Polyglutamylfolate synthesis in Neurospora crassa: changes in pool size following growth in glycine- and methionine-supplemented media

The effect of media supplements on total and polyglutamylfolate concentrations has been examined in Neurospora crassa wild type (FGSC 853), an ethionine-resistant mutant (FGSC 1212), and a methionine auxotroph (FGSC 1330) which lacks folylpolyglutamate synthetase. When the culture medium contained 1 mm glycine, folate concentrations in the wild type were increased by over 90% and more p-[³H]aminobenzoate was incorporated into folates. Growth in l-methionine-supplemented media (1–5 mm) decreased folate levels and labeling in all three strains. In the wild type, this effect of l-methionine was reversed on transfer to unsupplemented media but p-[³H]aminobenzoate pulse-chase experiments suggested that exogenous methionine did not increase the turnover of labeled folates. At 1 mm, d-methionine did not affect polyglutamylfolate labeling but l-methionine reduced ³H incorporation by 65% in the wild type. Ion-exchange chromatography showed that p-[³H]aminobenzoate was incorporated in formyl- and methyltetrahydrofolates which in the wild type, were principally hexaglutamyl derivatives. Glycine-supplemented growth yielded labeled folates that were 24% heptaglutamates but these and pentaglutamates were lacking when l-methionine was supplied. The specific activity of GTP cyclohydrolase was not significantly affected by culture in l-methionine-containing media. Dialysis and gel filtration both lowered enzyme activities and product formation was not changed when up to 10 μmol of l-methionine was added to the reaction system. The data suggest that methionine or its metabolic products exerts some control over folate production which is distinct from the established inhibition of methylenetetrahydrofolate reductase by AdoMet.     

Isolation and identification of 25-hydroxy-24-oxocholecalciferol: A metabolite of 25-hydroxycholecalciferol

A metabolite of 25-hydroxycholecalciferol has been isolated in pure form from chicken kidney homogenates. It has been identified as 25-hydroxy-24-oxocholecalciferol by means of ultraviolet absorption spectrophotometry, mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and specific chemical reactions.     

Cyclic GMP metabolism in macrophages. I. Regulation of cyclic GMP levels by calcium and stimulation of cyclic GMP synthesis by NO-generating agents

Levels of guanosine 3′,5′-cyclic monophosphate (cGMP) were determined by radioimmunoassay in adherence-purified, oil-induced guinea pig peritoneal exudate macrophages, after extraction of the cells with perchloric acid, purification on Dowex AG1-X8, and acetylation. We found that: (i) Basal cGMP levels were strictly dependent on the concentration of extracellular Ca²⁺ (0.33 ± 0.03 pmol/mg macrophage protein in Ca²⁺-free medium and 2.49 ± 0.42 pmol/mg in 1.8 mM Ca²⁺). (ii) The stimulatory effect of Ca²⁺ on cGMP levels was prevented by tetracaine. (iii) The cGMP content of macrophages was not elevated by incubation with the ionophore A23187 at extracellular Ca²⁺ concentrations varying between 0 and 1.8 mM. (iv) Macrophage cGMP levels were increased markedly (up to 40-fold) by incubation of the cells with the nitric oxide (NO)-generating agents, sodium azide, hydroxylamine, sodium nitrite, and sodium nitroprusside. (v) Stimulation of cGMP accumulation by NO-generating agents occurred within 30 sec, was Ca²⁺-independent, and developed in the presence and absence of the phosphodiesterase inhibitor, isobutyl-methylxanthine. (vi) A minimal elevation in the macrophage cGMP level (less than 2-fold) was induced by ascorbic acid but no significant increases were induced by the following agents, found effective in other cells: serotonin, acetylcholine, carbamylcholine, phorbol myristate acetate, arachidonic acid, Superoxide dismutase, and nitrate reductase.     

Morphological and functional peculiarities of mesenchymal cells in the pars tuberalis of the pituitary gland

Summary Following injection of high doses of horseradish peroxidase (HRP), mesenchymal cells distributed in the perisinusoidal space of the pars tuberalis of the hypophysis in cats, rabbits and Japanese quails, sequester the exogenously administrated peroxidase intensively. These cells are designated by the authors as horseradish peroxidase-uptake cells (HRP-uptake cells or HUC). HRP-uptake cells constitute a system of macrophages in the pars tuberalis of mammals and birds, and are located around the hypophysial portal veins. HRP-uptake cells differ in morphological and functional characteristics from similar cells in other parts of hypophysis. They are thought to play a role in the hypothalamic control of adenohypophysial secretion.Supported by grants (No. 144022, 237002) from the Ministry of Education, Science and Culture, Japan     

Hepatic microsomal epoxide hydrase. Chemical evidence for a single polypeptide chain.

Highly purified hepatic microsomal epoxide hydrase, which had been purified in the presence of proteolytic enzyme inhibitors, was subjected to carboxypeptidase Y digestion, automated Edman degradation, and carbohydrate analysis. Carboxypeptidase Y digestion resulted in the near stoichiometric release of leucine, the COOH-terminal amino acid. Automated Edman degradation permitted the identification of the first 20 amino acid residues of epoxide hydrase. Methionine was identified as the NH2-terminal residue. The NH2-terminal region of epoxide hydrase is similar in hydrophobicity to the NH2-terminal precursor segments of several secretory proteins and the NH2-terminal regions of several microsomal cytochromes P-450. Carbohydrate analyses of the enzyme revealed the presence of 0.5 to 1.0 mol of mannose/50,000 g of protein. These results provide evidence for the presence of a single polypeptide chain in our purified enzyme preparations and suggest that there may be only one enzymic form of epoxide hydrase in microsomes from phenobarbital-treated rats.     

Correlative Inhibition of Lateral Bud Growth in Phaseolus vulgaris L. Ethylene and the Physical Restriction of Apical Growth

Restriction of apical growth in Phaseolus by enclosing the upperpart of the shoot in sealed or ventilated tubes induced developmentof axillary buds beneath the enclosure. Enclosed parts of shootsshowed a reduction of leaf growth and, in experiments wherethe tubes were sealed, of internode extension. Enclosure ofthe shoots in large vessels that did not restrict leaf expansion,but which contained 0?5 vols 10^–6 ethylene, similarlyinduced axillary bud growth. Analysis of the gaseous extractof physically restricted shoots showed a 2?5-fold increase inethylene concentration. The results suggest involvement of ethylenein the release of correlative inhibition brought about by physicalrestriction of apical growth.     

Slow equilibration of a denatured protein: comparison of the cluster model with the proline isomerization model

The slow equilibration of the denatured state after rapid unfolding of a globular protein is examined by the cluster model of protein folding (Kanehisa &; Tsong, 1978). The detection of this process in ribonuclease A and its acid catalysis have been considered evidence for the proline isomerization model. Our calculation shows that similar kinetic behavior is also expected for the cluster model.     

Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution

The crystal structure of the complex of subtilisin BPN′ (EC 3.4.21.14) with its protein inhibitor (Streptomyces subtilisin inhibitor) was solved at 4.3 Å resolution, thus establishing the following. (1) Two subtilisin BPN′ molecules (2E) associate with one dimeric inhibitor molecule (I₂) to form the complex molecule E₂I₂. (2) The conformation of neither the inhibitor nor subtilisin BPN′ undergoes any detectable change at this resolution upon complex formation. (3) The inhibitor binds to subtilisin to form an antiparallel β-sheet, as in the case of trypsin/ trypsin inhibitor complexes. (4) The scissible bond of the inhibitor is between Met73′ and Val74′, as proposed earlier (Ikenaka et al., 1974). (5) The protein inhibitor and the substrates bind to subtilisin BPN′ in essentially the same way.