Potential involvement of vicinal sulfhydryls in stimulus-induced rabbit platelet activation

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.     

Ligand recombination to the alpha and beta subunits of human hemoglobin

The rebinding of CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to isolated alpha and beta chains and intact hemoglobin at pH 7, 20 degrees C was examined both during and after a 30-ns dye laser pulse. The resultant absorbance changes were analyzed in terms of a linear three-step reaction scheme: Hb + X in equilibrium with C in equilibrium with B in equilibrium with A or HbX, where A is the final bound state, and C and B are geminate states. Rate constants were assigned for each of the transitions in this mechanism using fitting procedures described previously for analyzing ligand rebinding to sperm whale myoglobin at room temperature (Gibson, Q. H., Olson, J. S., McKinnie, R. E., and Rohlfs, R. J. (1986) J. Biol. Chem. 261, 10228-10239). Five major conclusions were obtained. First, initial geminate recombination phases for the NO and O2 complexes of hemoglobin and its isolated subunits exhibit half-times equal to approximately 12 and approximately 440 ps, respectively. These values are in excellent agreement with more direct, picosecond measurements of the geminate recombination of HbNO (Cornelius, P. A., Hochstrasser, R. M., and Steele, A. W. (1983) J. Mol. Biol. 163, 119-128) and HbO2 (Friedman, J. M., Scott, T. W., Fisanick, G. J., Simon, S. R., Findsen, E. W., Ondrias, M. R., and MacDonald, V. W. (1985) Science 229, 187-229) following extremely short laser pulses. Second, the correspondence between our nanosecond measurements and the published picosecond data suggests strongly that the intrinsic photochemical yield of all ferrous, hexacoordinate heme complexes approaches one. Third, the major differences between the isolated alpha and beta chains involve the rate of ligand migration to the solvent, kC----X and the extent of recombination from the second geminate state, C, as measured by the ratio kC----B/kC----X. Fourth, for both isolated chains and intact hemoglobin, the rate and equilibrium constants for the formation of the initial O2 geminate state starting from ligand in the solvent (i.e. kX----B and KX----B) are 5-10 times greater than the corresponding parameters for the formation of the first CO geminate state. Fifth, the rate-limiting step for NO, O2, and isonitrile binding to hemoglobin and its isolated subunits is ligand migration up to the initial geminate state (i.e. kX----B). In the case of CO binding, both migration to state B and iron-ligand bond formation (kB----A) affect the overall, bimolecular association rate constant.     

Relative affinity of Ca(II) and Mg(II) ions for human and bovine prothrombin and fragment 1

Equilibrium dialysis results are presented for Ca(II) and Mg(II) ion binding to human and bovine prothrombin and fragment 1. Ca(II) ions bind cooperatively, Mg(II) does not.     

The majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

The BC3Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins [Olson, E. N., Towler, D. A., & Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790]. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages [Olson, E. N., & Spizz, G. (1986) J. Biol. Chem. 261, 2458-2466]. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, we examined the subcellular localization of the major fatty acylated proteins in BC3Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [3H]palmitate and [3H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins.     

Acceleration of surface-dependent autocatalytic activation of blood coagulation factor XII by divalent metal ions

The effect of divalent metal ions on the rate of dextran sulfate dependent autocatalytic activation of human blood coagulation factor XII was studied at pH 7.4 and 25 degrees C. Zn2+ and Cu2+, but not Co2+, increased the rate of factor XII activation induced by dextran sulfate with optimum effects at approximately 5 and 1 microM, respectively, while Ca2+ acceleration required much higher concentrations (millimolar). Further investigation of the effect of Zn2+ on factor XII activation demonstrated a complete dependence on the presence of dextran sulfate, lack of inhibition by soybean trypsin inhibitor, the appearance of alpha-XIIa as the primary reaction product, and reaction kinetics characteristic of an autocatalytic process. These results were consistent with Zn2+ affecting only the rate of surface-mediated factor XII autoactivation. The initial turnover velocity of dextran sulfate induced factor XII autoactivation increased linearly with factor XII concentration in the absence of Zn2+ up to 0.9 microM factor XII but showed saturation behavior over this same concentration range in the presence of 5 microM Zn2+, indicating that Zn2+ increased the reaction rate primarily by lowering the apparent Km. Comparison of the kinetics of autoactivation at mu = 0.15 and 0.24 revealed that the enhancement in the apparent kcat/Km brought about by Zn2+ increased from 19-fold to 520-fold, respectively, due to a differential dependence of the Zn2+-stimulated and unstimulated reactions on ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of nucleolin by a nucleolar type NII protein kinase

Nucleolin [C23 or 100 kilodaltons (kDa)] is the major nucleolar phosphorylated protein in exponentially growing Chinese hamster ovary cells. A nucleolar cyclic nucleotide independent protein kinase copurified with nucleolin in a complex which could be dissociated by hydroxyapatite chromatography. The kinase was stimulated by spermine and inhibited by heparin and presented most of the properties of nuclear casein kinase NII. Kinetic analyses showed the apparent Km value for nucleolin (7 X 10(-4) mg/mL) to be lower than those for other casein kinase II substrates such as nuclear protein HMG 14 (0.15 mg/mL), topoisomerase I (0.025 mg/mL), or topoisomerase II (0.04 mg/mL). Similarly, Vmax values were higher for nucleolin than for other substrates. Nucleolin thus appears to be a natural preferential substrate of nucleolar casein kinase NII. The kinase phosphorylated nucleolin in vitro at serine residues in a 29-kDa CNBr fragment located near the amino terminus of the molecule. The enzyme labeled typical casein kinase II sites. These sites were found predominantly in two highly acidic tryptic fragments designated A (residues 21-49) and C (residues 180-221) which contained serines having at least two acidic residues on their carboxyl-terminal sides. These results demonstrate the existence in the nucleolus of a type of NII protein kinase that uses a protein involved in ribosome assembly as preferential substrate.     

The kinetics of milk coagulation: IV. The kinetics of the gel-firming process

A mechanistic kinetic model of gel firmness development during milk gel formation is presented. The model correctly accounts for the influence of enzymatic kappa-casein hydrolysis on the rate of firmness development in renneted milk gels. The model used is based on two first-order reactions occurring in series. The first reaction is enzymatically controlled and corresponds to the formation of gel crosslink sites by kappa-casein hydrolysis. The second reaction is nonenzymatic and corresponds to the process of crosslink formation and depletion of active sites. The model successfully predicts gel firmness development in the temperature range 31-45 degrees C for a variety of initial enzyme concentrations.     

下丘脑外侧区注射TRH对大鼠胃酸分泌的影响

本文采用连续收集胃腔灌流法,观察下丘脑外侧区(LHA)注射促甲状腺激素释放激素(TRH)对大鼠胃酸分泌的影响,并分析TRH在LHA促进胃酸分泌的作用机制。结果表明:(1)LHA注射TRH(1μg)明显地刺激胃酸分泌;(2)预先向LHA注射酚妥拉明(10μg)、美多心安(5μg)及胃泌素抗体1μl(1:640)并不影响TRH的泌酸作用,如预先向LHA注射阿托品(5μg)则可消除TRH的泌酸效应;(3)垂体摘除及肾上腺切除均不影响TRH的泌酸作用;(4)隔下迷走神经切断后,LHA注入TRH的泌酸效应仍然出现,但持续时间显著缩短;腹腔交感神经节摘除后,TRH仍能促进胃酸分泌,但分泌量少而平稳。以上结果提示:LHA是TRH中枢泌酸效应的有关结构之一,其中枢机制是通过胆碱能M受体中介的,腹腔交感神经节和膈下迷走神经是TRH泌酸效应的传出途径。前者引起的泌酸反应出现较早且引起泌酸高峰,但持续时间短;后者则引起低平的持续分泌。     

A Chronobiologic Approach to Ethanol and Acidified Aspirin Injury of the Gastric Mucosa in the Rat

Several models of erosive peptic disease have used drug-induced lesions to examine protective mechanisms of the gastric mucosa. Physiological processes such as acid secretion, motility, or epithelial cell turnover have circadian rhythms which may modulate the susceptibility of the gastric mucosa to injury. In this review are described recent studies which demonstrated that susceptibility to gastric mucosal injury by acidified aspirin and absolute ethanol varied with the phases of the light-dark cycle. Acidified aspirin caused significantly more gastric mucosal lesions when administered early in the light phase compared to administration early in the dark phase. The differences in susceptibility were not altered by pretreatment conditions such as immobilization or length of the fasting period. Absolute ethanol also caused significantly greater gastric mucosal injury when administered in the light than in the dark phase, but this difference was only evident in rats immobilized during the pretreatment fasting period. Further studies are needed to correlate circadian susceptibility to drug-induced gastric mucosal injury with physiological defense mechanisms. Careful attention to circadian timekeeping may allow us to refine therapy to optimize physiological defense mechanisms in the stomach.     

Biotransformation of mercury by bacteria isolated from a river collecting cinnabar mine waters

One hundred six strains of aerobic bacteria were isolated from the Fiora River which drains an area of cinnabar deposits in southern Tuscany, Italy. Thirty-seven of the strains grew on an agar medium containing 10g/ml Hg (as HgCl₂) with all of these strains producing elemental mercury. Seven of the 37 strains also degraded methylmercury. None of 106 sensitive and resistant strains produced detectable monomethylmercury although 15 strains produced a benzene-soluble mercury species. Two strains of alkylmercury (methyl-, ethyl- and phenylmercury) degrading bacteria were tested for the ability to degrade several other analogous organometals and organic compounds, but no activity was detected toward these compounds. Mercury methylation is not a mechanism of Hg resistance in aerobic bacteria from this environment. Growth of bacteria on the agar medium containing 10g/ml HgCl₂ was diagnostic for Hg detoxification based on reduction.