Profilin Enhances Cdc42-Induced Nucleation of Actin Polymerization

We find that profilin contributes in several ways to Cdc42-induced nucleation of actin filaments in high speed supernatant of lysed neutrophils. Depletion of profilin inhibited Cdc42-induced nucleation; re-addition of profilin restored much of the activity. Mutant profilins with a decreased affinity for either actin or poly-l-proline were less effective at restoring activity. Whereas Cdc42 must activate Wiskott-Aldrich Syndrome protein (WASP) to stimulate nucleation by the Arp2/3 complex, VCA (verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP) constitutively activates the Arp2/3 complex. Nucleation by VCA was not inhibited by profilin depletion. With purified N-WASP and Arp2/3 complex, Cdc42-induced nucleation did not require profilin but was enhanced by profilin, wild-type profilin being more effective than mutant profilin with reduced affinity for poly-l-proline. Nucleation by the Arp2/3 complex is a function of the free G-actin concentration. Thus, when profilin addition decreased the free G-actin concentration, it inhibited Cdc42- and VCA-induced nucleation. However, when profilin was added with G-actin in a ratio that maintained the initial free G-actin concentration, it increased the rate of both Cdc42- and VCA-induced nucleation. This enhancement, also seen with purified proteins, was greatest when the free G-actin concentration was low. These data suggest that under conditions present in intact cells, profilin enhances nucleation by activated Arp2/3 complex.

Chitin synthase in encysting Entamoeba invadens

Although the cyst wall of Entamoeba invadens contains chitin, synthesis of this structural polymer during encystation has not been described before. Here we report that conditions which stimulate encystation of the parasite lead to increased chitin synthase (ChS) activity, measured by incorporation of [3H]GlcNAc ([3H]N-acetylglucosamine) from UDP-GlcNAc. The radiolabelled product was precipitable by trichloroacetic acid or ethanol and identified as chitin because it was digested by purified chitinase to radioactive chitobiose and GlcNAc. Cell fractionation indicated that approx. 60% of the enzyme is in the high-speed supernatant. pH-activity profiles showed that soluble ChS has an optimum at 6.0, whereas particulate ChS has a peak at pH 7.0-7.5. Both the activities were dependent on bivalent metal ions, especially Mn2+ and Mn2+ plus Co2+. In contrast with the ChS of other organisms, neither the particulate nor the soluble ChS of E. invadens was activated by trypsin treatment. Soluble and particulate ChS were also stimulated by digitonin and phosphatidylserine, whereas phosphatidylethanolamine stimulated only the soluble ChS. The enzyme activities were inhibited by UDP, UDP-glucose and UDP-GalNAc, but not by the analogues Polyoxin-D or Nikkomycin. This is the first report of an enzyme which is developmentally regulated during encystation of the primitive eukaryotic genus Entamoeba.

Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver

The finding that N-glycoloylneuraminic acid (Neu5Gc) in pig submandibular gland is synthesized by hydroxylation of the sugar nucleotide CMP-Neu5Ac [Shaw & Schauer (1988) Biol. Chem. Hoppe-Seyler 369, 477-486] prompted us to investigate further the biosynthesis of this sialic acid in mouse liver. Free [14C]Neu5Ac, CMP-[14C]Neu5Ac and [14C]Neu5Ac glycosidically bound by Gal alpha 2-3- and Gal alpha 2-6-GlcNAc beta 1-4 linkages to fetuin were employed as potential substrates in experiments with fractionated mouse liver homogenates. The only substrate to be hydroxylated was the CMP-Neu5Ac glycoside. The product of the reaction was identified by chemical and enzymic methods as CMP-Neu5Gc. All of the CMP-Neu5Ac hydroxylase activity was detected in the high-speed supernatant fraction. The hydroxylase required a reduced nicotinamide nucleotide [NAD(P)H] coenzyme and molecular oxygen for activity. Furthermore, the activity of this enzyme was enhanced by exogenously added Fe2+ or Fe3+ ions, all other metal salts tested having a negligible or inhibitory influence. This hydroxylase is therefore tentatively classified as a monooxygenase. The cofactor requirement and CMP-Neu5Ac substrate specificity are identical to those of the enzyme in high-speed supernatants of pig submandibular gland, suggesting that this is a common route of Neu5Gc biosynthesis. The relevance of these results to the regulation of Neu5Gc expression in sialoglycoconjugates is discussed.

GENETIC ANALYSIS OF MUTANTS WITH A REDUCED Ca2+-DEPENDENT K+ CURRENT IN PARAMECIUM TETRAURELIA

ABSTRACT Two mutants of Paramecium tetraurelia with greatly reduced Ca2+-dependent K+ currents have been isolated and genetically analyzed. These mutants, designated pantophobiac, give much stronger behavioral responses to all stimuli than do wild-type cells. Under voltage clamp, the Ca2+-dependent K+ current is almost completely eliminated in these mutants, whereas the Ca2+ current is normal. The two mutants, pntA and pntB, are recessive and unlinked to each other. pntA is not allelic to several other ion-channel mutants of P. tetraurelia. The microinjection of a high-speed supernatant fraction of wild-type cytoplasm into either pantophobiac mutant caused a temporary restoration to the wild-type phenotype.

The ability of adenosine to decrease the concentration of fructose 2,6-bisphosphate in isolated hepatocytes. A cyclic AMP-mediated effect

The presence of adenosine (25-250 microM) or of 2-chloroadenosine (2.5-100 microM) in the incubation medium caused a marked decrease in the concentration of fructose 2,6-bisphosphate in isolated hepatocytes. This effect was accompanied by an increase in the concentration of cyclic AMP, an activation of phosphorylase and of fructose 2,6-bisphosphatase, and an inactivation of pyruvate kinase and of 6-phosphofructo-2-kinase. As a rule, the changes in the fructose 2,6-bisphosphate-modifying system were slower but more persistent than those in the activities of phosphorylase and pyruvate kinase. The effect of the nucleoside to decrease the concentration of fructose 2,6-bisphosphate was not affected by an inhibitor of adenosine transport and could not be obtained in a liver high-speed supernatant. These data indicate that the effect of adenosine to decrease the concentration of fructose 2,6-bisphosphate is mediated by the stimulation of adenylate cyclase, secondary to the binding of adenosine to membranous receptors. Like glucagon, 2-chloroadenosine stimulated gluconeogenesis in isolated hepatocytes, whereas adenosine had an opposite effect.

Novel prostaglandin dehydrogenase in rat skin

Present evidence suggests that skin is an important organ of prostaglandin metabolism. To clarify its role, the basic kinetics of 15-hydroxyprostaglandin dehydrogenase (PGDH) from rat skin were investigated with either NAD+ of NADP+ as co-substrate. Prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) were used as substrates and preliminary studies were made of the inhibitory effects of the reduced co-substrates NADH and NADPH. A radiochemical assay was used in which [3H]PGF2 alpha or [14C]PGE2 were incubated with high-speed supernatant of rat skin homogenates. The substrate and products were then extracted by solvent partition, separated by t.l.c. and quantified by liquid-scintillation counting. At linear reaction rates and at an NAD+ concentration of 10 mM the mean apparent Km for PGF2 alpha was 24 microM with a mean apparent Vmax. of 9.8 nmol/s per litre of reaction mixture. For PGE2 the mean apparent Km was 8 microM, with a mean apparent Vmax, of 2.7 nmol/s per litre of reaction mixture. With NADP+ as a co-substrate at a concentration of 5 mM a mean apparent Km of 23 microM was obtained for PGF2 alpha with a mean apparent Vmax. of 5.2 nmol/s per litre. For PGE2 values of 7.5 microM and 3.0 nmol/s per litre were obtained respectively. These results show that skin contains NAD+- and NADP+-dependent PGDH. An important finding was that the NADP+-linked enzyme gave Km values for PGE2 that were considerably lower than those reported for NADP+-linked PGDH from other tissues. Furthermore, preliminary inhibition studies with the NAD+-linked PGDH system indicate that this enzyme is not only inhibited by NADH, but also by NADPH, a property not previously reported for NAD+-linked PGDH derived from other tissues.

Plasma-membrane location of phosphatidylinositol hydrolysis in rabbit neutrophils stimulated with formylmethionyl-leucylphenylalanine

Rabbit peritoneal neutrophils, disrupted by sonication, were separated into three subcellular fractions by sucrose-step-gradient centrifugation and these were analysed with respect to biochemical markers. They comprised a high-speed supernatant containing the cytosol, a light particulate fraction enriched in Golgi and plasma membranes and a heavy particulate fraction enriched in granules and nuclei. The light particulate fraction was further separated into its components, which were identified as Golgi membranes (galactosyltransferase activity) and plasma membranes ((radioactivity derived from labelling intact cells with [125I]di-iodosulphanilic acid diazonium salt and [3H]formylmethionyl-leucylphenylalanine ([3H]fMet-Leu-Phe) binding)). In cells prelabelled with [3H]glycerol, the hydrolysis of phosphatidylinositol due to cell stimulation with fMet-Leu-Phe (10 nM) was shown to occur in the light particulate fraction. The [32P]Pi-labelling of phosphatidate, which is an early consequence of phosphatidylinositol hydrolysis, also occurred in this fraction. Analytical sucrose-gradient centrifugation of the light particulate fraction showed that the stimulated increment in [32P]phosphatidate (and thus by implication the initial phosphatidylinositol breakdown) was localized in the plasma membrane.

Discrete primary locations of a tyrosine-protein kinase and of three proteins that contain phosphotyrosine in virally transformed chick fibroblasts.

We have studied the localization of three abundant cellular proteins which are substrates for tyrosine protein kinases in virally transformed chicken embryo fibroblasts. The primary location of each substrate is unaltered by transformation with Rous sarcoma virus (RSV). The tyrosine-phosphorylated species is localized with the nonphosphorylated species. Two of the proteins, of about 46,000 and 28,000 daltons, have a similar location. They are present in the high speed supernatant of cells homogenized in hypotonic buffer, and are soluble in nonionic detergent. The third protein, of about 39,000 daltons, is particulate when cells are homogenized in hypotonic buffer containing divalent cations, but approximately 30% is free in the high-speed supernatant when divalent cations are absent. This protein appears to be associated with the detergent-insoluble matrix when adherent cells are gently lysed in nonionic detergent in situ, but is soluble when the same cells are extracted with nonionic detergent in suspension. This suggests that one of the proteins are tightly associated with detergent-insoluble cytoskeletal structures, unlike the RSV transforming protein itself, which is the main tyrosine protein kinase known to be active in RSV-transformed cells.