CTP-dependent lipid kinases of yeast

Membrane fractions from yeast Saccharomyces cerevisiae catalyzed a transfer of gamma-phosphate from [gamma-32P]CTP into membranous lipids. Phosphorylated compounds were identified as phosphatidic acid and dolichyl phosphate (DolP). The membrane fraction also catalyzed phosphorylation of the exogenous dolichol. The activity of the phosphorylating enzymes could be modified by the yeast growing conditions; i.e., the enzyme from yeast grown aerobically favored the synthesis of phosphatidate over dolichyl phosphate in the ratio of 3:1, whereas the membrane fraction from anaerobically grown yeast synthesized PA and DolP in the ratio of 0.5:1. The activity of the phosphorylating enzymes could also be modified by divalent cations and the concentration of detergents. Phosphorylation of lipids does not occur in the presence of [gamma-32P]ATP and is not influenced by the presence of UTP or GTP. This result points to the specific role of CTP as a gamma-phosphate donor for the synthesis of phosphatidate and dolichyl phosphates in the yeast system.     

Recapitulation in the development of the components of the counterflow system in the vertebrate metanephros

The investigation has been performed on 107 renal preparations obtained from persons of various age (from 5-month-old fetuses up to 45 years of age), certain representatives of other classes of the Vertebrata are also included: fish, amphibia, reptile and mammalia at various stages of pre- and postnatal periods of ontogenesis by means of preparing graphic and plastic reconstructive models, histological investigation and microdissection. The complexity of the intrarenal branching of derivatives of the mesonephric duct diverticulum, development and structure of the canalicular part in nephrons directly depend on the phylogenetic position of the animal. Complexity of the nephron architectonics occurs along the progressive line of taxonomic groups of higher Vertebrata. The nephron loop becomes longer, thin segment of the nephron canalicular part increases in its length and, at last, in mammalia a cone-shaped fasciculus appears as a structural-functional unit of the osmoregulating apparatus of the constant kidney. In the comparative anatomical and comparative embryological aspects recapitulation is observed concerning certain morphological signs of derivatives of the metanephric duct and nephron.     

Phenolsulphonphthalein conjugation and biliary excretion in the rat: influence of phenobarbital and 3-methylcholanthrene

The effect of phenobarbital and 3-methylcholanthrene pretreatment on the biliary excretion of phenolsulphonphthalein (PSP) was investigated in male Wistar rats. The dye was injected at a single dose of 200 mumol/kg body wt. About 20% of the compound was excreted as a glucuronide in the controls, the liver UDP-glucuronyltransferase activity toward PSP being 0.064 +/- 0.005 nmol.min-1.mg protein-1. Treatment for two weeks with phenobarbital (354 mumol.kg body wt-1.day-1) caused a transient increase in conjugated and unconjugated PSP excretion, but glucuronyltransferase activity was not modified. 3-Methylcholanthrene pretreatment for 4 days (75 mumol.kg body wt-1.day-1) also enhanced biliary excretion of the dye, but the increase corresponded only to the glucuronide and glucuronyltransferase activity was significantly enhanced by 20%. Our data indicate that not only the rate of biotransformation but also other factors could be responsible for increased PSP biliary excretion following administration of microsomal enzyme inducers.     

Molecular and catalytic properties of a butyrylesterase from human red cells and brain

A butyrylesterase from human red cells was prepared to homogeneity using DEAE-cellulose, Ultrogel ACA-34, DEAE-Sephacel, and precipitation with 1.5 M (NH4)2SO4. The yield was 25-35% relative to the enzyme activity of the hemolysate. Because of its preference for butyric acid esters the enzyme was designated a butyrylesterase. With alpha-naphthyl butyrate the Km was 7.6 microM and the kcat, 48 s-1. The molecular weight was 340,000 and the subunit weight 85,000, indicating a tetrameric structure. The isoelectric pH was 4.0. The enzyme preparation did not contain cystine. Sialic acid or other carbohydrate components could not be detected. The enzyme was irreversibly inhibited by organophosphate esters and the second-order rate constant was 192 M-1 s-1 for diethyl p-nitrophenyl phosphate. For the brain enzyme the constant was 206 M-1 s-1. The enzyme was irreversibly inhibited by sulfhydryl reagents, indicating that the enzyme is a sulfhydryl-dependent serine esterase. The enzyme was identical to the butyrylesterase from human brain, and the two enzymes were immunochemically identical. An amino acid ester has been shown to be split at a higher rate than butyric acid esters; however, the specificity constant (kcat/Km) was lower for the amino acid ester than for the butyric acid ester. The enzyme did not exhibit amidase activity.     

Non-protein-mediated transfer of phosphatidic acid between microsomal and mitochondrial membranes

The transfer of phosphatidic acid between rat liver microsomes loaded with [32P]-phosphatidic acid and rat liver mitochondria was studied in the absence of added lipid transfer proteins. It was found that during 1 h at 37 degrees C in the medium containing 100 mM KCl, 20-30% of phosphatidic acid but only 2.5% of phosphatidylcholine were transferred. This spontaneous transfer of phosphatidic acid remained the same after pretreatment of microsomes and mitochondria with 125 mM KCl or microsomes alone with 1 mM Tris, pH 8.6, procedures reported to remove adsorbed lipid transfer proteins. This transfer was insensitive to thiol-blocking reagents. The initial rate of this non-protein-mediated transfer of phosphatidic acid was virtually independent of the concentration of the acceptor membranes (mitochondria), thus indicating that it occurs by diffusion of the phospholipid through the aqueous phase rather than by membrane collision. About 80% of phosphatidic acid synthesized in the outer mitochondrial membrane was recovered in the inner membrane after a 1-h incubation, pointing to a high rate of the intermembrane transfer of this phospholipid within intact mitochondrion.     

Chemical characterization by protein sequence analysis of the bovine estrogen receptor

Tryptic peptides generated from bovine estrogen receptor have been fractionated and purified using microbore column high performance liquid chromatography. Sequence analysis performed on six of these peptides, derived from diverse structural regions of the receptor protein, yielded 73 unique assignments corresponding to approximately 12% of the molecule. The amino acid sequences of these peptides displayed a high degree of similarity with corresponding sequences from estrogen receptors of mammalian origin, but were only moderately conserved in receptors from non-mammalian species. The sequenced residues of one tryptic peptide, positioned in the estrogen binding domain, were fully conserved in all estrogen receptors.     

Isolation and characterization of three protein proteinase isoinhibitors from the granular fraction of horse neutrophilic granulocytes

Three cathodically migrating protein protease isoinhibitors were isolated from the granule-rich fraction of equine neutrophilic granulocytes by means of FPLC chromatography, in addition to two previously described anodically migrating inhibitors. The three isoinhibitors had an identical enzyme specificity which was equal to the two previously described isoinhibitors; they inhibited exclusively proteinase K and subtilisin. The inhibitors retained their activity between pH 1 and 12. They also were heat stable at 100 degrees C for 20 min. Neither the biological function of isoinhibitors nor the fundamental role of granular protease inhibitors of such narrow and peculiar enzyme specificity are known.     

Localization of glycoconjugates in tissues of mammals revealed by means of labeled lectins

Histochemical peculiarities on binding of castor-oil plant, soybean and lentil lectins with tissues of the mucous membrane in the stomach, small and large intestine have been studied in the human being, rat, mouse, as well as the lectins mentioned and the maize agglutinin with the nervous tissue of the rat cerebral tissue. The reactions are carried out with nonfixed cryostat and deparaffinized histological slices. Lectins labelled with horseradish peroxidase are used. Certain specific peculiarities and differences concerning the lectin binding with tissues of the organs studied are determined. Predominant binding is noted of the soybean lectin with parietal and mucin-producing cells of the stomach, with epitheliocytes of the duodenal glands, with the brush border of the epithelial cells of the intestinal villi. The lentil and castor-oil plant lectins make contours of the basal membrane epithelium in the stomach and intestine. The lentil lectin also reacts with the germinative centers of the stomach lymphatic nodules and the castor-oil plant agglutinin--with the brush border of the small intestine epitheliocytes. The lectins used are predominantly bound with neurons of the subcortical formations of the rat brain and cerebral cortex. By means of labelled lectins of lentil, soybean, and castor-oil plant it is possible to reveal certain modifications of the rat small intestine glycoconjugates produced by means of the immortelle extract.     

Periodic cleavage of poly(dA) by oligothymidylates covalently linked to the 1,10-phenanthroline-copper complex

1,10-Phenanthroline (OP) was covalently attached to the 3'-terminus of two oligothymidylates via different linkers [abbreviated as T8-(OP) and T6-(OP)]. In the presence of Cu2+ and 3-mercaptopropionic acid (MPA), these reagents induce a hybridization-dependent cleavage of poly(dA) and of a 27 nucleotide long oligodeoxynucleotide containing an A8 sequence. The principal cleavage sites on the 27-mer span four residues located near the 3'-terminal phosphate group of T8-(OP). When poly(dA) was degraded by T6-(OP) and T8-(OP), a series of bands were obtained corresponding to a repeat unit of six and eight nucleotides, respectively. This periodicity reflects the cooperative binding of oligothymidylate-OP to the polynucleotide matrix and the localized nicking sites.