a decrease in G6PDH activity as compared with collagen alone. RA pretreatment of human blood platelets resulted in an increase in the activities of catalase and GPx, two important radical scavenging enzymes, with significant decrease in MDA formation when compared with ADP alone. It is suggested that RA has a significant effect on the antioxidant defence system in ADP stimulated platelets but not in the collagen stimulated platelets.     

Differential effect of retinoic acid on ADP and collagen induced platelet aggregation

Retinoic acid (RA) was found to inhibit ADP induced but not collagen induced aggregation of human platelets and the differential action is related to intraplatelet Ca2+ reflux. RA was active at concentrations as low as 10(-7) M and required 20 min prior incubation with platelet suspension in order to inhibit aggregation by ADP. All the steps in ADP induced but not collagen induced platelet activation, viz. hydrolysis of phosphatidyl inositol, phosphorylation of 20, 47 and 250 kDa proteins as well as increased association of actin with Triton X-100 insoluble cytoskeletal matrix were inhibited by RA. RA when used as an agent for differentiation induction of cell progenitor is likely to affect the platelet aggregation and thereby the haemostatic process.     

Intracellular fate of fibrinogen B beta chain expressed in COS cells

Full-length fibrinogen B beta cDNA was subcloned into an expression vector, pBC12BI, and transfected into COS cells. B beta chain expression was measured by pulse-labelling cells with L-[35S]methionine, immunoprecipitating the B beta chain with antibody to fibrinogen and separating the nascent radioactive protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). B beta chain was expressed in transfected COS cells but was not secreted into the medium. Treatment with endoglycosidase H showed that non-secreted B beta chain contains mannose-rich carbohydrates rather than the complex form of carbohydrate which occurs in plasma fibrinogen and indicates that B beta chain is not transported to the Golgi apparatus. In transfected COS cells, antibody to fibrinogen co-immunoprecipitated B beta chain and 78 kDa immunoglobulin-binding protein (BiP) and antibody to BiP immunoprecipitated BiP and nascent B beta chains. Non-secreted B beta chain was degraded intracellularly with a half-life of 5 h by enzymes which were not affected by incubating transfected cells with NH4Cl, which indicates a non-lysosomal pathway of degradation. These studies indicate that B beta chain by itself does not contain the signal for fibrinogen secretion and that non-secreted B beta chain is associated with BiP and degraded in the rough endoplasmic reticulum.     

Microtubule associated proteins of goat brain

The microtubule associated proteins of goat brain were separated from tubulin on the basis of their thermostability and then fractionated by chromatography on Sepharose 4B column. Analysis of the fractions by SDS-Polyacrylamide gel electrophoresis and assay of their tubulin-assembly-promoting activity indicate that this activity resides primarily in the tauproteins (mol. wt. 55,000–70,000) and a class of even lower molecular weight (25,000–35,000) proteins. Electrophoresis of the microtubule associated protein fractions separated from tubulin by phosphocellulose chromatography are in agreement with the results obtained from fractionation on Sepharose 4B columns.     

The crystal and molecular structure of d1-17β-hydroxy-8α-androst-4-en-3-one (8-isotestosterone)

The molecular conformation of d1-8-isotestosterone has been determined crystallographically. Crystals of the title compound belong to the space group P2₁/c with a = 11.449(4), b = 10.962(4), c = 25.860(5) , β = 100.95(4)⁰, with two molecules in the asymmetric unit. The structure has been refined to a final R value of 0.052 for 2227 reflections. Unlike testosterone, which is a flat molecule, its 8-isomer has a folded conformation. The conformations of the ring-B in the two crystallographically independent molecules (A and B) correspond to the twist form and differ significantly from one another.     

New coumarins from the umbels of Seseli sibiricum

Three new coumarins, seselinal, sesibiricol and sibirinol, and 12 known coumarins have been isolated from the umbels of Seseli sibiricum. The new coumarins have been characterized as 5, 7-dimethoxy-8-(2-methyl-2-formylpropyl)-2H-1-benzopyran-2-one, 5-(3-methylbut-2-enyloxy)-7-methoxy-8-(2-hydroxy-3-methylbut-3-enyl)-2H-1-benzopyran-2-one and 5,7-dimethoxy-8-(2-hydroxy-3-methylbut-3-enyl)-2H-1-benzopyran-2-one,respectively. The known ones were identified as sesibiricin, isosibiricin, osthol, coumurrayin, sesebrin, sesebrinol, sibiricin, imperatorin, bergapten, xanthotoxin, isopimpinellin and mexoticin.     

Alpha- and beta-adrenergic receptors of the rat salivary gland. Elevation after chemical sympathectomy.

Binding of [3H]dihydroergokryptine and [3H]dihydroalprenolol to membrane preparations from rat submaxillary gland was measured to characterize the alpha- and beta-adrenergic receptors, respectively. Kinetic analysis of the data revealed a high affinity binding site for each radioligand. Inhibition of binding at each site was stereospecific for the active isomer of the catecholamine used. The greater ability of a beta1 than beta2 specific beta-adrenergic antagonist to displace [3H]dihydroalprenolol binding indicated that this binding site was of the beta1 type. Chemical sympathectomy with reserpine or 6-hydroxydopamine resulted in a significant increase in both [3H]dihydroalprenolol and [3H]dihydroergokryptine binding in the rat submaxillary gland. 3scatchard analysis of the data indicated that these increases in binding were due to a change in total number of binding sites for [3H]dihydroergokryptine and [3H]dihydroalprenolol with little change in apparent affinities. This suggests that changes in alpha- and beta-adrenergic receptor density may be important in the development of supersensitivity in salivary glands after reserpine and 6-hydroxydopamine treatment.     

Characterization of an adenosine triphosphatase from myeloblasts infected with the avian myeloblastosis virus.

An adenosine triphosphatase (ATPase EC 3.6.1.3) was partially purified from myeloblasts of chicken infected with the avian myeloblastosis virus and some of its molecular, catalytic and immunological properties were compared with that of the ATPase purified from the virus. Both the enzymes possessed almost same electrophoretic mobility, molecular weight, S20,w value, substrate specificity, metal-ion requirement, apparent Km value and sensitivity to inhibitors and activator. Evidence also indicated immunological identity of the two enzymes. The insensitivity of this enzyme to rutamycin or ouabain and extreme sensitivity to most of the detergents, trypsin and mercurials are the remarkable properties of this enzyme.