Protein phosphorylation in human peripheral blood lymphocytes. Phosphorylation of endogenous plasma membrane and cytoplasmic proteins

Phosphorylation of endogenous proteins in subcellular fractions of human peripheral-blood lymphocytes was studied by one- and two-dimensional polyacrylamide-gel electrophoresis. Studies using extensively purified subcellular fractions indicated that the endogenous phosphorylating activity in the particulate fractions was derived primarily from the plasma membrane. Electrophoresis of (32)P-labelled subcellular fractions in two dimensions [O'Farrell (1975) J. Biol. Chem.250, 4007-4021] provided much greater resolution of the endogenous phosphoproteins than electrophoresis in one dimension, facilitating their excision from gels for quantification of (32)P content. More than 100 cytoplasmic and 20 plasma-membrane phosphorylated species were observed. Phosphorylation of more than 10 cytoplasmic proteins was absolutely dependent on cyclic AMP. In the plasma membrane, cyclic AMP-dependent phosphoproteins were observed with mol.wts. of 42000, 42000, 80000 and 90000 and pI values of 6.1, 6.3, 6.25 and 6.5 respectively. Phosphorylation of endogenous cytoplasmic and plasma-membrane proteins was rapid with t((1/2))=5-12s at 25 degrees C. Between 40 and 70% of the (32)P was recovered as phosphoserine and phosphothreonine when acid hydrolysates of isolated plasma-membrane phosphoproteins were analysed by high-voltage paper electrophoresis. The presence of cyclic AMP-dependent protein kinase and endogenous phosphate-acceptor proteins in the plasma membranes of lymphocytes provides a mechanism by which these cells might respond to plasma-membrane pools of cyclic AMP generated in response to stimulation by mitogens or physiological modulators of lymphocyte function.     

Chromosome 4 controls potential water use efficiency ({delta}13C) in barley

By combining the approaches of whole-shoot carbon discriminationand genetic analysis, we found that chromosome 4 controls potentialwater use efficiency (     

Plasmid-mediated resistance to fosfomycin in Staphylococcus epidermidis

Staphylococcus epidermidis strain BM2641, isolated from a patient, was resistant to penicillin G, methicillin, aminoglycosides, chloramphenicol, macrolide, lincosamide and streptogramin B-type (MLS) antibiotics, and to high levels of fosmycin. Resistance to forsfomycin and/or to MLS was lost at low frequencies either spontaneously or after curing with novobiocin. The plasmid DNA from BM2641 and its cured derivatives was purified, analyzed by agarose gel electrophoresis and transferred to a nitrocellulose sheet. Comparative analysis of the resistance phenotypes with the plasmid content of the strains indicated that fosfomycin and MLS resistance were encoded by plasmids pIP1842 (2.5 kb) and pIP1843 (2.6 kb), respectively. Southern hybridization with a probe specific for gene fosA of Serratia marcescens showed that the fosfomycin resistance determinant in Staphylococcus is not homologous to that of Gram-negative bacteria.     

A new bacterial alcohol dehydrogenase active on degraded lignin and several low molecular weight aromatic compounds

A new intracellular bacterial dehydrogenase has been purified. It was active in the reversible reduction by NADH of conjugated carbonyl groups in partially degraded lignin. It was also active on various aromatic aldehydes such as vanillin, syringaldehyde and cinnamaldehyde, but had no effect on acetovanillone and lignin models carrying a conjugated ketone. It is proposed that this enzyme functions as a broadly specific lignin dehydrogenase at the level of aldehydic groups that are present in the lignin preparations.     

Adenosine metabolism: Modification by S-adenosylhomocysteine and 5′-methylthioadenosine

To elucidate potential toxic properties of S-adenosylhomocysteine and 5′-methylthioadenosine, we have examined the inhibitory properties of these compounds upon enzymes involved with adenosine metabolism. S-Adenosylhomocysteine, but not S-adenosylmethionine, was a noncompetitive inhibitor of adenosine kinase with K_i values ranging from 100 to 400 μm. Methylthioadenosine competitively inhibited adenosine kinase with variable adenosine below 1 μm with a K_i of 120 μm, increased adenosine kinase activity when the adenosine concentration exceeded 2 μm, and did not appear to be a substrate for adenosine kinase. Methylthioadenosine inactivated S-adenosylhomocysteine hydrolase from erythrocytes, B-lymphoblasts, and T-lymphoblasts with K_i values ranging from 65 to 117 μm and “k₂” from 0.30 to 0.55 min^?1. Adenosine deaminase was not inhibited by 5′-methylthioadenosine up to 1000 μm. To clarify how 5′-methylthioadenosine might accumulate, 5′-methylthioadenosine phosphorylase was evaluated. This enzyme was not blocked by up to 500 μm adenosine, deoxyadenosine, S-adenosylhomocysteine, or S-adenosylmethionine and was not decreased in erythrocytes from patients with adenosine deaminase deficiency, purine nucleoside phosphorylase deficiency, or hypogammaglobulinemia. These observations suggest that the inhibitory properties of 5′-methylthioadenosine upon adenosine kinase and S-adenosylhomocysteine hydrolase may contribute to the toxicity of the exogenously added compound. The toxicity resulting from S-adenosylhomocysteine accumulation intracellularly may be related to adenosine kinase inhibition in addition to disruption of transmethylation reactions.     

Insufficient glucose supply is linked to hypothermia upon cold exposure in high-fat diet-fed mice lacking PEMT

Mice that lack phosphatidylethanolamine N-methyltransferase (Pemt^−/− mice) are protected from high-fat (HF) diet-induced obesity. HF-fed Pemt^−/− mice show higher oxygen consumption and heat production, indicating that more energy might be utilized for thermogenesis and might account for the resistance to diet-induced weight gain. To test this hypothesis, HF-fed Pemt^−/− and Pemt^+/+ mice were challenged with acute cold exposure at 4°C. Unexpectedly, HF-fed Pemt^−/− mice developed hypothermia within 3 h of cold exposure. In contrast, chow-fed Pemt^−/− mice, possessing similar body mass, maintained body temperature. Lack of PEMT did not impair the capacity for thermogenesis in skeletal muscle or brown adipose tissue. Plasma catecholamines were not altered by Pemt genotype, and stimulation of lipolysis was intact in brown and white adipose tissue of Pemt^−/− mice. HF-fed Pemt^−/− mice also developed higher systolic blood pressure, accompanied by reduced cardiac output. Choline supplementation reversed the cold-induced hypothermia in HF-fed Pemt^−/− mice with no effect on blood pressure. Plasma glucose levels were ∼50% lower in HF-fed Pemt^−/− mice compared with Pemt^+/+ mice. Choline supplementation normalized plasma hypoglycemia and the expression of proteins involved in gluconeogenesis. We propose that cold-induced hypothermia in HF-fed Pemt^−/− mice is linked to plasma hypoglycemia due to compromised hepatic glucose production.     

DNA Polymerase α Subunit Residues and Interactions Required for Efficient Initiation Complex Formation Identified by a Genetic Selection

Biophysical and structural studies have defined many of the interactions that occur between individual components or subassemblies of the bacterial replicase, DNA polymerase III holoenzyme (Pol III HE). Here, we extended our knowledge of residues and interactions that are important for the first step of the replicase reaction: the ATP-dependent formation of an initiation complex between the Pol III HE and primed DNA. We exploited a genetic selection using a dominant negative variant of the polymerase catalytic subunit that can effectively compete with wild-type Pol III α and form initiation complexes, but cannot elongate. Suppression of the dominant negative phenotype was achieved by secondary mutations that were ineffective in initiation complex formation. The corresponding proteins were purified and characterized. One class of mutant mapped to the PHP domain of Pol III α, ablating interaction with the ϵ proofreading subunit and distorting the polymerase active site in the adjacent polymerase domain. Another class of mutation, found near the C terminus, interfered with τ binding. A third class mapped within the known β-binding domain, decreasing interaction with the β₂ processivity factor. Surprisingly, mutations within the β binding domain also ablated interaction with τ, suggesting a larger τ binding site than previously recognized.     

Bacterial production in freshwater sediments: Cell specific versus system measures

Estimates of bacterial production based on total trichloroacetic acid (TCA)-precipitable [methyl-³H]thymidine incorporation and frequency of dividing cell (FDC) techniques were compared to sediment respiration rates in Lake George, New York. Bacterial growth rates based on thymidine incorporation ranged from 0.024 to 0.41 day^–1, while rates based on FDC ranged from 1.78 to 2.48 day^–1. Respiration rates ranged from 0.11 to 1.8mol O₂·hour^–1·g dry weight sediment^–1. Thymidine incorporation yielded production estimates which were in reasonable agreement with respiration rates. Production estimates based on FDC were 4- to 190-fold higher than those predicted from respiration rates.