Molecular and enzymatic properties of 1,2-α-d-mannosidase fromAspergillus saitoi

The molecular properties, such as molecular weight, N-and C-terminal amino acids, amino acid composition, and circular dichroism, of 1,2--mannosidase isolated from the culture filtrate ofAspergillus saitoi were determined.The enzyme had aK _m of 0.67 mM andk _cat of 1.27/s with mannobiose at pH 50.0 and 30°C. The anomeric configuration of the reaction products of the enzyme was examined by studying the -anomer. A single Manl2Man linkage in intact Taka-amylase A fromAspergillus oryzae was hydrolyzed, producing free mannose.     

Sea urchin (Anthocidaris crassispina) egg zinc-binding protein. Cellular localization, purification and characterization

Gel-filtration analysis of cytosol fraction obtained from unfertilized sea-urchin (Anthocidaris crassispina) eggs on Sephadex G-75 revealed the presence of two Zn-binding-protein fractions. The major Zn-binding protein fraction had a low molecular weight and a low absorbance at 280 nm, properties similar to those of the metallothionein found in the regenerating rat liver. These fractions were further purified by DEAE-cellulose and Sephadex G-50 chromatography. Homogeneity of the Zn-binding protein was judged by polyacrylamide-disc-gel electrophoresis and gel-permeation chromatography in the presence of 6 M-guanidinium chloride. The molecular weight determined by gel-permeation chromatography was 3900. This value is in good agreement with the minimum molecular weight calculated from the amino acid composition, which was 3655. Zn-binding protein is composed of 36 amino acid residues and the distinctive features include an extremely high content of cysteine, which accounted for one-third of the total amino acid residues, and a complete absence of aromatic amino acids, as well as of methionine, histidine and arginine. Zn-binding protein contained 4.1 g-atoms of zinc per mol and a trace of cadmium, but no copper, iron or calcium. The molar ratio of reactive thiol groups to metal ion was calculated to be 2.73:1. Possible roles of this Zn-binding protein in the homoeostasis of zinc in unfertilized sea-urchin eggs are discussed.     

Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections

Summary The mechanism of intracellular maturation and sorting of herpes simplex virus type I glycoproteins is not known in details. To elucidate the intracellular sorting of viral glycoproteins and their possible interaction with the cytoskeleton, a method for simultaneous immunogold staining of three antigens in ultrathin cryosections is described. Each antigen is stained by an indirect technique using mouse monoclonal IgG as first layer, rabbit antimouse IgG as second and gold-conjugated goat anti-rabbit IgG as third layer antibody. After each staining cycle the paraformaldehyde vapour at 80° C for 30 min. This destroys the free antigen combining sites of the second and the third layer IgG and abolish contaminating staining. Simultaneous triple-staining is documented with three mouse monoclonal antisera specific for 1) herpes simplex virus type 1 glycoprotein C, 2) glycoprotein D and 3) - and -tubulin as primary antibodies. Labelling for virus glycoproteins was found in some Golgi vesicles and close to the cytoplasmic microtubules as well as on the cell surface and on intracytoplasmic and extracellular virus particles.Presented in part at the 9th European Congress on Electron Microscopy, York, England, September 4–9, 1988     

Synthetic inositol trisphosphate analogs and their effects on phosphatase, kinase, and the release of Ca2+

A series of inositol 1,4,5-trisphosphate (IP3) analogs and positional isomers was examined to explore the structure-activity relationships among IP3 5-phosphatase, IP3 3-kinase, and the release of Ca2+. All analogs with additional groups on the 2nd position of IP3 inhibited the hydrolysis of [5-32P]IP3 catalyzed by erythrocyte ghosts, with a lower Ki value than seen with IP3. IP3 dehydroxylated at the 2nd position also had a lower Ki, while 2,4,5-IP3 or cyclic(1:2), 4,5-IP3 had higher Ki values. Among these compounds 2-deoxy-IP3 was as potent as IP3 in inhibiting the phosphorylation by [3H] IP3-3-kinase in rat brain cytosol. The other compounds, except for 2,4,5-IP3 inhibited the phosphorylation, however, 2-30 times higher concentrations were required. By lowering free Ca2+, the concentrations required for half-maximal inhibition were low, while those of IP3, 2-deoxy-IP3, and positional isomers remained unchanged. These compounds acted as full agonists in releasing Ca2+ from permeabilized macrophages, although 1.6-50-fold higher concentrations than IP3 were required. These compounds also inhibited the binding of [3H]IP3 to rat cerebellum and bovine adrenal cortex microsomes, but the potencies were 2.9-33 times less than that of IP3. Thus, the 2nd position of IP3 can be modified with only a slight loss of biological activity.     

Using microparticle labeling and counting for attomole-level detection in heterogeneous immunoassay.

A new heterogeneous "sandwich" immunoassay utilizing microparticles as labels to realize high sensitivity is described. In this method, antibody fixed on the microparticles reacts with antigen previously trapped on a microplate surface, which makes the antigen molecules visible and countable with an inverted optical microscope. The method is highly sensitive because the reacted single microparticle, therefore single antigen molecule, can be detected. The sensitivity depends both on the reaction efficiency of the immunoreaction and on nonspecific adsorption of the microparticles on the microplate surface. Therefore, the protocol for preparing microparticle having antibody on the surface and a microplate having capture antibody was investigated to realize high sensitivity. Carboxylated microparticles of 0.76 microns in diameter were conjugated with affinity-purified antibody using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. It was determined that 1 g microparticles had 880 micrograms antibody (approximately 1100 antibody molecules per 1 microparticle). The immunoreaction efficiency reached 18% at 1 x 10(-13) mol/liter antigen concentration. The lower detection limit was 3.1 x 10(-14) mol/liter (1.6 amol) using human alpha-fetoprotein as a model antigen.     

Gene expression and immunohistochemical localization of basic fibroblast growth factor in renal cell carcinoma.

Renal cell carcinoma is known as a neoplastic condition of renal tubular cells and usually shows a hypervascular tumor in angiographic examination. We examined the presence of basic fibroblast growth factor (bFGF) in human renal cell carcinoma. To determine if alterations in bFGF gene expression are present in human renal cell carcinoma, paired samples of normal and neoplastic renal tissue from 6 patients were analyzed for bFGF mRNA content by Northern blot hybridization. In 4 out of 6 patients, tumor tissue expressed bFGF mRNA 2 to 4 times greater than corresponding normal tissue. Two patients showed minimal elevation of tumor bFGF mRNA. The localization of bFGF in the renal cell carcinoma tissue was also examined using immunohistochemical staining, and it was found that bFGF was positively stained at the nuclei of tumor cells and the cell surface. These results suggest that increased expression of bFGF may be associated with neoplastic growth in renal tubular epithelial cells and neovascularization.     

A point mutation in exon 3 (His 107→Tyr) in two unrelated Japanese patients with carbonic anhydrase II deficiency with central nervous system involvement

We have analyzed two unrelated Japanese patients with carbonic anhydrase II deficiency born to consanguineous parents. We have identified the same mutation as that reported to be homozygous in a Belgian family and compound heterozygous in an American family. It comprises to C-to-T transition that results in the amino acid substitution of Tyr (TAT) for His (CAT) at position 107. This point mutation creates an AccI site that can be conveniently screened by the polymerase chain reaction/restriction fragment length polymorphism method using a restriction enzyme for gene tracking. Our patients exhibit severe mental retardation, not seen in the Belgian and American patients. Received: 23 November 1994 / Revised: 22 May 1995