Identification of a novel amino acid, o-bromo-L-phenylalanine, in egg-associated peptides that activate spermatozoa

Eight sperm-activating peptides containing a novel amino acid were isolated from the egg jelly of the sea urchin Tripneustes gratilla. Accurate mass measurement of the peptide in FAB mass spectrometry showed that the mass of the novel amino acid residue was 224.978. On the basis of the isotopic ion distribution and the degree of unsaturation, the mass value indicated that the elemental composition of the amino acid residue was C9H8O1N1Br1, suggesting that the novel amino acid was bromophenylalanine. Proton NMR spectroscopy, amino acid analysis, and RP-HPLC with three synthetic isomers of bromophenylalanine demonstrated that o-bromophenylalanine was the novel amino acid. Derivatization of the amino acid with Marfey's reagent, (1-fluoro-2,4-dinitrophen-5-yl)-L-alanine amide (FDAA), further indicated that the amino acid was the L-isomer. In other sperm-activating peptides isolated from the egg jelly of the sea urchin, both m- and p-bromophenylalanines were discovered. The presence of m-bromophenylalanine has not been previously reported in natural products, while p-bromophenylalanine is found in theonellamide F, an antifungal bicyclic peptide from a marine sponge.     

The influence of glycosylation on the fate of renin expressed in Xenopus oocytes

It has been recently reported that, in Xenopus oocytes injected with the mRNA for human renin, this secretory renal glycoprotein acquires phosphomannosyl residues on its asparagine-linked oligosaccharide chains, remains intracellular and undergoes a proteolytic cleavage which removes the prosegment. To understand the influence of glycosylation on the fate of renin in Xenopus oocytes and whether it is specific for human renin, we have expressed human renin and mouse Ren1 renin, which are glycosylated at two and three selected asparagine residues, respectively, and mouse Ren2 renin, which is not glycosylated, in Xenopus oocytes. The majority of human and Ren1 renins remained intracellular and underwent proteolytic cleavage, whereas mouse Ren2 renin was secreted efficiently. When human and Ren1 renins were expressed in oocytes treated with tunicamycin, both were secreted efficiently. A mutant of human renin, which had amino-acid substitutions at both glycosylation sites, was also secreted efficiently, whereas that mutated at one of the two sites was not. These results indicate that the majority of all of the glycosylated renin molecules remain intracellular and undergo proteolytic cleavage, probably due to the acquisition of phosphomannosyl residues, and the human renin remains intracellular if it is only glycosylated at one of the two sites.     

Inhibition of epidermal growth factor-induced DNA synthesis by tyrosine kinase inhibitors

We prepared methyl 2,5-dihydroxycinnamate as a stable analogue of erbstatin, a tyrosine kinase inhibitor. This analogue was about 4 times more stable than erbstatin in calf serum. It inhibited epidermal growth factor receptor-associated tyrosine kinase in vitro with an IC₅₀ of 0.15 μg/ml. It also inhibited in situ autophosphorylation of epidermal growth factor receptor in A431 cells. Methyl 2,5-dihydroxycinnamate was shown to delay the S-phase induction by epidermal growth factor in quiescent normal rat kidney cells, without affecting the total amount ofDNA synthesis. The effect of erbstatin on S-phase induction was smaller, possibly because of its shorter life time.     

Some common properties of lectins from marine algae

Twelve kinds of lectins isolated from four species of marine algae, Boodlea coacta (Chlorophyta) and Hypnea japonica, Carpopeltis flabellata and Solieria robusta (Rhodophyta), were compared for their chemical and biological properties. These lectins were proteins or glycoproteins, similar to terrestrial plant lectins. However, unlike most terrestrial plant lectins, they had a small molecular size (4,200 to 25,000 daltons), were mostly monomeric, and had no affinity for monosaccharides. They strongly agglutinated trypsin-treated rabbit erythrocytes, and their activities commonly were inhibited by glycoproteins bearing N-glycans. From hemagglutination-inhibition tests with various glycoproteins and related compounds, it was found that B. coacta lectins recognize high-mannose N-glycans; H. japonica lectins complex N-glycans, and C. flabellata and S. robusta lectins recognize both types of N-glycans.     

Application of 15N-nuclear magnetic resonance (N.M.R.) spectroscopy for the study of nitrogen metabolism of a parasite: transamination in Angiostrongylus cantonensis eggs

In an attempt to study nitrogen metabolism in a parasite, we applied 15N-nuclear magnetic resonance (NMR) technology with a stable isotope, a 15N-labeled compound, for the study of the transamination system in A. cantonensis eggs, and demonstrated that 15N-aspartic acid can serve as an amino group donor for both the 2-oxoglutaric-glutamic acid and the pyruvic acid-alanine transamination systems in the eggs.     

Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonshivering thermogenesis and cold resistance during seasonal acclimatization in the Djungarian hamster

Summary The absence of juvenile hormone (JH) at the time of head capsule slippage during the molt to the fifth (final) instar of the tobacco hornworm was found to cause ommochrome (primarily dihydroxanthommatin) synthesis in the epidermis during the first two days after ecdysis. Then synthesis decreased until its transient reappearance during the wandering stage. Either JH-I (ED₅₀=8x10^–4 g) or methoprene (ED₅₀=1.4x10^–2 g) applied at this critical time during the molt prevented the first synthesis. A comparison of developmental profiles of tryptophan and its metabolites, kynurenine and 3-hydroxykynurenine, in normal and allatectomized wild type larvae showed that JH at this critical time prevented both the conversion of kynurenine to 3-hydroxykynurenine and 3-hydroxykynurenine to ommochromes. A similar study in normal and methoprene-treatedblack mutant larvae showed that only the latter conversion was inhibited by JH. The accumulation of 3-hydroxykynurenine in the epidermis of the JH-treatedblack mutant is thought to be due to the altered tryptophan metabolism in these mutants in previous instars due to lower JH levels. Neither starvation of theblack mutant nor injection of 3-hydroxykynurenine significantly affected ommochrome synthesis by the epidermis. Preliminary studies of the enzymes involved showed that JH at the critical period suppressed the later activity and/or production of kynurenine 3-hydroxylase in the wild type larva, but had little effect on the particulate ommochrome synthetase activity of the epidermis.Abbreviations CA corpora allata - JH juvenile hormone - PTTH prothoracicotropic hormone     

Kinetic Properties of Bovine Pineal Tryptophan-5-Monooxygenase Activated by an Endogenous Activating Substance

Abstract: We previously reported that an endogenous activating substance different from bovine serum albumin, phospholipids and heparin, exists in the extract from bovine pineal glands and that this substance interacts with tryptophan-5-monooxygenase under reducing conditions with sulfhydryl reagents, to stimulate monooxygenase activity. The present paper reports that the activating substance is of peptide nature; that it is sensitive to trypsin-digestion; and that it does not change the apparent K _m's for substrates, L-tryptophan and oxygen, and coenzyme, reduced biopterin or DMPH₄: but that it increases the V _max 1.5- to 2.3-fold. These results suggest that an activating protein, present in some particles of the cell structure, activates tryptophan-5-monooxygenase under the regulation of a sulfhydryl compound. The apparent K _m's for reduced biopterin and DMPH₄ were 77.2μM and 294 μM, respectively. The apparent K _m's for L-tryptophan and oxygen with reduced biopterin were 15.0 μM and 4.7%, respectively: with DMPH₄, they were 11.0 μM and 8.5%, respectively. Significant inhibition of both L-tryptophan and oxygen was observed with reduced biopterin, but not with DMPH₄ (at the tested concentrations of up to 0.5 MM and 20%, respectively).