Effects of tryptic digestion on the enzymatic activites of chicken gizzard myosin.

Tryptic digestion of gizzard myosin resulted in the degradation of the 20K light chain (G1) to its 17K fragment, which could not be phosphorylated. The rapid loss of Ca2+-dependent activation of actomyosin ATPase activity accompanied the degradation of G1. Increase in the Ca2+-ATPase activity and decrease in the EDTA-ATPase activity of myosin accompanied the degradation of myosin heavy chain, but not the cleavage of G1.     

The Pro to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro¹¹⁷ is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.     

Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E₂) and estradiol 17-sulfate (E₂-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E₂. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E₂ was converted to such metabolites as estrone and those having a hydroxyl group at positions 6β, 15 or 16, each production of which was estimated to be catalyzed by single or multiple P-450s.     

Kinetics of the reconstitution of hemoglobin from semihemoglobins α and β with heme

Kinetics of the reconstitution of hemoglobin from semihemoglobins and with hemin dicyanide have been investigated using three kinds of stopped-flow technique (Soret absorption, fluorescence quenching of tryptophan, and Soret CD). The semihemoglobins and are occupied by heme in the and chains, respectively, the other chain being heme-free. Based on the kinetic results, the following scheme for the reconstitution is proposed; First, hemin dicyanide enters the pocket-like site of the apo chains. Second, in semihemoglobin , the CN-ligand in the fifth coordination position of iron is replaced by the imidazole ring of the proximal His immediately after the heme insertion. In contrast, semihemoglobin changes its conformation after the heme insertion, and this is followed by the ligand replacement. Finally, the partial structure changes induced by the ligand replacement propagate onto the whole molecule and the final conformation is attained. The results indicate that semihemoglobin retains a more rigid and organized structure, and more closely approaches its final structure than does semihemoglobin . Correspondence to: Y. Kawamura-Konishi     

Impact of whitefish on an enclosure ecosystem in a shallow eutrophic lake: changes in nutrient concentrations,phytoplankton and zoobenthos

Large bag-type (75 m³) and tube-type (105 m³) enclosures were set up in the shallow eutrophic Lake Suwa and were each stocked with exotic planktivorous whitefish (Coregonus lavaretus maraena). The release of whitefish caused the increase in nutrient concentration in the tube-type enclosure whereas no such increase was observed in the bag-type enclosure. Bottom sediment seemed to be an important source of chironomid food for whitefish. The proportion of phytoplankton measuring<10μm and 20–40μm, which respectively corresponded toOchromonas spp. andCryptomonas sp., were lower in the fish enclosures than in the control, which might have been caused by high grazing pressure by rotifers. The predation by whitefish might have affected the species composition of phytoplankton through reducing copepod predation on rotifers, not through reducing the densities of cladocerans which directly feed on phytoplankton as many investigators have reported. The phytoplankton biomass was not affected much by the release of fish. Possible reasons are that the increase in density of rotifers reduced the biomass of available phytoplankton and also that inedible Cyanophyceae were in the decreasing phase of their seasonal succession and could not increase successfully in spite of elevated nutrient levels.     

Antiserum to Nitrogenase Generated from an Amplified DNA Fragment from Natural Populations of Trichodesmium spp

A fragment of the nifH gene was amplified from natural populations of Trichodesmium spp. and cloned into a maltose-binding protein (MBP) expression vector. The peptide product of the amplified 359-bp fragment of nifH was cleaved from the fusion protein, purified, and used to generate a specific antibody to the Fe protein of nitrogenase. The antiserum recognized the MBP-nitrogenase fusion protein and the cleaved nif peptide product but not MBP. The antibody cross-reacted with nitrogenase from natural populations of Trichodesmium spp. from the Caribbean Sea and with a cultured isolate from the Kuroshio waters (Trichodesmium sp. strain NIBB1067). The same nifH fragment was amplified, cloned, and sequenced from Trichodesmium sp. strain NIBB1067 and was found to be 98% identical at both the protein and DNA levels to nifH from the Caribbean populations. Three of the six nucleotide differences between the Trichodesmium sp. strain NIBB1067 and the Trichodesmium spp. nifH sequence had also been found in a second sequence from the natural populations, indicating either that there is more than one strain of Trichodesmium sp. in natural assemblages or that there are multiple copies of nifH in the genome. This DNA fragment, which is easily amplified with the polymerase chain reaction, may provide a good indicator of species relatedness without requiring extensive cloning or sequencing. Furthermore, the use of the polymerase chain reaction in combination with a MBP protein fusion vector provides a rapid method for production of highly specific sera, starting with a small amount of DNA.     

A role of H+ flux in active Ca2+ transport into sarcoplasmic reticulum vesicles. II. H+ ejection during Ca2+ uptake

Proton efflux during Ca2+ transport into sarcoplasmic reticulum vesicles was examined. Although a rapid H+ ejection was observed during the initial phase of Ca2+ uptake and the amount of the liberated H+ was more than that due to hydrolysis of ATP, generation of a pH difference as a result of the H+ efflux could not be detected by direct pH measurement with a pH meter. Alkalinization of the inside of the vesicles during Ca2+ uptake was more precisely examined by flow dialysis assay and a significant uptake of acetate or salicylate into the vesicles was found, suggesting the generation of a small pH difference across the SR membrane. From these results, it was concluded that counter-transport of H+ was operative in Ca2+ uptake but that only a relatively small pH difference was generated as a result of the H+ efflux. The intrinsic buffering capacity of sarcoplasmic reticulum vesicles was measured and a relatively large value (130 nmol H+/pH unit/mg at pH 6.2) was obtained.     

Fluorescent tracer method for protein SH groups: III. Use ofN-(7-dimethylamino-4-methyleoumarinyl) malelmide as a tracer of cysteine-containing peptides

We studied whetherN-(7-dimethylamino-4-methyleoumarinyl) maleimide (DACM) could be used as a fluorescent tracer for the purification and analysis of cysteine-containing peptides. An addition product of DACM with SH compound was stable at room temperature in the solution of pH 9.0 and in 50% acetic acid. However, it was hydrolyzed when heated at 110°C for 48 h in 6n HCl. On filter paper, 1 pmol of the addition product in a spot 3 mm in diameter was visible under ultraviolet illumination. The addition product was also stable on filter paper for at least several months after spotting. The elution velocity of the addition products with low molecular weight SH compounds in the Sephadex G-25 column was low considering their molecular weights. However, in general, the elution velocity increased with an increase in the molecular weight of the addition product. The addition product with a peptide having cysteinyl residue at an N-terminal showed another abnormally retarded peak in elution profile which presumably corresponded to a cyclic compound, a thiazane derivative. However, it was shown on the C-terminal tryptic peptide of actin (Cys Phe) that the conversion to a thiazane derivative could be avoided by hydrolyzing the succinimide portion of DACM at pH 9.0 before digestion. The R_f values on paper chromatography for the addition products also depended on their molecular weights. However, the hydrophobicity of the coumarin portion of DACM and of the side chain of amino acid residues also affected the value. It was concluded that DACM was a valuable reagent for the purification and analysis of cysteine-containing peptides.