Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.

To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.     

Cold-induced hemolysis in a hypertonic milieu

Summary Suspension of human erythrocytes at 37° C in an environment made hypertonic by increasing concentrations of sodium chloride and sucrose was followed by hemolysis when the temperature was lowered to 0° C. Two distinct stages were involved in this hemolytic phenomenon, the first being incubation with hypertonic solute at some temperature above 20° C with an increasing effect up to 45° C, and the second stage consisting of lowering the temperature below 15° C with increasing hemolysis down to 0° C. The rate of cooling was not an important factor, but the presence of ions reduced the extent of cold-induced hemolysis in hypertonic sucrose. No significant release of membrane phospholipid and cholesterol accompanied this hemolysis. The solubilization of membrane protein components was investigated, with some differences appearing on sodium dodecyl sulfate polyacrylamide gel electrophoresis between hypertonic and isotonic supernatants. Spectrin could not be identified in solubilized form. Correlation of the temperatures of note in these studies with results from the literature on other biological effects of temperature-induced phase transitions in membrane lipids strongly points to the conclusion that such transitions are involved in the mechanism of cold-induced hypertonic hemolysis. It is postulated that the hypertonic milieu has resulted in membrane-protein alteration damage which prevents normal adaption to the new physical state of the membrane lipids during cooling.     

gamma-Vinyl GABA (4-amino-hex-5-enoic acid), a new selective irreversible inhibitor of GABA-T: effects on brain GABA metabolism in mice

Abstract— γ-Vinyl GABA (4-amino-hex-5-enoic acid, RMI 71754) is a catalytic inhibitor of GABA-T in vitro. When given by a peripheral route to mice, it crosses the blood-brain barrier and induces a long-lasting, dose-dependent, irreversible inhibition of brain GABA transaminase (GABA-T). Glutamate decarboxylase (GAD) is only slightly affected even at the highest doses used. γ -Vinyl GABA has little or no effect on brain succinate semialdehyde dehydrogenase, aspartate transaminase and alanine transaminase activities. GABA-T inhibition is accompanied by a sustained dose-dependent increase of brain GABA concentration. From the rate of accumulation of GABA it was estimated that GABA turnover in brain was at least 6.5 μmol/g/h. Based on recovery of enzyme activity the half-life of GABA-T was found to be 3.4 days, that of GAD was estimated to be about 2.4 days. γ -Vinyl GABA should be valuable for manipulations of brain GABA metabolism.     

The use of percoll gradients, elutriator rotor elution, and mithramycin staining for the isolation and identification of intraerythrocytic stages of plasmodium berghei

Intraerythrocytic parasites of Plasmodium vinckei and Plasmodium berghei were separated according to their developmental stages using discontinuous Percoll gradients. Contaminating nucleated blood cells such as leukocytes were removed by elutriation centrifugation. The stages were unequivocally identified in smears using a newly developed DNA-specific staining procedure with mithramycin and fluorescence microscopy. This stain can also be used to detect parasites in human blood of very low parasitemias. The combination of methods described has many possible applications in immunologic and biochemical parasite research.     

Uptake of neutral and acidic amino acids by Lemna gibba correlated with the H+-electrochemical gradient at the plasmalemma

Uptake rates of L-alanine, L-serine and L-aspartate and trans-membrane electrical potentials (Δψ) were determined for a pH range in the external medium between 3.5 and 9.0. The proton electrochemical gradients (     ) were calculated from Δψ, pH of the medium, and an assumed cytoplasmic pH of 7.5. At external amino-acid concentrations of 0.1 mol m⁻³, where carrier-mediated uptake dominates total uptake, a linear correlation between uptake rates and     is obtained, which extrapolates to zero uptake at zero     . This corroborates the contention that neutral and acidic amino acids are taken up by Lemna gibba L. by H⁺-cotransport.     

Amino acid uptake by Lemna gibba by a mechanism with affinity to neutral L-and D-amino acids

Earlier work suggested that amino acid uptake by Lemna gibba cells is a H⁺-cotransport mechanism driven by a proton-electrochemical gradient at the plasmalemma. The present investigations of the transient membrane depolarizations elicited by amino acids and tracer-uptake experiments show that all neutral -L-amino acids, D-alanine and analogues, like -alanine and p-fluorophenylalanine, are transported by the same system. It remains to be seen if there are separate mechanisms for the uptake of acidic and basic amino acids.     

Effect of alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on polyamine levels in rat tissues.

α-Difluoromethylornithine (RMI 71.782), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (E.C. 4.1.1.17) $\text{in vitro}$, causes a rapid, long-lasting, dose-dependent decrease of ornithine decarboxylase activity in prostate and, to a lesser extent, in thymus and testis of rats when injected intraperitoneally. Repeated doses (100 mg/kg or 1 g/kg) of α-difluoromethylornithine given to rats for two weeks markedly decreased polyamine concentrations in several rat tissues and selectively slowed down growth of ventral prostate and of thymus.     

Swelling and contraction of potato mitochondria

Mitochondria isolated from potato tubers fail to undergo passive osmotic swelling when suspended in isotonic Na⁺ acetate or phosphate, in NaCl following addition of tripropyltin, or in Na⁺ nitrate following addition of an uncoupler. Swelling under each of these conditions in mitochondria from other sources has been attributed to the inward movement of Na⁺ on an endogenous Na⁺/H⁺ exchanger. Such a monovalent cation/H⁺ exchanger has also been implicated in respiration-dependent cation extrusion and contraction of swollen mitochondria. Potato mitochondria swollen in chloride and nitrate salts extrude ions and contract when respiration is initiated. The contraction reaction is slower and less efficient than that in beef heart mitochondria, but like the latter, is sensitive to uncouplers and stimulated by nigericin, butacaine, and Mg²⁺. These comparative studies suggest that a cation⁺/H⁺ exchanger is present in potato tuber mitochondria, but that it functions exclusively as a cation-extruding mechanism. They further suggest that cation⁺/H⁺ exchange activity is not identical in mitochondria from different sources and that these exchange components may have a directionality and regulatory features which differ with the metabolic needs of the source tissue.     

The interaction of methemoglobin and modified poly-(hydroxyethylmethacrylates) studied by spectroscopic methods

The interaction between methemoglobin (MetHb) and macroporous matrices on the basis of polymethacrylates was investigated by means of optical and e.p.r. spectroscopy. The spectroscopic data show that the adsorption of MetHb to imidazole-containing matrices occurs by complex formation between matrix-bound imidazole and the iron of the prosthetic group, with all 4 polypeptide chains of the MetHb molecule being included in the interaction. The adsorption to hydrophobic side chains containing matrices leads, via the protein-matrix interaction, to considerable disturbances of iron protoporphyrin IX in equilibrium or formed from protein-contacts, which are of general importance with respect to the functional variablity and control, respectively, of iron porphyrins in hemoproteins. In case of matrix containing n-hexyl groups deoxyHb is oxidized by O2 to MetHb, instead of being oxygenated to HbO2. Not all prosthetic groups are able to bind N-3. With the increase in hydrophobicity of the matrix a conformational change is enforced leading in the beta-chains to the direct interaction between iron and sulfur of cysteine (beta-cys 92), as it is proved in all cytochrome P-450 and other model compounds.