Retarded nuclear migration in Drosophila embryos with aberrant F-actin reorganization caused by maternal mutations and by cytochalasin treatment

Three X-linked mutations of Drosophila melanogaster, gs(1)N26, gs(1)N441 and paralog, had a common maternal-effect phenotype. Mutant embryos show reduced egg contraction that normally occurs at an early cleavage stage in wild-type embryos. In addition, the mutants exhibited retarded nuclear migration while synchronous nuclear divisions were unaffected. The retarded migration causes nuclei to remain in the anterior part of the embryo retaining their spherical distribution even in a late cleavage stage. This consequently results in an extreme delay in nuclear arrival in the posterior periplasm. A mutant phenocopy was induced in wild-type embryos that were treated with cytochalasin B or D at a very early cleavage stage. Remarkable differences were noticed in the organization of cortical F-actin between the mutants and the wild type throughout the cleavage stage: obvious F-actin aggregates were dispersed in the cortex of mutant embryos, in contrast to the wild type where the cortical F-actin layer was smooth and underlying F-actin aggregates were smaller than those in the mutants; the transition of the distribution pattern of F-actin in the yolk mass, from the centralized to the fragmented type, occurred later in the mutants than in wild type. The results suggest that these mutations affect the mechanism underlying establishment and transition of F-actin organization required for normal egg contraction and nuclear migration in the cleavage embryos.     

Further fractionation of the glycoprotein families of porcine zona pellucida by anion-exchange HPLC and some characterization of the separated fractions

The glycoproteins of porcine zonae pellucidae have been fractionated into three families (PZP1-3) by gel filtration HPLC [Nakano et al. (1987) Biochem. Int. 14, 417-423]. However, they still comprise heterogeneous molecular species differing in electric charge. We found that sulfate, but not phosphate, is contained in PZP1-3 by a simple and rapid method for microanalysis of the anionic groups. These families were efficiently separated into many fractions by anion-exchange HPLC. When elution was performed by stepwise increase in NaCl concentration in 8 M urea/20 mM Tris-HCl, pH 8.0, a single distinctive peak emerged for each step. The analyses of amino acids, monosaccharides, and anions of the eight separated fractions of the major family, PZP3, showed that larger amounts of sulfated lactosamine linked to the constituent proteins are present in the fractions that are eluted later: the chain length and/or the chain number of these polylactosamines and the sulfate content increased with stepwise increase in NaCl concentration. Composition analyses also revealed that twice as much N-glycolylneuraminic acid is present as N-acetylneuraminic acid in all fractions. The contents of these sialic acids in the fractions slightly increased in the order of elution. These results together with those of the analyses of endo-beta-galactosidase digests showed that the charge heterogeneity of the porcine zona proteins is due mainly to differences in the amount of sulfated lactosamine, which is predominantly distributed in the non-reducing regions of the sugar chains.     

Purification to homogeneity and properties of glucosidase II from mung bean seedlings and suspension-cultured soybean cells

Glucosidase II was purified approximately 1700-fold to homogeneity from Triton X-100 extracts of mung bean microsomes. A single band with a molecular mass of 110 kDa was seen on sodium dodecyl sulfate gels. This band was susceptible to digestion by endoglucosaminidase H or peptide glycosidase F, and the change in mobility of the treated protein indicated the loss of one or two oligosaccharide chains. By gel filtration, the native enzyme was estimated to have a molecular mass of about 220 kDa, suggesting it was composed of two identical subunits. Glucosidase II showed a broad pH optima between 6.8 and 7.5 with reasonable activity even at 8.5, but there was almost no activity below pH 6.0. The purified enzyme could use p-nitrophenyl-alpha-D-glucopyranoside as a substrate but was also active with a number of glucose-containing high-mannose oligosaccharides. Glc2Man9GlcNAc was the best substrate while activity was significantly reduced when several mannose residues were removed, i.e. Glc2Man7-GlcNAc. The rate of activity was lowest with Glc1Man9GlcNAc, demonstrating that the innermost glucose is released the slowest. Evidence that the enzyme is specific for alpha 1,3-glucosidic linkages is shown by the fact that its activity on Glc2Man9GlcNAc was inhibited by nigerose, an alpha 1,3-linked glucose disaccharide, but not by alpha 1,2 (kojibiose)-, alpha 1,4(maltose)-, or alpha 1,6 (isomaltose)-linked glucose disaccharides. Glucosidase II was strongly inhibited by the glucosidase processing inhibitors deoxynojirimycin and 2,6-dideoxy-2,6-imino-7-O-(beta-D- glucopyranosyl)-D-glycero-L-guloheptitol, but less strongly by castanospermine and not at all by australine. Polyclonal antibodies prepared against the mung bean glucosidase II reacted with a 95-kDa protein from suspension-cultured soybean cells that also showed glucosidase II activity. Soybean cells were labeled with either [2-3H]mannose or [6-3H]galactose, and the glucosidase II was isolated by immunoprecipitation. Essentially all of the radioactive mannose was released from the protein by treatment with endoglucosaminidase H. The labeled oligosaccharide(s) released by endoglucosaminidase H was isolated and characterized by gel filtration and by treatment with various enzymes. The major oligosaccharide chain on the soybean glucosidase II appeared to be a Man9(GlcNAc)2 with small amounts of Glc1Man9(GlcNAc)2.     

Potato and rabbit muscle phosphorylases: comparative studies on the structure,function and regulation of regulatory and nonregulatory enzymes

Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.     

Stimulative effect of serum on the growth of HeLa cells suppressed in a K+-deficient chemically defined medium

Growth of HeLa cells cultured with a chemically defined medium was slightly stimulated in the presence of 5% dialyzed calf serum. The growth-promoting action of serum was more conspicuous when cell growth was suppressed in the same medium, in which K⁺ was replaced by Rb⁺ to various ratios. The growth-promoting factors(s) of serum was heat-labile. Upon addition of dialyzed serum, passive K⁺ or Rb⁺ influx was increased, whereas the active cation uptake was unaffected and cell K⁺ was rather decreased. The serum did not alter uptake of [³H] amino acids. Also, protein synthesis inhibited in the Rb⁺-substituted medium was not stimulated significantly, except that observed only when the external K⁺/Rb⁺ ratio was $\text{1}\text{5}$. From the distinct effects of serum on cell growth and protein synthesis, we conclude that (i) the serum-induced stimulation of cell growth, which is suppressed in the Rb⁺-substituted medium, is not a result of the direct effect of serum on synthesis of bulk protein, but a reflection of the effect on another mechanism(s) required for cell growth; and that (ii) this action is basically identical with the growth-promoting action on cells cultured in the normal medium.     

Nδ-benzoyl-l-ornithine and Nδ-benzoyl-l-γ-hydroxyornithine from Vicia pseudo-orobus

Two new non-protein amino acids, N^δ-benzoyl-l-ornithine and N^δ-benzoyl-l-γ-hydroxyornithine have been characterized from the seeds of Vicia pseudo-orobus.     

Isolation of cis-3-Amino-l-Proline from Cultured Mycelia of Morchella esculenta Fr

cis-3-Amino-l-proline, identified once as a nonprotein amino acid from the fruiting bodies of Morchella esculenta Fr., was isolated also from the growth medium and cultured mycelia of the same fungus.     

Asymmetrical radical formation in D- and L-alanines irradiated with yttrium-90β-rays

Radical formation in^{9 0}Y--irradiated D- and L-alanines was studied using ESR. It was observed that the relative radical concentration by -irradiation was distinguishably (13.9–21.5%) more in D-alanine than in L-alanine. Discussion was made on the possible mechanisms for the observed results.