Accumulation of fructose 1,6-bisphosphate in mutant cells of mucoid Pseudomonas aeruginosa as an evidence of phosphofructokinase activity

Phosphoglucose isomerase negative mutant of mucoid Pseudomonas aeruginosa accumulated relatively higher concentration of fructose 1,6-bisphosphate (Fru-1,6-P₂) when mannitol induced cells were incubated with this sugar alcohol. Also the toluene-treated cells of fructose 1,6-bisphosphate aldolase negative mutant of this organism produced Fru-1,6-P₂ from fructose 6-phosphate in presence of ATP, but not from 6-phosphogluconate. The results together suggested the presence of an ATP-dependent fructose 6-phosphate kinase (EC 2.7.1.11) in mucoid P. aeruginosa.Abbreviations ALD Fru-1,6-P₂ aldolse - DHAP dihydroxyacetone phosphate - F6P fructose 6-phosphate - G6P glucose 6-phosphate - Gly3P glyceraldehyde 3-phosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - PFK fructose 6-phosphate kinase - PGI phosphoglucose isomerase - 6PG 6-phosphogluconate     

Effects of 5'deoxy-5'-methylthioadenosine on the metabolism of S-adenosyl methionine

The treatment of transformed rat cells with micromolar amounts of 5'deoxy 5'methyl thioadenosine induces rapid effects on the rate of methylation of DNA concomitantly with alterations of intracellular pools of S-adenosyl methionine and S-adenosyl homocysteine. Pulse chase labelling experiments indicate that 5'deoxy 5'methylthioadenosine does not inhibit the degradation of S-adenosyl homocysteine but inhibits the consumption of S-adenosyl methionine. In vitro transmethylation assays performed with heterologous DNA show that low doses of the thioethernucleoside do not significantly affect the DNA methyltransferase activity of cellular extracts. The biological role of 5'deoxy 5'methylthioadenosine, a natural molecule formed during the synthesis of polyamines is discussed.     

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.     

Autophosphorylation of type 2 casein kinase TS at both its α- and β-subunits: Influence of different effectors

Rat liver casein kinase TS (Ck-TS) having quarternary structure α₂β₂, autophosphorylates at its 25 kDa, β-subunits, incorporating up to 1.2 mol P/mol enzyme. According to their effects on the autophosphorylation pattern the effectors of Ck-TS activity can be grouped into 3 classes: (i) inhibitors, like heparin, which also prevent the autophosphorylation of the β-subunit; (ii) stimulators possessing several amino groups (like spermine) which increase the autophosphorylation at the β-subunit; (iii) stimulators possessing several guanido groups, like protamines and related peptides, which prevent the phosphorylation of the β-subunit, while promoting the autophosphorylation of the 38 kDa α-subunit. In the presence of such polyarginyl effectors the 130 kDa Ck-TS is converted into forms with higher sedimentation coefficient.     

An enzymatic assay for acetate in spent bacterial culture supernatants

A method is presented for the rapid enzymatic determination of acetate in spent bacterial culture supernatants. The assay is based on a previously published assay for acetate kinase [Bergmeyer et al. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 1, pp. 425-426, Verlag Chemie-Academic Press, New York/London], and is sufficiently sensitive to detect acetate levels of 50 microM. The assay is cheaper than commercially available assays and is particularly useful for occasional use by laboratories not equipped for routine acetate analysis using gas chromatography. The application of the assay to the measurement of acetate in bacterial cultures is described, though it should also be applicable to other biological fluids and foodstuffs.     

Characterization of a fluorescent substrate for the adenosine 3',5'-cyclic monophosphate-dependent protein kinase

A synthetic tetradecapeptide derived from the phosphorylation site of the beta-subunit of phosphorylase kinase (Arg-Thr-Lys-Arg-Ser-Gly-Ser-Val-Tyr-Glu-Pro-Leu-Lys-Ile) is a highly efficient substrate for the cAMP-dependent protein kinase, exhibiting a 36% decrease in the intrinsic tyrosine fluorescence on phosphorylation. The fluorescence changes in continuous assays were monitored to demonstrate the roles of protein kinase effectors (cAMP, the type II regulatory subunit, and the 8000-Da heat-stable inhibitor) in the regulation of the enzyme and to determine Km and Vmax. The phosphorylation reaction requires 1 mol ATP/mol peptide. Amino acid analysis demonstrates the presence of phosphoserine in the phosphorylated peptide. Auxiliary experiments show that tyrosine phosphorylation can also be detected fluorometrically and distinguished from serine or threonine phosphorylation.     

Characterization, development, and localization of the deoxycytidine phosphorylating systems in mammalian brain

The accumulation of deoxycytidine by rabbit and mouse brain was studied in vitro. Brain slices from brain stem, cerebellum, and forebrain of rabbits of various ages (1 day to 2.5 years) and forebrain from adult mice were incubated for various times in artificial CSF containing 6 nM [3H]deoxycytidine at 37 degrees C under 95% O2/5% CO2. Rabbit and mouse brain slices of all ages accumulated [3H]deoxycytidine by a saturable system (IC50 = 4 microM) and converted it to [3H]deoxycytidine phosphates and [3H]DNA. When slices from all brain regions of 1-day-old rabbits were incubated in 6 nM [3H]deoxycytidine for 30 min, tissue-to-medium ratios of 3H were between 1.2 and 2.5 and declined with age, except in cortex; the percentages of total 3H in perchloric acid homogenates of brain slices as [3H]DNA were 10-24% and declined to low levels in middle age. However, at all ages and in all regions tested, 30-85% of the [3H]deoxycytidine within the slices was phosphorylated. After homogenization and subcellular fractionation of the brain slices incubated in [3H]deoxycytidine for 30 min, the highest percentage of [3H]deoxycytidine phosphates plus [3H]DNA was present in the nuclear and mitochondrial fractions of all brain regions. Deoxycytidine phosphates were synthesized from deoxycytidine in all brain regions tested into middle age.     

3',5'-Cyclic Adenosine Monophosphate- and Ca2+-Calmodulin-Dependent Endogenous Protein Phosphorylation Activity in Membranes of the Bovine Chromaffin Secretory Vesicles: Identification of Two Phosphorylated Components as Tyrosine Hydroxylase and Protein Kinase Regulatory Subunit Type II

Abstract: Membranes of the secretory vesicles from bovine adrenal medulla were investigated for the presence of the endogenous protein phosphorylation activity. Seven phosphoprotein bands in the molecular weight range of 250,000 to 30,000 were observed by means of the sodium dodecyl sulphate electrophoresis and autoradiography. On the basis of the criteria of molecular weight, selective stimulation of the phosphorylation by cyclic AMP (as compared with cyclic GMP) and immunoprecipitation by specific antibodies, band 5 (molecular weight 60,300) was found to represent the phosphorylated form of the secretory vesicle-bound tyrosine hydroxylase. The electrophoretic mobility, the stimulatory and inhibitory effects of cyclic AMP in presence of Mg²⁺ and Zn,²⁺ respectively, and immunoreactivity toward antibodies showed band 6 to contain two forms of the regulatory subunits of the type II cyclic AMP-dependent protein kinase, distinguishable by their molecular weights (56,000 and 52,000, respectively). Phosphorylation of band 7 (molecular weight 29,800) was stimulated about 2 to 3 times by Ca²⁺ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions.     

Evidence for Membrane-Associated Choline Kinase Activity in Rat Striatum

The distribution of choline kinase (EC 2.7.1.32) activity was investigated in subcellular fractions of rat striatum. Enzyme activity in the crude mitochondrial fraction, determined after dissolution in Triton X-100, was 5.90 mumol/g initial wet weight/h. When a crude mitochondrial preparation was hypoosmotically shocked and fractionated, followed by the addition of Triton X-100, choline kinase activity in the soluble and particulate fractions was 4.58 and 1.40 mumol/g initial wet weight/h, respectively. Enzyme activity in the particulate fraction was not detected in the absence of Triton X-100 or in the presence of NaCl (up to 1.5 M). Subcellular enzyme markers indicated that the membrane-associated activity was not attributable to mitochondrial or microsomal contamination. Kinetic analysis of the activity of soluble and membrane-solubilized choline kinase indicated Km values of 0.74 mM and 0.68 mM, respectively. Results indicate that choline kinase activity may be measured in both the soluble and the particulate fractions of rat striatum, the latter most likely involving enzyme associated with membrane through hydrophobic or covalent interactions. The specific function of the membrane-associated enzyme has not yet been determined.     

Feedback inhibition of homoserine kinase from radish leaves

Homoserine kinase is a potential control point in the biosynthetic pathway for threonine, isoleucine and methionine. The radish leaf enzyme was tested