Purification and properties of ATPase from an alkalophilic Bacillus

ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α₃β₃γδ?. The enzyme is activated by Mg²⁺ and Ca²⁺. The pH optima of the enzyme with 0.1 and 2.0 mm Mg²⁺ are 9 and 6, and those with 1 and 10 mm Ca²⁺ are 8–9 and 7, respectively. Ca²⁺-ATPase hydrolyzes only ATP, whereas Mg²⁺-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and K_m of the enzyme with ATP in the presence of 10 mm Ca²⁺ or 0.6 mm Mg²⁺ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg²⁺ is appreciably activated by HCO^?₃. Relationship of the ATPase to the active transport system in the bacterium is suggested.     

The development of functional neuromuscular junctionsin vitro: An ultrastructural and physiological study

Neuromuscular junctions were formedin vitro between rat spinal cord explants and myotubes. At various intervals after the spinal cord explants were added to the myotube culture (7 hr to 15 days of coculture), the presence of functional neuromuscular junctions was determined by recording miniature endplate potentials (mepps) from the myotubes contacted by a few neurites. Electron microscopical studies were conducted on identified myotubes in which mepps were recorded. Mepps were already found as early as 7 hr after coculture. The fine structure of these newly formed neuromuscular junctions was simple. No synaptic specializations were observed except the presence of a small number of synaptic vesicles in the nerve. The neuromuscular junctions differentiated during the coculture period. Synaptic vesicles formed a cluster at the prejunctional membrane with a localized density in the middle. Basal lamina started to form in 4-day-old cocultures and became continuous in cocultures of 10 days or longer. Clear postjunctional foldings were observed in 15-day-old cocultures. Higher mepp frequencies were correlated with more advanced ultrastructure.     

Colonization of R plasmid-bearing Escherichia coli strains in the mouse alimentary tract.

In order to examine the ability of R plasmid-bearing Escherichia coli strains to colonize in the mouse alimentary tract, an R plasmid-positive (R(+)) E. coli strain and its R plasmid-negative (R(-)) counterpart were together inoculated into the streptomycin-treated mouse alimentary tract, and the numbers of fecal E. coli strains were enumerated. The numbers of R(+) strains were always at the level similar to or lower than those of their counterparts and rapidly decreased in the fecal population. However, when R plasmids, which were originated from a cryptic plasmid of the host E. coli strain, were utilized, an R(+) strain dominated over its R(-) counterpart during the experimental period. These experimental results indicated that the relationship between the host strain and R plasmids affected the ability of the host strain to colonize in the alimentary tract.     

Chloride attachment negative-ion mass spectra of sugars by combined liquid chromatography and atmospheric pressure chemical ionization mass spectrometry

A method has been developed for the rapid determination of sugars, including molecular weight measurements, using high-performance liquid chromatography coupled with negative-ion, atmospheric-pressure chemical-ionization mass spectrometry. The chromatography was carried out on a 250 × 4 mm I.D. column packed with 7 μm NH₂-silica. The mobile phase consisted of a high percentage of methanol or acetonitrile with a small amount of chloroform. During the mass spectrometry, a strong base is formed from the polar solvent molecules by corona discharge, followed by ion—molecule reactions in the chemical ionization ion source (e.g. the methoxy anion is formed from methanol). This strong base reacts with the chloroform, generating chloride ions, which in turn react with the neutral sugar molecules as they elute from the chromatograph. The chloride ion and sugar interactions yield chloride-attachment ions, which are further stabilized by successive collisions. In this method, authentic monosaccharides and some oligosaccharides show dominant quasi-molecular ions, [M + Cl]⁻, with little fragmentation, and it is particularly useful for the molecular weight determination of sugars.     

Effects of inorganic iodide, epidermal growth factor and phorbol ester on hormone synthesis by porcine thyroid follicles cultured in suspension.

Porcine thyroid follicles cultured in suspension for 96 h synthesized and secreted thyroid hormones in the presence of thyrotropin (TSH). The secretion of newly synthesized hormones was assessed by determining the contents of thyroxine (T4) and triiodothyronine (T3) in the media and by paperchromatographic analysis of 125I-labelled hormones in the media where the follicles were cultured in the presence and absence of inhibitors of hormone synthesis. The hormone synthesis and secretion was modified by exogenously added NaI (0.1-100 microM). The maximal response was obtained at 1 microM. Thyroid peroxidase (TPO) activity in the cultured follicles with TSH for 96 h was dose-dependently inhibited by NaI. One hundred microM of NaI completely inhibited TSH-induced TPO activity. Moreover, both epidermal growth factor (EGF: 10(-9) and 10(-8) M) and phorbol 12-myristate 13-acetate (PMA: 10(-8) and 10(-7) M) inhibited de novo hormone synthesis. An induction of TPO activity by TSH was also inhibited by either agent. These data provide direct evidences that thyroid hormone synthesis is regulated by NaI as well as TSH at least in part via regulation of TPO activity and also that both EGF and PMA are inhibitory on thyroid hormone formation.     

Use of benzyl 2-acetamido-2-deoxy-3-O-(2-O-methyl-beta-D-galactosyl)-beta- D-glucopyranoside [2'-O-methyllacto-N-biose I beta Bn] as a specific acceptor for GDP-fucose: N-acetylglucosaminide alpha(1----4)-L-fucosyltransferase

A synthetic substrate, benzyl 2-acetamido-2-deoxy-3-O-(2-O-methyl-beta-D- galactopyranosyl)-beta-D-glucopyranoside, was demonstrated to be a specific acceptor for the Lewis blood group-specified alpha(1----4)-L-fucosyltransferase from human saliva and stomach mucosa. The fucosyl linkage of the product resulting from the use of this substrate isolated by paper chromatography was characterized by hydrolysis with specific alpha(1----3)/(1----4)-L- fucosidase. The product can be separated by adsorption onto the reverse-phase cartridge and recovered by one-step elution with methanol. The enzymatic properties of alpha(1----4)-L-fucosyltransferase from saliva and stomach mucosa have also been examined using this substrate.     

A factor in cell-free extracts inactivating sexual agglutinability of a mating type in Saccharomyces cerevisiae

Cell-free extracts of both a and a mating-type strains of Saccharomycescerevisiae contained a substance which irreversibly inactivatedsexual agglutinability of a cells, but not that of a cells. ¹ Present address: Department of Pharmacology, Osaka Collegeof Pharmacy, 2-10-65 Kawai-cho, Matsubara, Osaka 580, Japan. (Received January 9, 1976; )