Regulation of the amphibian oocyte plasma membrane ion permeability by cytoplasmic factors during the first meiotic division.

The denuded Rana pipiens oocyte depolarizes from 80–90 mV inside negative to 3–5 mV? inside positive during progesterone-induced meiotic maturation, apparently due to decreased K⁺ permeability. Depolarization is dependent upon protein synthesis and coincides with breakdown of the oocyte nucleus, but occurs even in the absence of the nucleus, suggesting cytoplasmic regulation of cation selectivity of the oocyte plasma membrane.     

Configurations of a polymeric antigen adsorbed to a B-cell membrane

For an unsynchronized cell population with several phases of DNA synthesis, the population distribution of tracer (thymidine) incorporation is derived. Compounding this with the Poisson probability for ³H disintegration, the distribution of autoradiographic grains over nuclei is obtained. The changes in distribution after various numbers of cell divisions are obtained on the assumption that the tracer is (approximately) equally partitioned between daughter cells during division. The parameters are expressed in terms of the number of phases of synthesis, specific activity of tracer, rate of incorporation, and autoradiographic exposure time. Application of the theory to experimental material is illustrated.     

Diurnal rhythmicity in plasma 24,25-dihydroxyvitamin D3: correlation with intestinal calcium transport and plasma minerals

Plasma 24,25-(OH)₂D₃ exhibits diurnal rhythmic variations in the adult rat. The decrease in 24,25-(OH)₂D₃ occurring at the onset of feeding during light-dark transition coincides with a decrease in plasma calcium but is inversely related to plasma phosphate and to increased intestinal calcium transport activity.     

Three new alpha-chains of collagen from a non-basement membrane source

Three new collagen α chains have been isolated from synovial membrane, gingiva and skin. Two of these have a similar chromatographic behaviour to the α[A] and α[B] chains described by Burgeson $\text{et al}$. (4) from a foetal basement membrane source but have been separated from another contaminating α chain, α[C]. The α[A] and α[B] chains are present in approximately equal amounts. They contain no detectable 3-hydroxyproline, are highly glycosylated and all sugar residues are present as the disaccharides. The percentage of hydroxylation of the lysine is of the order of 70%. Only a third of these hydroxylysine residues are glycosylated. The significance of these peptides, present in a tissue substantially free of basement membrane, is discussed.     

Isolation of non-myelin plasma membranes unique to white matter

A procedure is described for isolating two membrane fractions from rabbit spina-cord white matter enriched with 5′-nucleotidase, a nonspecific plasma membrane marker, 2′, 3′-cyclic nucleotide phosphohydrolase, an oligodendroglial plasma membrane marker, and acetylcholinesterase, an axonal plasma membrane marker. While the two membrane fractions exhibited similar enrichments with respect to cyclic nucleotide phosphohydrolase, enrichments of 5′-nucleotidase and acetylcholinesterase were significantly greater in the heavier membranes were not detected. Moreover, gray matter did not yield homologous membrane fractions in the gradient when subjected to the identical procedure, indicating that the two membrane fractions were unique to white matter. While electronmicroscopic examination revealed that both membrane fractions were contaminated with myelin, the heavier fraction was least contaminated and exhibited a fair degree of homogeneity with respect to single membrane vesicular profiles. It was concluded that both membrane fractions were enriched with oligodendroglial and axonal plasma membranes, with the heavier fraction containing significantly more axolemma.     

Interactions of cadmium and selenium in rat plasma in vivo and in vitro

⁷⁵Se and ¹⁰⁹Cd tracers were used to study the binding of Se and Cd to plasma proteins at various SeO₃^2? doses and times up to 24 h after the simultaneous subcutaneous administration of SeO₃^2? and CdCl₂ to adult male rats. The simultaneous injection of CdCl₂ and SeO₃^2? markedly increased both Se and Cd plasma levels over that in control animals. Gel permeation chromatography of plasma indicated that at all times up to 24 h Cd and Se were bound in an atomic ratio of approx. 1 : 1 in 330 000 and 130 000 dalton fractions. From 4 to 24 h, Cd and Se appeared in the 420 000 dalton fraction, also with an atomic ratio of approx. 1 : 1. The 330 000 dalton molecules appeared to have a maximal binding capacity for the Cd-Se complex at a concentration of approx. 30 μmol/ml of plasma, while the 130 000 and 420 000 dalton molecules show a higher binding capacity. Studies in vitro revealed that SeO₃^2? does not interact directly with Cd and plasma proteins. It is metabolized by erythrocytes to a form that interacts in an atomic ratio of 1 : 1 with Cd to form a protein-bound complex of 130 000 daltons.     

Self-regulation of membrane fluidity The effect of saturated normal and methoxy fatty acid supplementation on Tetrahymena membrane physical properties and lipid composition

Tetrahymena cells elongated and desaturated massive supplements of palmitic or lauric acid at nearly twice the rates employed by unfed cells, thereby maintaining constant the physical properties of their membrane lipids. However, when a mixture of the 9- and 10-monomethoxy derivatives of stearic acid was administered, these compounds were incorporated without further metabolism. The marked fluidizing effect of the phospholipid-bound methoxy-fatty acids elicited an immediate reduction in fatty acid desaturase activity, the pattern of change very similar to that induced by supplements of polyunsaturated fatty acids. The modulation of fatty acid desaturase activity by methoxy-acids clearly seems to be governed by embrane fluidity rather than by some form of end product inhibition of the type which might have been postulated to explain the similar effect caused by polyunsaturated fatty acids.     

Properties of 3H-cimetidine binding in rat brain membrane fractions

In an attempt to characterize the brain histamine H₂ receptor, experiments were undertaken to study the binding properties of (N-methyl-³H) -cimetidine, an H₂ receptor antagonist, in rat brain membranes. Using a centrifugation assay, ³H-cimetidine binding having a K_d of 0.40μM and a Bmax of 3.9 pmoles/mg protein was detected. Of fourteen anions and cations tested, one, Cu⁺⁺, dramatically increased specific ³H-cimetidine binding, the increase being due mainly to a change in Bmax. Studies of substrate specificity for ³H-cimetidine binding revealed that Cu⁺⁺, while not significantly affecting the potency of H₂ receptor agonists and antagonists, dramatically decreases the potency of H₁ receptor substances on the ³H-cimetidine binding site. In addition, both the relative and absolute potencies of various H₂ receptor agonistsv and antagonists in displacing the ligand in the presence of Cu⁺⁺ parallels their potencies in biological systems. These findings suggest that, under these conditions, ³H-cimetidine may be labelling a biologically relevant H₂ binding site in brain and that Cu⁺⁺ may regulate the substrate specificity for this site. The brain regional distribution and kinetic analysis of the binding suggest that it is not localized solely to the synaptic receptor for histamine, but may also be associated with histamine receptors at other neuronal, glial or vascular sites.     

Diurnal variations in inducibility of rat liver tyrosine aminotransferase activity.

The activity of tyrosine aminotransferase (TAT) was measured in the livers of rats which were entrained to eat for the first 2 hours of a daily 12 hour dark period (‘2+22’ schedule) and were treated with the synthetic glucocorticoid dexamethasone and with glucagon at several times of day. TAT activity in untreated animals varies diurnally with a maximum 4 to 6 hours after the beginning of feeding. In both fed and fasted rats there was a small diurnal variation in inducibility by dexamethasone: in fed rats induction was greatest near the beginning of the dark period, shortly after feeding; in fasted rats induction increased towards the end of the dark period. Glucagon induction showed a marked diurnal variation in fed rats with a decrease coincident with the decline in control TAT activity after its food-induced peak. This variation did not appear to be depemdent on food intake, however, since the decline in inducibility occurred in fasted rats at the same time as in fed rats. Co-treatment with dexamethasone did not affect the decrease in glucagon inducibility. The diurnal variation in TAT induction may reflect a diurnal rhythm in the components of the enzyme synthesizing system (e.g. in the availability of mRNA or in enzyme degradation).     

Simultaneous single isotope radioenzymatic assay of plasma norepinephrine,epinephrine and dopamine

Modification of the original single isotope radioenzymatic assay of Passon and Peuler (1) permits the direct and simultaneous analysis of norepinephrine, epinephrine and dopamine in plasma samples of 50 μl or less. Plasma or cerebrospinal fluid without prior extraction of catecholamines or deproteinization is added directly into a mixture of 100 μl. This catechol-O-methyl-transferase-catalyzed assay is sensitive to 1 pg (20 pg/ml of plasma) for norepinephrine and epinephrine and 6 pg (120 pg/ml) for dopamine. A rapid thin layer chromatographic separation of the three ³H-methylcatecholamines contributes to the excellent specificity of the differential assay of the three catecholamines. The differential analysis of 15–20 plasma samples can be completed easily within one day. A total assay which omits the chromatographic step and, thus, measures norepinephrine plus epinephrine at the same sensitivity can be completed in 20 samples in one-half a working day.     

A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts.

A radioisotope assay for the measurement of glutamine synthetase activity has been developed in which tandemly arranged ion-exchange columns of Dowex 1-acetate and Amberlite CG-50 (H⁺) are used to separate the product, [¹⁴C]glutamine, from unreacted [U-¹⁴C]glutamate and other labeled compounds, particularly γ-aminobutyrate, that are formed by competing reactions. The technique is sensitive, reproducible, and suitable for multiple determinations. The assay has been used successfully to measure glutamine synthetase activity in neural and nonneural tissues which contain appreciable amounts of glutamate decarboxylase activity.     

The effect of A13+ on the physical properties of membrane lipids in Thermoplasma acidophilum.

Using Electron Paramagnetic Resonance Spectroscopy, Al³⁺ was shown to produce a dramatic decrease of membrane lipid fluidity on the microorganism $\text{Thermoplasma}\text{acidophilum}$ at a pH > 2. The ability of Al³⁺ to alter lipid fluidity was enhanced with increasing pH (from 3 to 5). At pH 4, 10^?2 M Al³⁺ increased the lower lipid phase transition by 39°C, and a detectable change was observed with AlCl₃ concentrations as low as 10^?5 M. The ability of Al³⁺ to increase the lower lipid phase transition temperature of $\text{T.}\text{acidophilum}$ is the largest of any cation/lipid interaction yet reported.     

Assay,properties, and regulation of rat ovarian δ-aminolevulinic acid synthetase activity

δ-Aminolevulinic acid synthetase (EC 2.3.1.37) has been detected in homogenates of rat ovaries. Optimal substrate and coenzyme concentrations, and parameters for assay of ovarian δ-aminolevulinic acid synthetase have been determined. Subcellular fractionation studies have shown that enzyme activity is predominantly localized in the mitochondrial fraction. Fasting, which is known to increase enzyme activity in the adrenal and to have no effect on activity in the testis, had no effect on enzyme activity in the ovary. Administration of the hepatic inducer allylisopropylacetamide or the hormone progesterone failed to alter activity of the ovarian enzyme. The activity of the enzyme was significantly increased during the diestrus-1 phase of the estrus cycle, during pregnancy, and by human chorionic gonadotropin at 24 and 48 h, suggesting that ovarian δ-aminolevulinic acid synthetase and the synthesis of heme may be under hormonal control.     

Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.     

Avian fatty acid synthetase: Evidence for short-term regulation of activity

Liver biopsies were performed on starved chicks at 0 and 4 h after refeeding a fat-free diet. Fatty acid synthetase activity increased after refeeding, and administration of cycloheximide did not prevent the rise of enzyme activity. Incorporation of [carboxyl-¹⁴C]leucine into fatty acid synthetase was measured in enzyme purified from the livers of starved chicks, starved-refed (4 h) chicks, and starved-refed chicks injected with cycloheximide. The data suggest that the synthesis of enzyme protein was inhibited in starved and cycloheximide-treated refed chicks in comparison with refed chicks. Liver cytosol from fed or starved chicks was filtered through centrifuge ultrafiltration membranes and the residues were suspended in the same or opposite filtrates. Fatty acid synthetase activity in residues from starved chicks was stimulated when suspended in filtrates from fed chicks. The evidence is consistent with the hypothesis that a portion of the fatty acid synthetase in the liver of starved chicks is present as an inactive form which can be activated upon refeeding.     

Preparation of 125I-calmodulin with retention of full biological activity: Its binding to human erythrocyte ghosts

Of several methods employed for preparing ¹²⁵I-calmodulin, only the glucose oxidase-lactoperoxidase system under controlled conditions produced an iodinated derivative which retained complete biological activity. Unlabeled calmodulin and ¹²⁵I-calmodulin stimulated cyclic nucleotide phosphodiesterase from bovine brain interchangeably and both proteins displaced ¹²⁵I-calmodulin from high-affinity binding sites on human erythrocyte ghosts with equal effectiveness. This procedure yielded a labeling stoichiometry of 1.34. Scatchard plots of binding of ¹²⁵I-calmodulin to ghosts were consistent with the presence of a single class of high-affinity binding sites with the properties expected of (Ca²⁺ + Mg²⁺)-ATPase molecules. The binding showed positive cooperativity and occurred only in the presence of Ca²⁺. The maximum amount of binding seen in Scatchard plots corresponded to 4.1 × 10³ sites per ghost.     

Changes of gamma-glutamyltranspeptidase activity in the rat during development and comparison of the fetal liver, placental and adult liver enzymes

Changes in the activity of gamma-glutamyltranspeptidase (GGT, EC 2.3.2.2) during development in rats were investigated. The activity of GGT in fetal liver increased rapidly immediately before birth, reached a maximum at birth and then decreased rapidly within a week after birth to nearly the level in adult rat liver. In contrast, placental GGT showed higher activity at an early stage (from day 13 to day 15) of the gestation period, but the activity decreased in the last part of fetal life. The activity in the amniotic fluid increased significantly just before birth, in parallel with the increase of activity in the fetal liver. No change of activity in the uterine wall was observed throughout gestation. The kinetic and immunological properties of partially purified GGTs from fetal liver and placenta were almost identical with those of adult liver GGT. However, the activity of soluble GGT in fetal liver was less effectively inhibited by antibody against adult kidney GGT. Thus, it is likely that at least one isozyme of GGT is present in the soluble fraction of fetal liver.     

The influence of chilling temperature alteration of glyoxysomal succinate levels on isocitratase activity from germinating seedlings

Succinate inhibited isocitratase activity to a greater extent in isolated glyoxysomes of cold-sensitive cotton than in cold-tolerant castor bean seedlings. Exposure of either isolated glyoxysomes or intact seedlings prior to glyoxysome isolation to 5°C appeared to alter the permeability of the glyoxysomal membranes to succinate. Endogenous succinate accumulated in glyoxysomes from 5°C treated cotton seedlings but not in similarly treated castor bean seedlings.     

Cumene hydroperoxide and yeast cytochrome P-450: spectral interactions and effect on the genetic activity of promutagens.

Cells of $\text{Saccharomyces}\text{cerevisiae}$, harvested from log phase cultures, contain cytochrome P-450 and are capable of activating promutagens to products that are genetically active in the same cell. The effect of cumene hydroperoxide, a compound known to support cytochrome P-450-mediated reactions, on the activation of a variety of the promutagens was investigated. In all cases the genetic activity of the promutagens was increased. With dimethyl-nitrosamine as the promutagen, the increased rate of gene conversion was linear for at least 1 hr. Yeast cytochrome P-450 was stable in intact cells in the presence of cumene hydroperoxide. However, in microsomal preparations the cytochrome was rapidly destroyed. When cumene hydroperoxide was added to a suspension of intact yeast cells, a spectrum with a Soret maximum at 455 nm — indicative of an interaction with cytochrome P-450 — was observed.     

NADH-cytochrome c reductase activity in cultured human lymphocytes: Similarity to the liver microsomal NADH-cytochrome b5 reductase and cytochrome b5 enzyme system

A NADH-cytochrome c reductase activity was increased upon mitogen stimulation of human lymphocytes. The activity was not inhibited by antimycin A or rotenone but was specifically inhibited by antibodies elicited against rat liver NADH-cytochrome b₅ reductase or cytochrome b₅. The activity was linear with cellular homogenates up to 5.2 × 10⁶ cells/ml and had abroad pH optimum of 7.7. The presence of 3-methylcholanthrene in mitogen stimulation media had no effect on the NADH-cytochrome c reductase activity but differentially induced the benzo(a)pyrene hydroxylase (AHH) activity. The reductase activity was present in nonstimulated cells and appears not to be significantly increased in activity per cell upon mitogen-stimulation of the peripheral lymphocyte.