5′-Nucleotidases of retinal rod membranes

5′-Nucleotidase (EC 3.1.3.5) was solubilized from rod membranes with Ammonyx LO and purified by chromatographic methods. A highly sensitive radioassay was developed. The purified enzyme behaved as a homogeneous protein of 75,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as a protein of 79,000 in gel filtration. Thus, the enzyme does not contain subunits. The K_m values obtained were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin. Rabbit muscle G-actin formed a complex with the enzyme and inhibited its activity. The catalytic site of the enzyme was localized on the internal surface of the disk which, in terms of membrane sidedness, corresponds to the cell surface. A soluble 5′-nucleotidase was extracted from rod membranes with Tris buffer (pH 8.0) containing EGTA in the dark; less enzyme was extracted if the membranes had been exposed to light or incubated with Ca²⁺. The extracted enzyme was partially purified. The enzyme was unstable and lost 50% of its activity in 3 days at 3 °C. The K_m values were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by G-actin. A role for the soluble enzyme in the regulation of 5′-GMP in the rod outer segment was suggested.     

Chemical modification of coenzyme B12-dependent diol dehydrase with pyridoxal 5′-phosphate: Lysyl residue essential for interaction between two components of the enzyme

The apoenzyme of diol dehydrase was inactivated by modification with pyridoxal 5′-phosphate (pyridoxal-P). The inactivation was accompanied by appearance of a new peak at 425 nm which was shifted to 325 nm by reduction with NaBH₄. ?-N-Pyridoxyl lysine was detected by paper chromatography and paper electrophoresis from the hydrolysate of the NaBH₄-reduced enzyme-pyridoxal-P complex. The relationship of inactivation vs pyridoxal-P incorporation as well as kinetic experiments suggests that one lysyl residue per enzyme molecule was essential for catalytic activity, although two to three pyridoxal-P molecules were introduced into the almost completely inactivated enzyme molecule. Both 1,2-propanediol (substrate) and adenosylcobalamin (coenzyme) completely protected the enzyme from inactivation. The result of disc gel electrophoresis showed that the inactivation of diol dehydrase by pyridoxal-P results from irreversible dissociation of the enzyme into subunits upon pyridoxal-P modification. Therefore, it is suggested that this modifiable lysyl residue is essential for subunit interaction to form an active oligomeric enzyme. The inactivated enzyme restored activity by addition of excess component F, but not by S, suggesting that the essential lysyl residue is located in component F of the enzyme. Pyridoxal-P-modified enzyme was no longer able to bind cyanocobalamin (a competitive inhibitor of adenosylcobalamin).     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

An electron microscopic study on the mating tube formation in the heterobasidiomycete Tremella mesenterica

Ultrastructure of the mating tube formed in yeast haplont of the heterobasidiomycete Tremella mesenterica was studied by electron microscopy. Cell wall of the mating tube emerged as evagination of the inner layers, rupturing outer layers of the mother cell wall. Comparison with budding cells suggested that the tube emergence place at bud scar and the process of tube emergence was the same as that of bud emergence. Electron transparent vesicles of 0.1 m diameter were scattered in the cytoplasm of the mating tube. Nucleus-associated organelle was located at one side of the nuclear envelope which extended towards the mating tube. A few microtubules were detected in the mating tube, but their association with a nucleus was not clear. The cytoplasmic structure of the mating tube was discussed in comparison with that of hyphae of the filamentous fungi.     

Performance of fluidized-bed reactors utilizing magnetic fields

The performance of fluidized-bed reactors utilizing a magnetic field was determined by the use of magnetite-containing beads of immobilized unease. The reactors showed similar or higher conversions in comparison with fixed-bed reactors, although some aggregation of the beads in the magnetic field was observed. No effusion of the beads occurred up to a flow rate of 24 cm/min.     

Polynucleotides XLVII. Synthesis and properties of poly(2-methylthio- and 2-ethylthioadenylic acid). Formation of non-Watson-Crick type complexes.

Poly(2-methyl- and 2-ethylthioadenylic acid) were prepared by polymerization of corresponding diphosphates with Escherichia coli polynucleotide phosphorylase. These polynucleotides have relatively large hypochromicity of 30-35%. Acid titration of these polymers showed abrupt transition at pH 5.34-5.4, which may indicate that the introduction of alkylthio group at 2-position of adenine bases reduced their basicity. Thermal melting of these polymers showed no clear transition points at neutral pH, but in acidic media they have Tm values of 57 and 56 degrees C, somewhat lower than that of poly(A). Upon complex formation with poly(U), these poly(A) analogs showed only one poly(rs2A) . poly(U) type double-strand complexes, similar to that found in the case of poly(m2A) . poly(U).     

Application of photo-crosslinkable resin prepolymers to entrap microbial cells. Effects of increased cell-entrapping gel hydrophobicity on the hydrocortisone Δ1-Dehydrogenation

Summary Acetone-dried cells of Arthrobacter simplex, whose steroid ¹ activity had been previously induced, were entrapped by the use of photo-crosslinkable resin prepolymers. When the hydrophobicity of the cell-entrapping gel was increased by mixing a hydrophobic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polyethyleneglycol) (up to 30%), the hydrocortisone to prednisolone conversion rate of the immobilized cells increased significantly, attaining approximately 20% of that of the free cells. A 10% addition of organic solvents, such as methanol, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and to a lesser degree the reaction rate of the immobilized cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, stimulated the steroid conversion of the entrapped as well as the free cells. The stability of the entrapped cells over repeated reactions was improved by immobilization.     

Entrapment of microbial cells and organelles with hydrophilic urethane prepolymers

Summary Microbial cells and cellular organelles were immobilized by mixing aqueous suspensions of the biocatalysts with water-miscible urethane prepolymers. Thus immobilized preparations of acetone-dried cells of Arthrobacter simplex and thawed cells of Nocardia rhodocrous showed appreciable {ie351-1} activities in the transformation of hydrocortisone into prednisolone and 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione, respectively. The activities of catalase and alcohol oxidase were observed in the immobilized peroxisomes (microbodies) of a methanol-grown yeast Kloeckera sp. No. 2201. Yeast mitochondria entrapped with the prepolymer showed adenylate kinase activity. These results indicate the usefulness of the urethane prepolymers as convenient materials for entrapment of not only enzymes, but also organelles and microbial cells.     

Fatty acid beta-oxidation system in microbodies of n-alkane-grown Candida tropicalis.

Localization of fatty acid beta-oxidation system in microbodies of Candida tropicalis cells growing on n-alkanes was studied. Microbodies isolated from the yeast cells showed palmitate-dependent activities of NAD reduction, acetyl-CoA formation and oxygen consumption. When sodium azide, an inhibitor of catalase, was added to the system, palmitate-dependent formation of hydrogen peroxide was observed. Stoichiometric study revealed that two moles of NAD were reduced per one mole of oxygen consumed in the absence of sodium azide and the presence of the inhibitor doubled the oxygen consumption by microbodies without an appreciable change in NAD reduction. These results indicate that the yeast microbodies contain beta-oxidation system of fatty acid, and that catalase located in the organelles participates in the degradation of hydrogen peroxide to be formed at the step of dehydrogenation of acyl-CoA.