Phosphoglycerate mutase. Kinetics and effects of salts on the mutase and bisphosphoglycerate phosphatase activities of the enzyme from chicken breast muscle.

The steady state kinetics and effects of salts on chicken breast phosphoglycerate mutase have been examined. The enzyme can catalyze three phosphoryl transfer reactions: mutase, bisphosphoglycerate phosphatase, and bisphosphoglycerate synthase. The mutase rate was measured in the favorable direction (Keq = glycerate-3-P/glycerate-2-P approximately equal to 12) using [2T]glycerate-2-P as substrate. The bisphosphoglycerate phosphatase activity was studied in the presence of the activator, glycolate-2-P. The latter is an analog of the glycerate-P's and appears to act as an abortive mutase substrate. The kinetic pattern obtained with both activities is that of a ping-pong mechanism with inhibition by the second substrate occurring at a lower concentration than the Km value for that substrate. The kinetic parameters for the mutase determined in 50 mM N-[tris(hydroxymethyl)methyl-2-amino]ethanesulfonate (TES)/sodium buffer containing 0.1 M KCl, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.069 micron; Km glycerate-2-P, 14 micron; Km glycerate-3-P approximately 200 micron; Ki glycerate-2-P, 4 micron. The kinetic parameters for the phosphatase reaction in 50 mM triethanolamine/Cl- buffer, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.065 micron:Km glycolate-2P, 479 micron; Ki glycolate-2-P, 135 micron. The enzyme is sensitive to changes in the ionic environment. Increasing salt concentrations activate the phosphatase in the presence of glycolate-2-P by decreasing the apparent Km of glycerate-2,3-P2. The effects are due to the anionic component and Cl- greater than acetate greater than TES. The same salts are competitive inhibitors with respect to glycolate-2-P. With high levels of KCl that produce a 30-fold decrease in the apparent maximal velocity due to competition with glycolate-2-P, the Km of glycerate-2,3-P2 remains low. These observations lead us to postulate that each monophosphoglycerate substrate has a separate site on the enzyme and that glycerate-2,3-P2 can bind to either site. The binding of anions to one site of the nonphosphorylated enzyme allows an increase in the on and off rates of glycerate-2,3-P2 at the alternate site. Salts inhibit the mutase reaction. The Km of glycerate-2,3-P2 is increased as is that of glycerate-2-P. The effect on the Km of glycerate-2,3-P2 is attributed to an increase in the off rate/on rate ratio for glycerate-2,3-P2. The bisphosphoglycerate synthase reaction is shown to require added glycerate-3-P. The equilibrium between enzyme and glycerate-1,3-P2 is favorable (Kdiss less than or equal 7 X 10(-8) M) and suggests that in the absence of a separate synthase this reaction may have functional significance.     

Rates of phosphorylation and dephosphorylation of phosphoglycerate mutase and bisphosphoglycerate synthase.

Phosphoglycerate mutase and bisphosphoglycerate synthase (mutase) can both be phosphorylated by either glycerate-1,3-P2 or glycerate-2,3-P2 to form phosphohistidine enzymes. The present study uses a rapid quench procedure to determine if, for each enzyme, the formation of the phosphorylated enzyme and phosphate transfer from the enzyme can occur at rates consistent with the overall reactions. With bisphosphoglycerate synthase from horse red blood cells (glycerate-1,3-P2 leads to glycerate-2,3-P2) at pH 7.5, 25 degrees, phosphorylation of the enzyme appears rate-limiting, k = 13.5 s-1, compared with kcat = 12.5 s-1 for the overall synthase rate. Phosphoryl transfer from the enzyme to phosphoglycerate occurs at 38 s-1 at 4 degrees and was too fast to measure at 25 degrees. With chicken muscle phosphoglycerate mutase the half-times were too short to measure under optimal conditions. The rate of enzyme phosphorylation by glycerate-2,3-P2 at pH 5.5, 4 degrees, could account for the overall reaction rate of 170 s-1. The rate of phosphoryl transfer from the enzyme to glycerate-3-P was too rapid to measure under the same conditions. It is concluded that the phosphorylated enzymes have kinetic properties consistent with their participation as intermediates in the reactions catalyzed by these enzymes.     

Evolutionary insights on the origin of human T-cell lymphoma/leukemia virus type I (HTLV-I) derived from sequence analysis of a new HTLV-I variant from Papua New Guinea.

Recent studies have established the presence of human T-cell lymphoma/leukemia virus type I (HTLV-I) in Melanesia. An HTLV-I strain, PNG-1, has now been isolated from a healthy member of the Hagahai, a remote, recently contacted group in Papua New Guinea. To further characterize PNG-1, we employed polymerase chain reaction amplification with subsequent cloning and sequencing of amplified products. Sequence analyses of amplified regions of pol, env, and pX genes of this variant indicate marked heterogeneity (approximately 7%) from prototype HTLV-I. Based on available sequence data, PNG-1 is distinct from all other known HTLV-I strains and diverges from the common ancestor of HTLV-I prior to prototype isolates. The data also suggest, therefore, that HTLV-I originated in the Indo-Malay region rather than Africa.     

A 31P-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain,and the ionophore R02-2985 (X537A) in the isolated,perfused rat heart

1. Some metabolic effects of increased mechanical activity by the Langendorff-perfused rat heart have been characterized using ³¹P-NMR. Mechanical activity was increased by infusion of ouabain (0.9?7.0·10^?5 M), the ionophore R02-2985 (1·10^?5 M) or epinephrine (5·10^?8 M). 2. Similar metabolic changes accompanied infusion of each of the positive inotropic agents into hearts perfused with buffer containing 11 mM glucose as the substrate. In each case phosphocreatine concentrations decreased. During the period of epinephrine infusion the phosphocreatine began to recover its original concentration, although there were no significant changes in mechanical activity. 3. Comparisons of the metabolic changes accompanying the positive inotropic and chronotropic effects of epinephrine were made between hearts perfused with either glucose (11 mM), acetate (5 mM) or lactate (5 mM). A time-dependent decrease in phosphocreatine concentrations also accompanied infusion of epinephrine into hearts perfused with lactate as the sole exogenous substrate, but no statistically significant metabolite changes were observed after identical epinephrine infusions with acetate as the substrate. 4. Calculation of the concentration of free ADP assuming equilibrium in the creatine phosphokinase reaction allows estimation of the cytosolic phosphate potential ($\text{[}\text{ATP}\text{]}\text{[ADP][P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$), which appears to be dependent on a number of factors, including the nature of the exogenous substrate and the level of mechanical activity. 5. Thus, we conclude that there is no general correlation between the phosphate potential and the mitochondrial respiratory rate in the perfused rat heart.     

Sequence Similarity in Nuclear and Mitochondrial Gene Regions in Plants

A 114-basepair fragment at 5′-upstream of the gene oee2-A from Nicotiana tabacum encoding the 23kD polypeptide of oxygen-evolving complex of photosystem II was found, through database search, to have high sequence similarity with segments in the mitochondrial genome of Oenothera, watermelon and wheat. This observation is supported by the Southern hybridization data. Repeated sequences were also found in the nuclear-mitochondrial homologous region, However, the function of these repeats is yet to be determined.     

Chemical properties and NMR spectroscopic identification of certain fungal siderophores

Siderophores of six fungi viz. Aspergillus sp. ABp4, Aureobacidium pullulans, Penicillium oxalicum, P. chrysosporium, Mycotypha africana and Syncephalastrum racemosum were examined for their (1) electrophoretic mobilities to determine the acidic, basic or neutral charge; (2) Fe (III) binding nature viz., mono-, di-, or trihydroxamate; (3) amino acid composition; and (4) NMR (nuclear magnetic resonance) spectroscopy to determine their structure. Electrophoretic mobilities of siderophores of 3 fungi (P. oxalicum, P. chrysosporium, and M, africana) exhibited net basic charge, siderophores of 2 fungi (Aspergillus sp. ABp4 and S. racemosum) were acidic and 1 fungus (A. pullullans) was neutral. Electrophoresis of ferrated siderophore at pH 2 and colour of the spots indicated that siderophores of Aspergillus sp. ABp4 and P. oxalicum and A. pullulans were trihydroxamates, whereas siderophore of P. chrysosporium was dihydroxamate. Amino acid composition of siderophores purified by XAD-2 column chromatography, revealed the presence of asparagine, histidine, and proline in Aspergillus sp. ABp4, serine and alanine in P. chrysosporium, and valine in M. africana. The structure of purified siderophores as revealed by NMR spectroscopy identified siderophore of AB - 2670 (A. pullulans) as asperchrome F1, and AB-513 (M. africana) as rhizoferrin. The peak obtained for siderophore AB-5 (Aspergillus sp. ABp4) did not show resemblance to any known siderophore, therefore may be an exception.     

Calcium transport by isolated anterior and posterior Malpighian tubules of Drosophila melanogaster: roles of sequestration and secretion

Ca(2+) transport was examined in isolated Malpighian tubules (MTs) of adult Drosophila melanogaster. All segments of both anterior and posterior MTs have substantial capacity to transport Ca(2+) and to play a role, therefore, in calcium homeostasis and elimination of excess dietary Ca(2+). Approximately 85% of Ca(2+) which enters the tubule is sequestered, and approximately 15% is secreted in soluble form into the tubule lumen. Tubules secreting fluid at maximal rates can remove an amount of Ca(2+) equal to the whole animal calcium content in approximately 9 h. Distal segments of the pair of anterior MTs can sequester the same amount of Ca(2+) in <2 h. Functional advantages of high Ca(2+) turnover rates are discussed. Transepithelial Ca(2+) secretion is increased by treatments which depolarize the transepithelial potential (thapsigargin, high K(+)), or acidify the secreted fluids (bicarbonate-free salines). The effects of pharmacological reagents and variations in bathing saline ionic composition indicate that the processes of secretion and sequestration are controlled independently, and that diltiazem-sensitive Ca(2+) channels are an important component of sequestration. The contribution of some form of apical Ca(2+) pump is evaluated.     

Helium-neon laser preirradiation induces protection against UVC radiation in wild-type E. coli strain K12AB1157

We have observed that preirradiation with a helium-neon laser (632.8 nm) induces protection against UVC radiation in wild-type E. coli strain K12AB1157. The magnitude of protection was found to depend on the helium-neon laser irradiance, exposure time, and period of incubation between helium-neon laser exposure and subsequent UVC irradiation. The optimum values for dose, irradiance and interval between the two exposures were found to be 7 kJ/m(2), 100 W/m(2) and 1 h, respectively. The possible involvement of singlet oxygen in the helium-neon laser-induced protection is also discussed.