Regulatory and Structural Properties of the Cyanobacterial ADPglucose Pyrophosphorylases

ADPglucose pyrophosphorylase (EC 2.7.7.27) has been purified from two cyanobacteria: the filamentous, heterocystic, Anabaena PCC 7120 and the unicellular Synechocystis PCC 6803. The purification procedure gave highly purified enzymes from both cynobacteria with specific activities of 134 (Synechocystis) and 111 (Anabaena) units per milligram protein. The purified enzymes migrated as a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular mass corresponding to 53 (Synechocystis) and 50 (Anabaena) kilodaltons. Tetrameric structures were determined for the native enzymes by analysis of gel filtrations. Kinetic and regulatory properties were characterized for the cyanobacterial ADPglucose pyrophosphorylases. Inorganic phosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. The Synechocystis enzyme was activated 126-fold by 3-phosphoglycerate, with saturation curves exhibiting sigmoidicity (A_0.5 = 0.81 millimolar; n_H = 2.0). Activation by 3-phosphoglycerate of the enzyme from Anabaena demonstrated hyperbolic kinetics (A_0.5 = 0.12 millimolar; n_H = 1.0), having a maximal stimulation of 17-fold. I_0.5 values of 95 and 44 micromolar were calculated for the inhibition by inorganic phosphate of the Synechocystis and Anabaena enzyme, respectively. Pyridoxal-phosphate behaved as an activator of the cyanobacterial enzyme. It activated the enzyme from Synechocystis nearly 10-fold with high apparent affinity (A_0.5 = 10 micromolar; n_H = 1.8). Phenylglyoxal modified the cyanobacterial enzyme by inactivating the activity in the presence of 3-phosphoglycerate. Antibody neutralization experiments showed that anti-spinach leaf (but not anti-Escherichia coli) ADPglucose pyrophosphorylase serum inactivated the enzyme from cyanobacteria. When the cyanobacterial enzymes were resolved on sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoresis and probed with Western blots, only one protein band was recognized by the anti-spinach leaf serum. The same polypeptide strongly reacted with antiserum prepared against the smaller spinach leaf 51 kilodalton subunit, whereas the anti-54 kilodalton antibody raised against the spinach subunit reacted weakly to the cyanobacterial subunit. Regulatory and immunological properties of the cyanobacterial enzyme are more related to the higher plant than the bacterial enzyme. Despite this, results suggest that the ADPglucose pyrophosphorylase from cyanobacteria is homotetrameric in structure, in contrast to the reported heterotetrameric structures of the higher plant ADPglucose pyrophosphorylase.     

Chloroplast and extrachloroplastic starch-degrading enzymes in Pisum sativum L.

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [³H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [³H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.Abbreviations CV(s) coated vesicle(s) - Da dalton - ER endoplasmic reticulum - GSI,II glucan synthase I and II, respecitively Two whom correspondence should be directed. Address after February 1986:Department of Biology, Texas A&M University. College Station, TX 77843-3258, USA     

Linear, Single-Stranded Deoxyribonucleic Acid Isolated from Kilham Rat Virus

Kilham rat virus (KRV) was grown in a rat nephroma cell line and was purified by two isopycnic centrifugations in cesium chloride. The virus contains single-stranded deoxyribonucleic acid (DNA) with a molecular weight of approximately 1.6 x 10(6). The DNA was extracted from the virion by both phenol extraction and by 2% sodium dodecyl sulfate at 50 C. KRV DNA, extracted by both procedures, was observed in an electron microscope by using a cytochrome c or diethylaminoethyldextran monolayer. The DNA was also exposed to exonuclease I, an enzyme which hydrolyzes specifically linear, single-stranded DNA. Hydrolysis of 70 to 80% of the DNA was observed. Both the enzymatic and the electron microscope studies support the conclusion that extracted KRV DNA is a single-stranded, linear molecule. The length of the DNA was measured in the electron microscope and determined to be 1.505 +/- 0.206 mum.     

Electron Microscopic Observations on the Ribonucleic Acid of Murine Leukemia Virus

The ribonucleic acid (RNA) of murine leukemia virus (MLV) Rauscher strain was observed by the aid of electron microscopy with the use of the protein monolayer technique. RNA was observed directly after release from virus particles or after isolation by sedimentation in sucrose density gradients. Molecules were found in an extended linear form. Many of the RNA filaments released by detergent treatment contained curled regions, suggesting the linear filaments were originally coiled within the virus particle. The relationship of the curled areas to the containment of the RNA within the virus particle is discussed, and a mechanism for the inclusion of RNA in the budding virion is proposed. Treatment of the extended MLV-RNA with dimethyl sulfoxide resulted in the collapse of the molecule forming a tangled complex. Treatment with urea or heating at 50 C in 3 mm NaCl also produced this effect. Also under the conditions in which MLV-RNA was linear, RNA from Rous sarcoma virus also was linear, but Newcastle disease virus RNA and ribosomal RNA of rat liver had collapsed structures. The results indicated that the RNA of MLV, and perhaps other RNA-containing tumor viruses, has a specific unique conformation dependent upon hydrogen bonds.     

Alteration of plasmid DNA-mediated transformation and mutation induced by covalent binding of benzo[a]pyrene- 7,8-dihydrodiol-9,10-oxide in Escherichia coli

Plasmid-mediated transformation and mutagenesis induced by (±)-trans- benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BP-DEI) in recipient Escherichia coli (E. coli) have been studied. Because plasmid DNA is used, the system is entirely free from direct toxic effects of BP-DEI on the recipient cells. Plasmid pK0482 DNA, which has two dominant genes, β-lactamase (amp-r) and galactokinase (galK) was modified with BP-DEI prior to its transformation of E. coli N99, AB1157, AB2463(recA^?) and AB1886(uvrA^?). Transformants were selected by ampicillin resistance and mutations were analyzed simultaneously by the altered expression of the galK gene. (1) Approx. 3 molecules of BP-DEI per molecule of pK0482 DNA decreased the transformation efficiency to 37% in AB1157 and the mutation frequency in this strain was proportional to the amount of BP-DEI covalently bound to pK0482 DNA. (2) In AB1886(uvrA^?) a 37% transformation efficiency was produced by only 1 molecule of BP-DEI per molecule of pK0482 DNA, and the mutation frequency in this strain was higher than in AB1157. (3) In AB2463(recA^?), the transformation efficiency was similar to that obtained with AB1157, but mutagenesis was clearly suppressed. (4) Polyacrylamide gel patterns of restriction digests of the pK0482 mutated at the galK gene were indistinguishable from those of the unmutated plasmid DNA.     

Substrate regulation of calcium binding in Ca2+-ATPase molecules of the sarcoplasmic reticulum. I. Effect of ATP

The effect of ATP on calcium binding of the Ca2+-ATPase of the sarcoplasmic reticulum has not been clarified. By comparing the calcium dependence of the ATPase activity and of phosphorylation of the ATPase molecules with that of calcium binding in the absence of ATP, we show the existence of two types of regulatory site of the enzyme molecules at which ATP binding variously improves the calcium binding performance of the molecules depending on the aggregation state of the molecules and pH; the two regulatory sites bind ATP at submillimolar (0.25 mm) and millimolar (5 mm) ATP, respectively. The results are discussed based on a model of two conformational variants (A and B forms) of the chemically equivalent ATPase molecules (Nakamura, J., and Furukohri, T. (1994) J. Biol. Chem. 269, 30818-30821). For example, in the sarcoplasmic reticulum membrane at pH 7.40, submillimolar ATP converted the calcium binding manner of the A form from noncooperative (Hill number (n(H)) of approximately 1) to cooperative (n(H) approximately 2), concurrent with a decrease in the apparent calcium affinity (K(0.5)) from 2-6 to 0.1-0.3 microm. The binding of the A form became almost the same as that of the B form (n(H) approximately 2, K(0.5) approximately 0.2 microm), which was not affected by ATP. Millimolar ATP further decreased the K(0.5) of the cooperative binding of the two forms to approximately 0.05 microm. Regulation of the calcium binding performance by ATP is discussed in terms of monomeric and oligomeric pathway models.     

Insular Gray Matter Volume and Objective Quality of Life in Schizophrenia

Improving quality of life has been recognized as an important outcome for schizophrenia treatment, although the fundamental determinants are not well understood. In this study, we investigated the association between brain structural abnormalities and objective quality of life in schizophrenia patients. Thirty-three schizophrenia patients and 42 age-, sex-, and education-matched healthy participants underwent magnetic resonance imaging. The Quality of Life Scale was used to measure objective quality of life in schizophrenia patients. Voxel-based morphometry was performed to identify regional brain alterations that correlate with Quality of Life Scale score in the patient group. Schizophrenia patients showed gray matter reductions in the frontal, temporal, limbic, and subcortical regions. We then performed voxel-based multiple regression analysis in these regions to identify any correlations between regional gray matter volume and Quality of Life Scale scores. We found that among four subcategories of the scale, the Instrumental Role category score correlated with gray matter volume in the right anterior insula in schizophrenia patients. In addition, this correlation was shown to be mediated by negative symptoms. Our findings suggest that the neural basis of objective quality of life might differ topographically from that of subjective QOL in schizophrenia.     

Discovery of 4,5-diphenyl-1,2,4-triazole derivatives as a novel class of selective antagonists for the human V(1A) receptor

In the search for a novel class of selective antagonists for the human V(1A) receptor, high-throughput screening (HTS) of the Yamanouchi chemical library using CHO cells expressing the cloned human V(1A) (hV(1A)) receptor led to the discovery of 5-(4-biphenyl)-4-(2-methoxyphenyl)-3-methyl-1,2,4-triazole (3) which possessed the novel 4,5-diphenyl-1,2,4-triazole structure. Subsequent structure-activity relationships studies on a series of the 4,5-diphenyl-1,2,4-triazole derivatives related to 3 revealed that the 4,5-diphenyl-1,2,4-triazole structure played an essential role in exerting high affinity for the hV(1A) receptor and that introduction of a basic amine moiety to the methoxy part of the 4-phenyl ring was effective in the improvement of both affinity for the hV(1A) receptor and selectivity versus the hV(2) receptor. Compound 3 and the 2-(morphorino)ethoxy derivative (11b) were shown to be antagonists for the hV(1A) receptor, from their effects on AVP-induced [Ca(2+)](i) response in CHO cells expressing the hV(1A) receptor.