Binding of the transition metal ion [(H20)(NH3)5Ru(II)]2+ to nucleosomal core and internucleosomal DNA

The goal of this study is to establish the nature of pentammineruthenium(III) binding to DNA in intact mouse liver nuclei. Also, we wish to determine whether the nucleosomal organization of mouse chromatin has a substantial effect on the relative Ru(III) binding levels of internucleosomal and nucleosomal core DNA. These questions are important because ammineruthenium compounds share chemical and biological properties with the cis-dichlorodiammineplatinum(II) or cisplatin chemotherapeutic agent. Therefore, they represent a potential class of new chemotherapeutic agents. We find that in intact nuclei the predominant DNA binding site for pentammineruthenium(II), followed by air oxidation to pentammineruthenium(III), is N-7 guanine, as is the case with cisplatin. Also, the Ru(III) distribution between internucleosomal and nucleosomal core DNA was found to be nearly identical as probed with three non-specific deoxyribonucleases.     

Hexokinase-binding properties of the mitochondrial VDAC protein: Inhibition by DCCD and location of putative DCCD-binding sites

The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin. The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD). At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity. The stoichiometry of [¹⁴C]-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer. A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence. Both of these sites are located near the C-terminal end of yeast VDAC (residues 257–265 and 275–283). These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase.     

On the mechanism of cellular cadmium uptake

Review of the available evidence on the mechanism of cellular Cd uptake in the rat jejunum supports the concept that this process consists of nonspecific binding to anionic sites on the membrane, followed by a temperature-dependent and rate-limiting internalization step. Because temperature-sensitive transmembrane movement of Cd can be demonstrated also in isolated brush-border vesicles and in erythrocyte ghosts, it is not likely to result from pinocytosis but may be related directly to membrane fluidity. There is no need to assume the existence of saturable Cd carriers, or competition of Cd with essential polyvalent cations for their specific transport systems. Uptake of Cd by tubular epithelium in the kidney of the intact rabbit appears to resemble that described for the jejunum, with the internalization step limiting the rate of uptake.     

Cholesterol binding reserve of hyperlipemic rat serum lipoproteins in chronic ethanol administration

Chronic administration of ethanol in rats caused the reduction of serum cholesterol binding reserve. The very low density and high density lipoproteins, main serum cholesterol binding reserves, were slightly increased with corresponding increases in their lipid and protein components during initial stage of alcohol consumption. However, these capacities get deminished during reversal of hyperlipemia induced by prolonged action of ethanol. This situation may be an early indicator for the initiation of hepatic damage and a variety of secondary effects of ethanol.     

Unusually strong immunological cross-reaction between elongation factor Tu of Escherichia coli and Bacillus subtilis

Polyclonal antibodies were prepared against the purified elongation factor Tu (EF-Tu) of Escherichia coli and Bacillus subtilis. Using the methods of Western blotting and microcomplement fixation the cross-reactivities of EF-Tu of 19 different prokaryotes were determined. The immunological distance were compared with the results of 16S rRNA oligonucleotide analysis. An unexpectedly high cross-reactivity was revealed between the EF-Tu of B. subtilis and the antiserum against the EF-Tu of E. coli. A comparison of the predicted amino acid sequences from the tuf-genes of E. coli and B. subtilis yielded two identical peptide fragments that are likely candidates for antibody binding sites.Abbreviations EF-Tu elongation factor Tu - GDP guanosine 5-diphosphate - GTP guanosine 5-triphosphate - MCF microcomplement fixation - T type strain     

CD3/T-cell receptor coupling to a pertussis and cholera toxin-insensitive G-protein

We have analyzed the effect of CD3/T-cell receptor stimulation on GTP hydrolysis and GTP binding. We show that stimulation of Jurkat, T-cell, membranes with OKT3 results in a 50% increase in GTP hydrolysis which is specifically inhibited by GDP. Pretreatment of the membranes with neither pertussis toxin nor cholera toxin inhibited the GTP hydrolysis. We also show that stimulation with OKT3 increases the binding of GTPγS to Jurkat membranes. These data strongly implicate the involvement of a G-protein in CD3/T-cell receptor signalling.     

The effect of glucose-6-phosphate isomerase genotype onin vitro specific activity andin vivo flux inMytilus edulis

Four samples of the musselMytilus edulis were taken between 1984 and 1987 from Stony Brook, New York, and used to study the glucose-6-phosphate isomerase (GPI) polymorphism in this species.In vitro specific activity andin vivo flux measured in the same animals were found to be significantly correlated. A significant effect of GPI genotype on flux was observed in one of the samples; overall, significant evidence of effect of genotype on enzyme activity was also obtained. GPI activities of common genotypes tend to deviate less from the population mean than those of rare (frequency less than 5%) genotypes. This suggests the possibility that rare GPI genotypes are rare as a consequence of having biochemical properties that deviate from an optimum level and, therefore, having a lower fitness. In support of this hypothesis, we found in one of our samples that shell length is a concave function of GPI activity with an intermediate optimum activity level. The financial support provided to P.J.N.S. by the Luso-American Educational Commission (Fulbright Program), the Instituto Nacional de Investigacao Científica (Portugal), and the Faculdade de Ciências da Universidade de Lisboa during several stages of this research is gratefully acknowledged. Financial support from the Ministerio de Educatión y Ciencia (Spain) in the form of a postdoctoral Fulbright/MEC fellowship to M.S. is also gratefully acknowledged. Research was supported by National Science Foundation Grant BSR-8415060 to R.K.K. This is contribution No. 736 from the Program in Ecology and Evolution, State University of New York at Stony Brook. On leave from Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Campo Grande C2, Lisboa, Portugal.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

Effects of phospholipases,proteases and neuraminidase on γ-hydroxybutyrate binding sites

Summary -Hydroxybutyric acid (GHB) is a natural compound of mammalian brain synthesized from GABA. The characteristics of its synthesis, transport, release, distribution and turnover, in addition to the presence of a high affinity binding site for this substance in brain are in favor of a modulator role for GHB. The effects of hydrolytic enzymes on the specific binding capacity of GHB have been studied in the present work. Phospholipases A₂ and C, neuraminidase and Pronase markedly decrease GHB binding to crude synaptosomal membranes from rat brain. This effect is time and enzyme concentration dependent. Trypsin, under the conditions employed, is less active. The inhibitory effects of phospholipases is correlated with phospholipid hydrolysis. Lysophospholipids, in the absence of bovine fatty acid free serum albumin partially inhibit GHB binding. The action of neuraminidase has been followed by sialic acid release and modifications of the ganglioside profile. The effects of phospholipase C and of neuraminidase are completely different to those on GABA binding sites. These results represent further data concerning the molecular existence of specific GHB binding sites on rat brain membranes.Abbreviations GHB -hydroxybutyrate - LPC L--lysophosphatidylcholine - LPE Lysophosphatidylethanolamine - PC Phosphatidylcholine - PE Phosphatidylethanolamine - BSA Bovine Serum Albumin     

Digitalis receptor sugar binding site characteristics: A model based upon studies of Na+, K+-ATPase preparations with differing digitalis sensitivities

The structure-activity relationships of the genin moieties of digitalis glycosides are commonly elucidated by determining the inhibitory potency of a variety of genins toward the plasma membrane Na⁺, K⁺-ATPase; qualitatively these relationships appear to be fairly independent of the specific Na⁺, K⁺-ATPase preparation utilized for the analysis. To determine whether this is the case with regard to the sugar moieties of glycosides, the inhibitory effects of 12 monoglycosides of digitoxigenin toward four Na⁺, K⁺-ATPase preparations of different origin were measured. It was found that while recognition of the major structural determinants of sugar activity appeared to be independent of enzyme source, recognition of the minor structural determinants of activity showed some source dependence. It was also observed that the intrinsic sensitivity to sugar potentiation may be source dependent and unrelated to intrinsic sensitivity to inhibition by digitoxigenin. These observations are compatible with a model of the Na⁺, K⁺-ATPase sugar binding site(s) in which intrinsic sensitivity to sugar attachment as well as recognition characteristics (for sugar structural features) both determine the extent to which a sugar moiety may contribute to the activity of monoglycosides. Further, in these studies one of the Na⁺, K⁺-ATPase preparations employed was obtained from rat brain, a tissue known to contain a mixture of ouabain sensitive and insensitive isoforms. We have observed that the rigorous purification techniques employed appear to have selectively removed from or denatured the less ouabain sensitive al isoform found in this enzyme preparation.