Mathematical model of the activation of prothrombin by factor X a and factor V t

A mathematical model of prothrombin activation is being proposed which includes the feedback mechanism of thrombin and the alteration of factor V by thrombin. This model is in good agreement with experimental data for the dependence of the rate of thrombin formation on the concentrations of factors V and X _a . In particular, it correctly predicts the existence and location of a maximum in both of these cases.     

Interaction of protein kinase C with phosphatidylserine. 2. Specificity and regulation.

The roles of specific and nonspecific interactions in the regulation of protein kinase C by lipid have been examined. Binding and activity measurements reveal two mechanisms by which protein kinase C interacts with membranes: (1) a specific binding to the activating lipid phosphatidylserine and (2) a nonspecific binding to nonactivating, acidic lipids. The specific interaction with phosphatidylserine is relatively insensitive to ionic strength, surface charge, and the presence of nonactivating lipids. The two second messengers of the kinase, diacylglycerol and Ca2+, increase markedly the affinity of the kinase for phosphatidylserine. In contrast, the nonspecific interaction is sensitive to ionic strength and surface charge, and is unaffected by diacylglycerol. These results suggest that electrostatic interactions promote the binding of protein kinase C to membranes but the cooperative and selective binding of phosphatidylserine is the dominant driving force in a productive protein-lipid interaction.     

Methylated poly(L-lysine): conformational effects and interactions with polynucleotides

Methylated lysine, arginine and histidine residues are found in a number of proteins (for example, histones, non-histone chromosomal proteins, ribosomal proteins, calmodulin, cytochrome C, etc.). We are studying the effects of methylation on the conformations of poly(lysine) and of the effects of methylation of poly(lysine) and poly(arginine) on interactions with polynucleotides. The conformational properties of epsilon-amino-methylated poly(lysine) differ from those of unmodified poly(lysine). Methylation increases resistance to thermally-induced and NaCl-induced changes in the CD spectrum. Guanidinium chloride increases (proportional to the degree of methylation) the extent of approach to the conformation in dispute as to its being a random coil or an extended helix. Methylation enhances aggregation in the helix-inducing solvent 0.5 M Ca(ClO4)2. With increasing methylation of poly(lysine), the conformation in dodecyl sulfate changes from beta, to 50% alpha, to random coil at the maximum methylation. Increasing methylation of poly(lysine) weakens the interaction with polynucleotides in respect to dissociation by salt, linearly with methyl content. Complexes of (dAdT)n.(dAdT)n with the polypeptides are increasingly stabilized to heat denaturation by progressive methylation. However, with a series of synthetic double-stranded RNA's and DNA's a more complex situation exists, Tm increasing or decreasing, depending on the base composition, sequence and type of sugar. Methylation of poly(lysine) and poly(arginine) can have opposite effects on Tm based on results with complexes with (dI)n.(dC)n. Methylated poly(lysine) affects the CD spectrum of polynucleotides, in a manner dependent on base composition and sequence. In some cases large positive or negative psi-spectra are induced, which, in the case of (dGdC)n.(dGdC)n, can be positive or negative depending on the degree of methylation of the polypeptide and the salt concentration. It is suggested that the biological effects of methylation proteins may be evoke by salt changes in the cell cycle, and that methylation can affect local interactions with nucleic acids and larger scale structure, and interactions with lipids.     

Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions

Salmonella typhimurium histidinol dehydrogenase produces histidine from the amino alcohol histidinol by two sequential NAD-linked oxidations which form and oxidize a stable enzyme-bound histidinaldehyde intermediate. The enzyme was found to catalyze the exchange of 3H between histidinol and [4(R)-3H]NADH and between NAD and [4(S)-3H]NADH. The latter reaction proceeded at rates greater than kcat for the net reaction and was about 3-fold faster than the former. Histidine did not support an NAD/NADH exchange, demonstrating kinetic irreversibility in the second half-reaction. Specific activity measurements on [3H]histidinol produced during the histidinol/NADH exchange reaction showed that only a single hydrogen was exchanged between the two reactants, demonstrating that under the conditions employed this exchange reaction arises only from the reversal of the alcohol dehydrogenase step and not the aldehyde dehydrogenase reaction. The kinetics of the NAD/NADH exchange reaction demonstrated a hyperbolic dependence on the concentration of NAD and NADH when the two were present in a 1:2 molar ratio. The histidinol/NADH exchange showed severe inhibition by high NAD and NADH under the same conditions, indicating that histidinol cannot dissociate directly from the ternary enzyme-NAD-histidinol complex; in other words, the binding of substrate is ordered with histidinol leading. Binding studies indicated that [3H]histidinol bound to 1.7 sites on the dimeric enzyme (0.85 site/monomer) with a KD of 10 microM. No binding of [3H]NAD or [3H]NADH was detected. The nucleotides could, however, displace histidinol dehydrogenase from Cibacron Blue-agarose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sympathetic and adrenocorticoid influences on diet-induced thermogenesis and brown fat activity in the rat

Bilateral adrenalectomy markedly reduced body weight and energy gain and energetic efficiency of adult cafeteria-fed rats but enhanced the thermogenic response to food and stimulated brown fat activity. These changes were totally prevented by replacement of the animals with corticosterone (1 mg/rat/day). Unilateral denervation of the sympathetic nerves supplying the interscapular brown adipose tissue abolished the enhanced activity resulting from adrenalectomy and inhibited thermogenic activity in brown fat from cafeteria rats with intact adrenals, but had no effect in adrenalectomised animals treated with a high dose of corticosterone.     

Interaction of the serum amyloid A proteins with phospholipid

The serum amyloid A proteins (SAA) are transported in plasma in association with the high density lipoproteins. We have studied the solution properties of two of the polymorphic forms of SAA, SAA1 and SAA4, and compared the lipid-binding properties of SAA4 to those of the well characterized apolipoproteins, apo-A-I, apo-A-II, and apo-C-III. SAA4 was monomeric at pH 2.9 but considerable self-association was demonstrated at pH 8.2, even in the presence of 1.0 M guanidine HCl. SAA4 differed from the apolipoproteins in its ability to disrupt multilamellar dimyristoylphosphatidylcholine (DMPC) liposomes and generate bilayer discs. Apo-A-I, apo-A-II, and apo-C-III reduced the turbidity of DMPC dispersions at protein:lipid molar ratios of 1:200. SAA4, however, increased turbidity at molar ratios of 1:250 and 1:100 even when preincubated in guanidine HCl before addition to liposomes. Optical density decreased only at ratios of 1:50 and 1:25. At an SAA4:DMPC ratio of 1:50, discoidal particles (long axis, 28.1 nm; short axis, 4.4 nm) were formed which were similar to those produced by apo-C-III. Lipid binding induced changes in SAA4 conformation similar to those observed in the apolipoproteins. The alpha-helical content and intrinsic tryptophanyl fluorescence were increased and quenching of tryptophanyl fluorescence by acrylamide was reduced in the presence of DMPC. In addition, SAA4 as well as the apolipoproteins broadened the range and increased the temperature of the gel-liquid crystal transition temperature of DMPC.     

Tidal flows and sediment budgets for a salt-marsh system,Essex, England

Studies of tidal flows in salt-marsh creeks in Essex, England, show large variations in water velocity during different tidal cycles, particularly between tides below, at, and above marsh level. Water level, velocity and suspended sediment concentration have been monitored at 5-min intervals during 700 tidal cycles during the year March 1982–March 1983, and the data are being used to calculate sediment budgets for the creek system studied. Completed analyses for two of the tidal cycles show a large positive sediment flux. Because of the importance of velocity in controlling total discharge through a creek cross-section, and hence its effect on total sediment movement, we cannot extrapolate from these two below-marsh tides to any general conclusions about marsh erosion or accretion. We use these preliminary data both to demonstrate our methods and to indicate some of the complexities involved in the analysis.Acknowledgements: This work has been supported by the Natural Environment Research Council. We thank the Philip Lake Fund for financial assistance and the Department of Geography, Cambridge University, for much material support. Mr D. J. Fisher kindly gave access to his land, and Mr W. Bailey helped us greatly. We also thank Mr A. St Joseph for his help, Mr M. Diver for practical support, and Dr J. S. Pethick for discussion.