Interaction of DnaK with ATP: binding, hydrolysis and Ca+2-stimulated autophosphorylation

The autophosphorylation of DnaK from Escherichia coli using ATP as phosphate donor is markedly stimulated by Ca+2 and to a lesser degree by Mn+2. Mg+2 and other divalent ions are without effect in this reaction. Lanthanum, an agonist/antagonist of Ca+2, is also effective in stimulating the autophosphorylation. In contrast, Mg+2 but not Ca+2, markedly stimulates the hydrolysis of ATP catalyzed by DnaK. Also at 0 degrees, ATP forms a stable complex with DnaK without hydrolysis that is independent of cations. About 15% of the DnaK in E. coli is associated with membrane vesicles where it also can be phosphorylated in the presence of Ca+2.     

FAD and GSH participate in macrophage synthesis of nitric oxide.

Following partial purification of macrophage nitric oxide (NO) synthase, enzyme activity requires L-arginine, NADPH, and constitutive cytosolic factors, one of which is tetrahydrobiopterin (BH4) (Kwon, N.S., Nathan, C.F. and Stuehr, D.J. [1989] J. Biol. Chem. 264, 20496). Here we identify FAD and GSH as two additional cofactors needed for full enzyme activity. With all defined cytosolic cofactors in excess, NO synthesis was linear over 3 h and was approximately 50% dependent on exogenous FAD, approximately 50% on glutathione (GSH), 84% on tetrahydrobiopterin (BH4), 95% on NADPH, and 98% on L-arginine. The concentrations of added FAD, GSH, and BH4 required for optimal activity were consistent with their levels in macrophage cytosol. Kinetic studies showed that GSH (or DTT) had little or no effect on the rate of NO generation over the first 20-30 min of the reaction, but prevented a subsequent dropoff in rate. This effect was distinct from thiol participation in BH4 regeneration. In contrast, exogenous FAD doubled the rate of NO synthesis throughout the assay period, consistent with a cofactor role. The role of NADPH was not to regenerate BH4, furnish NADP+, nor form reactive oxygen intermediates. These findings demonstrate NO synthesis by a partially purified enzyme in an otherwise defined system, and suggest that an NADPH-utilizing FAD flavoprotein may participate in the reaction.     

Impact of predation by the robber fly Proctacanthus milbertii (Diptera: Asilidae) on grasshopper (Orthoptera: Acrididae) populations

The impact of predation by the robber fly Proctacanthus milbertii Macquart on populations of adult grasshoppers from grasslands of the Nebraska sandhills was estimated. Densities of P. milbertii were estimated at 437 individuals per hectare (2 se=122). Overall densities of 23 species of grasshoppers were estimated to be 64,000 individuals per hectare with the most abundant species (Ageneotettix deorum) having a population size of approximately 15,000 individuals per hectare. Based on three estimates of predation level (ranging from 0.5 to 2 prey per day per robber fly), P. milbertii may take from 0.5% to 2% of the adult grasshoppers per day. Species of grasshoppers were taken by P. milbertii in about the same proportion in which they occurred at the study site and no size-selective component of predation was detectable.     

Effects of glucagon and insulin on liver lysosomes

Recent experimental evidence has been obtained, principally in the laboratory of Glenn Mortimore, that hepatic lysosomes can act as a pool of amino acids during fasting. This pool is generated through $\text{autophagy}$, whereby intracellular proteins are somehow captured by the lysosomes and then rapidly hydrolyzed to free amino acids by the lysosomal proteinases. Two important metabolic fates of these lysosomal digestive products can be: 1) conversion of the glucogenic amino acids into glucose, and 2) conversion of trimethyl-lysine into carnitine. The latter metabolite is required to transfer fatty acids to the mitochondrial site of β-oxidation. Most interesting is the observation that glucagon appears to induce lysosomal autophagy and the resulting degradation of intracellular proteins by decreasing the size of amino acid pools in the perfused liver. This effect of the hormone may be directed at the single amino acid glutamine, since adding it alone to the perfusate can prevent the increase in autophagy caused by glucagon. Insulin also rapidly inactivates hepatic autophagy and its ensuing proteolysis. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for the rate of los of autophagic vocuoles from the insulin-treated liver (or animal) is approximately 8 min. Thus, glucagon and insulin actively control intracellular protein catabolism that takes place within hepatic lysosomes, and this regulation by the two hormones may be one of their major molecular effects on gluconegenesis in the liver.     

Properties of a novel ATPase enzyme in chromaffin granules

Membranes were isolated from mitochondria and chromaffin granules of bovine adrenal medullae. The cross-contamination between the two membranes was examined by comparing the radioactive bands on autoradiograms of gels after phosphorylation of the membranes with [-³²P]-ATP and decoration with [¹²⁵I]concanavalin A and [¹²⁵I]protein A with antibody that was raised against chromaffin-granule membranes. It was found that the membranes cross-contaminated each other by less than 10%. The technique of immunodecoration with antibodies against subunits of proton-ATPases from yeast mitochondria, spinach chloroplasts, andE. coli membranes was used for quantitative estimation of proton-ATPase complexes in chromaffin granules and mitochondrial membranes. It was found that chromaffin-granule membranes contain less than 10% of the amount of proton-ATPase complex in mitochondrial membranes. The specific ATPase activity of chromaffin-granule membranes was on the order of 30 to 50% of the mitochondrial membranes. The ATPase activity of the chromaffin-granule membranes was more sensitive to 4-acetamido-4-isothiocyano-2,2-disulfonic acid stilbene and 4-chloro-7-nitrobenzofurazan. It was much less sensitive than the mitochondrial membranes to antibody against subunit of proton-ATPase fromE. coli membranes. After solubilization of chromaffin-granule membranes by octyglucoside and cholate and subsequent centrifugation on sucrose gradient, two different ATPase enzymes were separated. The heavier enzyme was identical to the mitochondrial-ATPase complex, while the lighter enzyme was identified as a novel ATPase, which might be responsible for the special properties of the ATPase activity of chromaffin-granule membranes.Abbreviations DCCD dicyclohoxylcarbodiimide - NBD-Cl 4-chloro-7-nitrobenzofurazan - SITS 4-acetamido-4-isothiocyano-2,2-disulfonic acid stilbene - SDS sodium dodecyl sulfate - MES 2-(N-morpholino)ethane sulfonic acid - FITC fluorescein isothiocyanate     

Guanosine-5′-diphosphate-3′-diphosphate inhibits the in, vitro synthesis of β-lactamase from pBR322 DNA

The $\text{in}\text{,}\text{vitro}$ synthesis of β-lactamase directed by pBR322 DNA is inhibited by guanosine-5′-diphosphate-3′-diphosphate.     

Lysosomal pool of free-amino acids

Free (non-protein) amino acids were measured in whole rat liver and in unmodified lysosomes which were prepared from rat liver by the technique of free-flow electrophoresis. Significant intralysosomal pools of threonine, serine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and arginine were found. No efflux occurred from rat liver lysosomes in isotonic buffered sucrose at 0°C, but all amino acids showed various degrees of efflux at 20⁰ and 37⁰.     

Cooperativity of lectin binding to lymphocytes,and its relevance to mitogenic stimulation

The relationship between the binding patterns of soybean agglutinin, peanut agglutinin (both in their native (unaggregated) form and in their polymerized form), and of Phaseolus vulgaris leucoagglutinin, to neuraminidase-treated lymphocytes from different sources, and the mitogenic activity of these lectins, was studied. In all cases investigated, binding of a lectin to lymphocytes which resulted in stimulation was a positive cooperative process. Our findings support the assumption that clustering of receptors and conformational changes in membrane structure are prerequisites for mitogenic stimulation.