Two functional states of sarcoplasmic reticulum ATPase.

The "total" ATPase activity of rabbit sarcoplasmic reticulum (SR) vesicles includes a Ca2+-independent component ("basic") and Ca2+-dependent component ("extra"). Only the "extra" ATPase is coupled to Ca2+ transport. These activities can be measured under conditions in which the observed rates approximate maximal velocities. The "basic" ATPase is predominant in one of the various SR fractions obtained by prolonged density-gradient centrifugation of SR preparations already purified by repeated differential centrifugations and extractions at high ionic strength. This fraction (low dnesity, high cholesterol) has a protein composition nearly identical with that of other SR fractions in which the "extra" ATPase is predominant. In these other fractions the ratio of "extra" to "basic" ATPase activities is temperature dependent, being approximately 9.0 at 40 degrees C and 0.5 at 4 degrees C. In all the fractions and at all temperatures studied, similar steady-state levels of phosphorylated SR protein are obtained in the presence of ATP and Ca2+. Furthermore, in all cases the "basic" (Ca2+-independent) ATPase acquires total Ca2+ dependence upon addition of the nonionic detergent Triton X-100. This detergent also transforms the complex substrate dependence of the SRATPase into a simple dependence, displaying a single value for the apparent Km. The experimental findings indicate that the ATPase of rabbit SR exists in two distinct functional states (E1 and E2), only one of which (E2) is coupled to Ca2+ transport. The E1 in equilibrium E2 equilibrium is temperature-dependent and entropy-driven, indicative of its relation to the physical state of the ATPase protein in its membrane environment. Thenonlinearity of Arrhenius plots of Ca2+-dependent ("extra") ATPase activity and Ca2+ transport is explained in terms of simultaneous contribtuions from both the free energy of activation of enzyme catalysis and the free energy of conversion of E1 to E2. Thermal equilibrium between the two functional states is drastically altered by factors which affect membrane structure and local viscosity.     

Active oligomeric states of pyruvate decarboxylase and their functional characterization.

Homomeric pyruvate decarboxylase (E.C 4.1.1.1) from yeast consists of dimers and tetramers under physiological conditions, a K(d) value of 8.1 microM was determined by analytical ultracentrifugation. Dimers and monomers of the enzyme could be populated by equilibrium denaturation using urea as denaturant at defined concentrations and monitored by a combination of optical (fluorescence and circular dichroism) and hydrodynamic methods (analytical ultracentrifugation). Dimers occur after treatment with 0.5 M urea, monomers with 2.0 M urea independent of the protein concentration. The structured monomers are catalytically inactive. At even higher denaturant concentrations (6 M urea) the monomers unfold. The contact sites of two monomers in forming a dimer as the smallest enzymatically active unit are mainly determined by aromatic amino acids. Their interactions have been quantified both by structure-theoretical calculations on the basis of the X-ray crystallography structure, and experimentally by binding of the fluorescent dye bis-ANS. The contact sites of two dimers in tetramer formation, however, are mainly determined by electrostatic interactions. Homomeric pyruvate decarboxylase (PDC) is activated by its substrate pyruvate. There was no difference in the steady-state activity (specific activity) between dimers and tetramers. The activation kinetics of the two oligomeric states, however, revealed differences in the dissociation constant of the regulatory substrate (K(a)) by one order of magnitude. The tetramer formation is related to structural consequences of the interaction transfer in the activation process causing an improved substrate utilization.     

Pathology and physiology of microbes. III. Criteria for assessing functional states

The study of different characteristics of the vital activity of microorganisms, made on type B Clostridium perfringens taken as an example, has disclosed the difficulty of evaluating the functional state of microorganisms only on the basis of the traditionally used characteristics of the population activity: the specific growth rate, the toxicity of the culture fluid and the antigenic properties of the culture. The economic and metabolic coefficients have been found to be the most suitable criteria for such evaluation; the state of the maximum physiological activity of the population is characterized by the maximum values of the economic coefficient and the minimum values of the metabolic coefficient. The minimum values of the economic coefficient and the maximum values of the metabolic coefficient are characteristic of the pathological state of the population.     

Osteoblasts during various functional states

At the electron microscopical investigation of osteoblasts in different zones of osteogenesis (enchondral foci, metaphyses, endosteum) in the rat and rabbit femoral bone it has been revealed that their population is heteromorphic. As demonstrate cytochemical data and radioautography, using 3H-uridine, 3H-glycine, 35S-sulfate, 45Ca, results of measurements in osteoblast population, 4 morpho-functional states (or types) are defined. In areas of an intensive osteoplastic process there are young osteoblasts (I type), mature functionally active osteoblasts (II type), osteoblasts with a hypertrophic endoplasmic network (cell-depots of the secrete--III type). In preosteoblasts and osteoblasts of the I type a higher than in osteoblasts of other types intensity of 35S-sulfate incorporation and alkaline phosphatase activity is revealed. In osteoblasts of the II type processes of biosynthesis of collagenous proteins predominate. Osteoblasts of the III type are subjected to destruction during secretion process. In the areas where osteopoesis is dying away, osteoblasts of the I and II types transform into a poorly active state, concerning specific biosynthesis (osteoblasts of the IV type). Presence of osteoblasts having various functional states in the areas of intensive osteopoiesis, depends on certain asynchronity of specific processes of biosyntheses, that occur in them. The morpho-functional states described are regarded as a specific peculiarity in function of collagene-producing cells.     

Ultrastructural characteristics of osteoclasts in different functional states

By electron microscopy and autoradiography, a study was made of osteoclasts in the metaphyseal zones of the femora of 1-7-day old rabbits and rats. It was established that depending on the intensity of resorptive processes osteoclasts noticeably differed in their morphology (shape, size, number of nuclei, degree of development of intracellular organelles, "brush border"), as well as in the level of biosynthetic activity registered by incorporation intensity of 3H-uridine and 3H-methionine in these. According to these indices, osteoclasts were classified as young, mature functionally active, and non-active osteoclasts, as well as perishing ones. The defined morpho-functional states represent successive stages of the life cycle of osteoclasts.     

Nonstationarity and duration of the R-R-segment in the diagnostics of functional states of operators

The effect of the nonstationarity of R-R interval series on the diagnostics of functional states of operators has been analyzed. The functional states were diagnosed by means of a factor model of heart rate variability. The heart rate was recorded in the supine position, before the performance of an important task, and after its completion. A high resistance of the diagnostics of functional states to nonstationarity was found for all periods. Indices of heart rate variability resistant to nonstationarity were defined. Also, the effect of R-R segment duration on functional states diagnostics was explored. The results obtained allow one to conclude that the diagnostics of functional states based on the three-factor model of heart rate variability can be used on short segments within a range of 256 divided 32 R-R intervals. The indices of the factor model of heart rate variability must be normalized for corresponding R-R segment duration before diagnostics. In addition, the effect of the duration of R-R segment on the indices of heart rate variability was analyzed for different functional states. The indices resistant to the duration of R-R segments and conditions necessary for heart rate recording were defined.     

Isozymic patterns and functional states of cell lines ofDrosophila melanogaster cultured in vitro. II

Summary Our previous isoenzyme investigation ofDrosophila melanogaster cell lines in vitro has been completed with twelve further enzyme systems. The enzyme profiles seem to be in good agreement with a previous hypothesis concerning the precise origin of these cell lines (probably from imaginal discs or nervous tissues). Our results have been summarized with reference to the biochemical genetic map ofDrosophila melanogaster in order to consider a possible functional organization of the genome.Abbreviations NAD nicotine adenine nucleotide - NADP nicotine adenine nucleotide phosphate - NBT nitroblue tetrazolium - PMS phenazine methosulfate - EDTA ethylene diamine tetraacetic acid - GOT Glutamate-oxaloacetate transaminase - PGK Phosphoglycerate kinase - GPDH -glycerophosphate dehydrogenase - MDH Malate dehydrogenase - PGM Phosphoglucomutase - Aph Alkaline phosphatase - MDH-NADP Malic enzyme - Lap Leucine Amino-Peptidase - LDH Lactate dehydrogenase - -1-OHDH L-3-hydroxyacid dehydrogenase - ADH Alcohol dehydrogenase - Aldox Aldehyde oxydase - 6PGD 6 Phosphogluconate dehydrogenase - G6PD Glucose-6-Phosphate dehydrogenase - Hex3 Fructokinase - IDH Isocitrate dehydrogenase - Est 6 Esterase 6 - Est C Esterase C - ODH Octanol dehydrogenase - XDH Xanthine dehydrogenase - AcPh Acid Phosphatase 1     

Nitric oxide and NO-synthases in mammals in different functional states

This review deals with the physical and chemical properties of nitric oxide as well as with the mechanisms and enzymes synthesizing this compound in animals including humans. The cytotoxic, vasodilatory, neuromediatory, and other properties of NO are analyzed. Polyfunctionality of NO in the norm and in pathologies of different genesis is shown. It is suggested that the analysis of the mechanisms of cyclic conversion of nitric oxide and the elucidation of the role of all NO metabolism products in living organisms would allow us to approach a more profound understanding of the NO problem in biology and medicine. It is reasonable to think the knowledge obtained in the course of the studies will permit an elaboration of the strategy and tactics of medical treatment of many diseases occurring on the background of disturbance in the mechanisms of formation and utilization of this compound.     

Structural studies of the archaeal MCM complex in different functional states

The primary candidate for the eukaryotic replicative helicase is the MCM2-7 complex, a hetero-oligomer formed by six AAA+ paralogous polypeptides. A simplified model for structure-function studies is the homo-oligomeric orthologue from the archaeon Methanothermobacter thermoautotrophicus. The crystal structure of the DNA-interacting N-terminal domain of this homo-oligomer revealed a double hexamer in a head-to-head configuration; single-particle electron microscopy studies have shown that the full-length protein complex can form both single and double rings, in which each ring can consist of a cyclical arrangement of six or seven subunits. Using single-particle techniques and especially multivariate statistical symmetry analysis, we have assessed the changes in stoichiometry that the complex undergoes when treated with various nucleotide analogues or when binding a double-stranded DNA fragment. We found that the binding of nucleotides or of double-stranded DNA leads to the preferred formation of double-ring structures. Specifically, the protein complex is present as a double heptamer when treated with a nucleotide analogue, but it is rather found as a double hexamer when complexed with double-stranded DNA. The possible physiological role of the various stoichiometries of the complex is discussed in the light of the proposed mechanisms of helicase activity.     

Electroorientation effect of isolated mitochondria in different functional states

For the first time, the electroorientation effect of isolated rat liver mitochondria has been investigated in a nonuniform alternating electric field at frequencies from 50 to 10(7) Hz. Two relaxation regions of mitochondria electroorientation have been revealed; they are due to the presence of low-frequency alpha - and high-frequency beta-dispersion. It has been shown that incubation of mitochondria in a medium with a low electroconductivity as well as in the presence of ionophores (valinomycin, 2,4-dinitrophenol) results in a decreased electroorientation value in the region of the high-frequency structural polarization. This is explained by a marked lowering of the effective electroconductivity of mitochondria due to changes in the permeability of the inner membrane of organelles. Upon the addition of ionophores, the decrease in the electroorientation effect value becomes more pronounced. Inhibition of respiration of mitochondria by antimycin A resulted in a slight diminution of the effect, followed by time-dependent stabilization, probably due to a lowered electroconductivity of the mitochondrial matrix, induced by the reduction of pH in the intramitochondrial space, change in the ionization of inner enzyme structures, and a partial limitation of the inner ion mobility.