Subcellular distribution of selenium during uptake and its influence on mitochondrial oxidations in germinatingVigna radiata L

The metabolic significance of Se in plants is not well documented, though the presence of many selenoenzymes in bacteria and the essentiality of Se in higher animals is established. Since germination is an active process in plant growth and metabolism, the effect of Se was investigated in germinatingVigna radiata L, a nonaccumulating Sedeficient legume. Growth and protein were enhanced in seedlings supplemented with selenium (Se) as sodium selenite in the medium up to 1 μg/mL. The pattern of uptake of⁷⁵Se in the differentiating tissues and the subcellular distribution were investigated. The percentage of incorporation of⁷⁵Se was greater in the mitochondria at the lowest level (0.5 μg/mL) of Se supplementation compared to higher levels of Se exposure. Proteins precipitated from the postmitochondrial supernatant fractions, when separated by means of polyacrylamide gel electrophoresis (PAGE), indicated a major selenoprotein in the seedlings germinated at 2.0 μg/mL Se. In seedlings grown with supplemented Se, enhanced respiratory control ratio and succinate dehydrogenase activity were observed in the mitochondria of tissues, indicative of a role for Se in mitochondrial membrane functions.     

The effect of Arg306-->Ala and Arg506-->Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S.

Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg306, Arg506, and Arg679. Site-directed mutagenesis of human factor V cDNA was used to substitute Arg306-->Ala (rfVa306A) and Arg506-->Gln (rfVa506Q). Both the single and double mutants (rfVa306A/506Q) were constructed. The activation of these procofactors by alpha-thrombin and their inactivation by APC were assessed in coagulation assays using factor V-deficient plasma. All recombinant and wild-type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVaPLASMA) in HBS Ca2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild-type factor Va (rfVaWT) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVaPLASMA. The rfVa306A and rfVa506Q mutants were each inactivated at rates slower than rfVaWT and fVaPLASMA. Following a 90-min incubation with APC, rfVa306A and rfVa506Q retain approximately 30-40% of the initial cofactor activity. The double mutant, rfVa306A/506Q, was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90-min incubation in the presence of APC. Recombinant fVaWT, rfVa306A, rfVa506Q, and rfVa306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVaWT and rfVa306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg506 is not affected by protein S. The initial rate of rfVa506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg306. Overall, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg306 and Arg506 are required for efficient factor Va inactivation.     

Prostaglandin receptors in rat kidney

The physiological effects of prostaglandins (PGs) are mediated through their interactions with specific binding sites (receptors) on effector cells. Since such receptors potentially regulate the action of PGs on the kidney, the distribution and properties of renal PG receptors in the rat were examined. The distribution of PGE2, PGE1, and PGF2 alpha receptors along the nephron was not uniform; the outer medulla had by far the greatest density of sites, followed by the inner medulla and cortex. Receptors were found exclusively in the particulate fractions, of which the 40,000g pellet had the highest specific activity. In the outer medulla, receptor density calculated from Scatchard plots was 2.12 pmol/mg for PGE2, 1.12 for PGE1, and 0.44 for PGF2 alpha; the KD's were similar for all prostaglandins. The conditions for optimal in vitro binding of PGE2 and PGF2 alpha by outer medullary membranes were investigated. In vivo administration of 16,16'-dimethyl-PGE2 resulted in a dose-dependent "down" regulation of PGE2 binding to outer medullary membranes due to changes in both the number and affinities of receptors. Changes in the numbers and/or properties of PG receptors may be an important mechanism for regulating the effects of PGs and renal function under normal and pathologic conditions.     

Phosphorylation of the androgen receptor by a nuclear cAMP-independent protein kinase

The androgen receptor was purified from rat ventral prostate. The purified receptor migrated as a single band of mol. wt. 87000 on SDS-polyacrylamide gels, had a kd for R-1881 (17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one) binding as 6 nM, and sedimentation coefficient of 4.5 S. Phosphorylation of the purified receptor was studied by incubating it with [gamma-32P]ATP in the presence of several purified protein kinases including cAMP-dependent protein kinase, and four cAMP-independent protein kinases (which were active towards substrates such as phosvitin and casein). Phosphorylation of the 87000 mol. wt. androgen receptor protein occurred only in the presence of a nuclear cAMP-independent protein kinase (of the N2 type). No auto-phosphorylation of the receptor was detected. The results indicate that the androgen receptor is a phosphoprotein. Further, phosphorylation of the androgen receptor by only a specific nuclear cAMP-independent protein kinase may be important in determining the dynamics of its function.     

A spectrophotometric method for the determination of the kinetic parameters of NH+4 uptake by yeast cells

Abstract The kinetic parameters of NH⁺₄-uptake in yeast cells were determined by a method that is based on the following changes in the external NH⁺₄ concentration in cell suspensions by using NADH-dependent glutamate formation from NH⁺₄ and 2-oxoglutarate. The kinetics of the observed NADH oxidation were analyzed by computer and enabled an estimation of V _max and K _m of the NH⁺₄-uptake system of the cells.     

Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review

A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed.     

The effect of inorganic phosphate on sodium fluxes in dog red blood cells

The effect of extracellular inorganic phosphate on Na⁺ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na⁺ efflux increases by 2- to 3-fold and Na⁺ influx increases approximately 2-fold. This enhancement of Na⁺ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4′-iso-thiocyantostilbene-2,2′-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na⁺ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na⁺ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.     

Modification of an essential arginine of carbamate kinase

Carbamate kinase from Streptococcus faecalis is inactivated by butanedione in borate buffer, which implies the presence of an essential arginine at the active site of the enzyme. The inactivation reaction is first order in [butanedione] and a replot of the inactivation rate data infers that one arginine is modified. The enzyme is protected against inactivation by ADP, ATP, the metal-nucleotides and carbamyl phosphate but not by carbamate. Amino acid analyses reveal that one of three arginines is modified by butanedione in the absence of protecting agents, and the binding of ADP to the enzyme prevents modification. Thus, analysis of the data suggest that (i) substrate binding to arginine and (ii) protein conformational changes at the active site are responsible for protection of an essential arginine against modification by butanedione.     

Kinetics of formation of ferrioxamine B

Stopped-flow kinetic studies of the formation of ferrioxamine B were performed. Formation of the complex follows the rate law

where K_a is the acid dissociation constant of the iron(III) aquo species in 0.1 M formate buffer. At 25°C k₁ = 3.94 × 10²M^?1 sec^?1, k₂K_a = 1.18 × 10^?1 sec^?1, k₃ = 3.6 × 10^?1 sec^?1. Activation parameters for k₁ are ΔH^≠ = 11.7 kcal mole^?1 and ΔS^≠ = ?8 cal K^?1 mole^?1. An associative mechanism is proposed. Attachment of the first chelate ring is the slow step and favorably positions the second chelate ring for attachment. Coordination of two chelate rings favorably positions the third chelate ring for attachment. These results are compared to kinetics of formation of model complexes and to a previous study of the formation of ferrioxamine B in which attachment of the third chelate ring was proposed as the slow step