Isolation and identification of two protein-protein crosslinks within the two subunits of Bacillus stearothermophilus ribosomes

Bacillus stearothermophilus 30S and 50S ribosomal subunits were isolated and crosslinked with diepoxybutane. The crosslinked proteins were extracted with LiCl or with 67% acetic acid and purified by a combination of different high performance liquid chromatography techniques. The protein fractions were analysed by two-dimensional and diagonal polyacrylamide gel electrophoresis and by immunological methods. Two crosslinked protein pairs, one from the large and one from the small subunit, consisting of proteins L23-L29 and S13-S19 respectively, were isolated in milligram amounts for determination of the crosslinked amino acids.     

Studies of enzyme-ligand complexes using dynamic fluorescence anisotropy. II. The coenzyme-binding site of malate dehydrogenase

The coenzyme-binding site in mitochondrial malate dehydrogenase from pig heart was studied using dynamic fluorescence anisotropy decay. The dynamics of the fluorescent ligands NADH and 6-cyano-7-hydroxy-4,8-dimethylcoumarin were used to detect conformational changes at the dihydronicotinamide-and at the adenosine-binding sites, respectively. Addition of the natural substrate L-malate to the complex from enzyme and NADH does not influence the complete immobilization of the dihydronicotinamide group, whereas the stereoisomer D-malate and the substrate-analogue hydroxymalonate form ternary complexes with highly mobile dihydronicotinamide. The dynamics of the fluorescent adenosine-analogue are not influenced by formation of complexes with substrate and substrate-analogues. Thus the conformational changes at the dihydronicotinamide-binding site remain local and are not transmitted to the adenosine-binding site.     

"Allosteric regulation" of calcium-uptake in rat liver mitochondria

During investigations of calcium uptake by rat liver mitochondria, at a buffered free calcium concentration of 2 microM, a considerable acceleration of calcium uptake was occasionally observed. From the following experiments it can be concluded that the acceleration occurred when mitochondria had become anaerobic, and hence deenergized, because they had been stored in the refrigerator for a while. Mitochondria which had become transitorily deenergized by blocking the respiratory chain with KCN, rotenone or antimycin showed an accelerated calcium uptake when the membrane potential necessary for calcium uptake was regenerated. This acceleration of calcium uptake was also seen when a potassium diffusion potential was induced by valinomycin in previously deenergized mitochondria. The velocity of calcium uptake in transitorily deenergized mitochondria increased irrespective of the presence of magnesium in the incubation medium. The activation of the Ca uniporter was reversible, and both processes, activation and deactivation, were time-dependent and developed within a time span of minutes. Oligomycin strongly inhibited the deactivation of the uniporter by ATP, hence the membrane potential is intrinsically effective and does not act via ATP. The altered kinetics of the Ca uniporter were responsible for the acceleration of calcium uptake which was measured at low calcium concentration with previously deenergized mitochondria. The dependence of the rate of calcium uptake on the concentration of calcium in the medium is hyperbolic in transitorily deenergized mitochondria [Km = 6.7 microM; V = 455 nmol/(min X mg protein)] and sigmoidal in normal ones. It is additionally independent of the presence of magnesium ions. We found Hill coefficients of 3.47 and 2.94 in experiments with and without magnesium, respectively. Correspondent kinetics, hyperbolic in deenergized and sigmoidal in normal mitochondria, were obtained when calcium uptake was not driven by the system of respiratory chain, but by the potassium diffusion potential induced by valinomycin. The alteration in the kinetics of the Ca uniporter has consequences in the range of physiological calcium levels, but mainly in pathological states of liver cells. These points are discussed.     

Inorganic pyrophosphatase activity of the mouse spleen in the immune response and after treatment with bis-phosphonates

The inorganic pyrophosphatase activity was determined in different tissues of mice. The immunization of mice by sheep erythrocytes increased the inorganic pyrophosphatase activity of the spleen. The in vivo administration of bisphosphonates (40 mg per 1 g of mass), which are structural analogs of inorganic pyrophosphate (methylene bisphosphonic acid--MBPA, hydroxyethylidene bisphosphonic acid--HEBPA and aminomethylene bisphosphonic acid--AMBPA), inhibited the inorganic pyrophosphatase activity only by MBPA in the thymus and spleen but not in liver. The addition of MBPA, HEBPA as well as of phosphonoacetic acid, imidobisphosphate, bis(phosphonomethyl)-phosphonic acid, MBPA and phosphoric acid monoanhydride to cytosol from the mouse spleen led to the competitive (relative to the [Mg (PPi)2-] complex) inhibition of the inorganic pyrophosphatase activity. AMBPA didn't possess the analogous effect.     

Effect of thiochrome capronate on lipid metabolism in rats

Thiochrome caproate modified by the oxyethyl radical of the thiochrome derivative was studied for its effect on different indices of lipid metabolism in the liver and blood of albino rats. It was shown that when animals were fed on a standard laboratory diet, thiochrome caproate changed the amount of total and free fatty acids in the studied tissues and the fatty acid composition in the liver to a greater extent than thiochrome and hydroxythiamine.     

Non-specific binding of protein-stabilized gold sols as a source of error in immunocytochemistry

The observation that protein-A conjugated gold sols bound to fibronectin-collagen (FNC) fibres in human fibroblast cultures prompted a series of studies on the binding of gold particles stabilized in various ways (Staphylococcal protein A, bovine serum albumin, avidin, streptavidin, gelatin, hemoglobin, polyethylene glycol (MW 20 000), methylcellulose and the nonionic detergent Tween 20) to cell and tissue components, to protein dot blots and SDS-PAGE blots on nitrocellulose paper. We found that binding of gold particles to certain cell and tissue components and to various immobilized proteins did occur irrespective of the stabilizing agent. We argue that, albeit gold sols are stabilized against salt coagulation by adsorption of proteins and other stabilizing agents, "naked areas" are (constantly or intermittently) present on particle surfaces, available for interaction with cell and tissue components that have a high electrostatic affinity for the charged gold surface under prevailing experimental conditions. Non-specific binding may be reduced or abolished by competing proteins (i.e. proteins with a higher affinity for gold than any component in the object studied) provided the proteins and the gold conjugate are present concomitantly during incubation. We found gelatin (Bloom number 60-100) to be an effective competitive protein probably due to its high affinity for gold over a wide pH range. Further, gelatin did not appreciably inhibit the specific interaction in dot blots between SpA and IgG except at very low IgG concentrations. A protocol for the use of gold-protein conjugates to circumvent the hazards of unspecific gold binding is suggested.     

Preparation and P content of non-pregnant and pregnant human uterine myosin

Myosin content and phosphorus (P) concentration of myosin preparations were measured in non-pregnant and pregnant human myometrial tissue specimens. It was found that the amount of myosin gained from 1 g of minced myometrial tissue is 0.5 mg in the early follicular phase of the menstrual cycle, 0.6-0.7 mg in the late luteal phase, and 6-7 mg during pregnancy. Considering the different functional stages of the myosin sources and the performance characteristics of the methods, the estimated myosin content of non-pregnant myometrium is 1.0-1.5 mg, while 10-15 mg in pregnant myometrial tissue. A considerable amount of P is bound to the preparations. It is the smallest in the post-menstrual period and increases towards the end of the cycle. The largest amount of P is gained from fresh pregnant uterine samples. Analysis of the alkaline hydrolysate showed that the phosphate group was bound to amino acids, in the largest amount to arginine, less to histidine and the smallest amount to lysine and serine. As a function of the duration of storage, especially the P-Arg concentration was decreasing. The prolonged hydrolysis time decreases again the concentration of P-Arg with a consecutive increase of No. 1 and 2 P-containing peaks in the chromatographic profile of alkaline hydrolysate.     

Effects of arginine-vasopressin and ACTH 4-10 on acquisition of active avoidance response in rats with alcohol pretreatment in prenatal and adult age

Alcohol administration in drinking water to mother rats during gestation resulted in a permanent learning deficit of the offspring when behavioural reactions were tested in active avoidance conditioned reflex situation in adult age. A similar deficit of learning capacity was observed in adult rats following alcohol administration for two weeks; the acquisition of active avoidance response was tested later. Administration of arginine-vasopressin and ACTH 4-10 during behavioural test led to a significant improvement of learning ability of the animals pretreated with alcohol. The observations indicate that the ethanol-induced deficit of learning capacity involves peptidergic mechanisms and the behavioural manifestations following chronic alcohol treatment are not irreversible processes.