Shoji Sasaki

The cis/trans isomerization of the double bond of a fatty acid as a strategy for adaptation to changes in ambient temperature in the psychrophilic bacterium, Vibrio sp. strain ABE-1

The major phospholipid, phosphatidylethanolamine (PE), of Vibrio sp. strain ABE-1 contains a unique trans-unsaturated fatty acid, 9-trans-hexadecenoic acid (16:1(9t], at the sn-2 position of the glycerol moiety. The major molecular species of PE that contain 16:1(9t) at the sn-2 position have either 9-cis-hexadecenoic acid (16:1(9c] or hexadecanoic acid (16:0) at the sn-1 position. The transition temperatures of the liquid-crystal to the gel phase of 16:1(9c)/16:1(9t)-PE and 16:0/16:1(9t)-PE were -3 degrees C and 38 degrees C, respectively, temperatures that were 31 degrees C and 18 degrees C higher than the corresponding temperatures for 16:1(9c)/16:1(9c)-PE and 16:0/16:1(9c)-PE. The proportion of 16:1(9c)/16:1(9t)-PE and 16:0/16:1(9t)-PE increased significantly in cells grown at 20 degrees C over that in cells grown at 5 degrees C. When cells grown at 5 degrees C were incubated at 20 degrees C in the presence of cerulenin, an inhibitor of the synthesis de novo of fatty acids, the level of 16:1(9t) increased almost in parallel with a concomitant decrease in the level of 16:1(9c) at the sn-2 position. These results suggest that 16:1(9c) is converted to 16:1(9t) by the cis/trans isomerization of the double bond in the fatty acid. This conversion is discussed as a possible strategy for adaptation by bacteria to changes in temperature.

Purification and properties of aldehyde dehydrogenase from Proteus vulgaris

NADP-linked aldehyde dehydrogenase (aldehyde : NADP+ oxidoreductase, EC 1.2.1.4) was purified from Proteus vulgaris to the stage of homogeneity as judged by ultracentrifugation and polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 130000 by gel filtration. The enzyme which was crystallized from ammonium sulfate solution, lost its activity. The enzyme did not require coenzyme A, and the reaction was completely dependent on ammonium ions which could be partially replaced by Rb+ or K+. The optimum pH was about 9. Broad substrate specificity was observed and Km values for propionaldehyde, acetaldehyde and isovaleraldehyde were 1.7 - 10(-5), 4 - 10(-5) and 3 - 10(-5) M, respectively. The physiological role of the enzyme in living cells is obscure, but might account for another degradative pathway of L-leucine in P. vulgaris differing from the established pathway.

Synthesis in vitro of very long chain fatty acids in Vibrio sp. strain ABE-1

The activity of fatty acid synthetase (FAS) from Vibrio sp. strain ABE-1 required the presence of acyl carrier protein and was completely inhibited by thiolactomycin, an inhibitor specific for a type II FAS. These observations indicate that this enzyme is a type II FAS. Analysis by gas-liquid chromotography of the reaction products synthesized in vitro from [2-14C]malonyl-CoA by the partially purified FAS revealed, in addition to 16-and 18-carbon fatty acids which are normal constituents of this bacterium, the presence of fatty acids with very long chains. These fatty acids were identified as saturated and mono-unsaturated fatty acids with 20 up to as many as 30 carbon atoms. The longest fatty acids normally found in this bacterium contain 18-carbon atoms. These results suggest that the FAS from Vibrio sp. strain ABE-1 has potentially the ability to synthesize fatty acids with very long chains.

Purification and properties of NADP pyrophosphatase from Proteus vulgaris

Abstract 1. 1. NADP pyrophosphatase was purified about 700-fold from Proteus vulgaris by a procedure consisting of solubilization by detergents, fractionation by ammonium sulfate and cold ethanol, and column chromatography using DEAE-cellulose, hydroxylapatite and phosphocellulose. 2. 2. The purified enzyme was slightly activated by Mg 2+ and Mn 2+ and inhibited by Co 2+ and Zn 2+ . When the enzyme was treated with 5 mM EDTA and dialyzed against 10 mM Tris-HCl buffer, pH 7.0, the activity almost disappeared and the enzyme was reactivated strongly by Co 2+ and slightly by Mn 2+ . 3. 3. The optimum pH is 7.0. K m value for NADP + in Tris-HCl buffer at pH 7.0 was about 25 μM. 4. 4. Both NADP + and NADPH were cleaved rapidly at almost the same rate. NADH was also cleaved at half the rate of NADP + cleavage, while NAD + was cleaved at only 7% of this rate. ADP and UDP were also cleaved to release inorganic orthophosphate. 5. 5. NADP + cleavage by the enzyme was competitively inhibited by CoA, FAD, ATP, GTP, UTP and CTP. Among them CoA, GTP and CTP were very effective inhibitors. RNA, DNA and denatured DNA also inhibited NADP pyrophosphatase activity as well as boiled extracts of P. vulgaris , but these inhibitors were not competitive with NADP + .

[80] Aldehyde dehydrogenase from Proteus vulgaris

Publisher Summary This chapter describes the assay method, purification, and properties of aldehyde dehydrogenase isolated from Proteus vulgaris . The activity of aldehyde dehydrogenase is monitored by measuring the production of nicotinamide adenine dinucleotide phosphate dehydrogenase (NADPH) spectrophotometrically at 340 nm with isovaleraldehyde as a substrate. Isovaleraldehyde is replaced with propionaldehyde or acetaldehyde, which are also good substrates for this enzyme. The steps involved in the purification are (1) protamine sulfate fraction, (2) acetone fraction, (3) ammonium sulfate fraction, (4) hydroxyapatite column chromatography, (5) diethylaminoethyl (DEAE)-cellulose column chromatography, and (6) crystallization. All procedures are performed at 0–4°C, unless otherwise stated. Washed cells are suspended in an appropriate amount of 20 m M potassium phosphate buffer, pH 7.0, to give about 8 g/40 ml. The aliquots of 40 ml of the cellular suspension are treated by sonic oscillation at 20 kc for 8 min at 0– 2°C to avoid thermal inactivation. The supernatants from similar aliquots obtained by centrifugation at 15,000 g for 30 min are combined.

Low-temperature-dependent ribonuclease in chromatin of winter wheat seedlings

Summary A kind of ribonuclease having an optimun temperature of about 15°C was detected in chromatin of seedlings of a species of winter wheat, besides another kind having an optimum temperature of about 30°C. The optimum pH was about 8.0. The activity of ribonuclease was inhibited by bontonite and DNA, and was stimulated by cyclic AMP. The low-temperature-dependent activity was thermolabile.

Temperature-Dependent Alteration in CIS and Trans Unsaturation of Fatty Acids in Vibrio SP. Strain ABE-1

The trans configuration has been regarded as a nonphysiological double-bond of fatty acids. However, a considerably high level of Δ9-trans-hexadecenoic acid (9-trans- 16:1) was found in a marine psychrophilic bacterium, Vibrio sp. strain ABE-1 (Vibrio ABE-1), which has an optimum and maximum growth temperature at 15°C and 20°C, respectively (1). The phase transition temperature of the membrane phospholipids from Vibrio ABE-1 depends on growth temperature (2). Since the phospholipid composition of Vibrio ABE-1 is unaffected by growth temperature (2), the changes in phase transition temperature of the phospholipids are likely to be associated with the temperature-dependent alteration in their fatty acid composition.

Studies on pH of shoyu koji. Part I. Relationship between pH and citric acid content in shoyu koji.

醤油麹のpHに及ぼすクエン酸量の影響,麹菌のクエン酸代謝について調べ,次の結果を得た. (1) 麹のクエン酸量は麹pHと負の相関関係にあった. (2) 麹のクエン酸量は散水量,培養湿度,菌株によって大きく異なった.クエン酸は低散水量,低温培養麹ではクエン酸が生成,蓄積され,高散水量,高温培養麹では消費,減少する傾向があった. (3) トレーサー実験より,A. oryzaeを使用した100%散水,23°C培養で,添加したリンゴ酸,ピルビン酸よりクエン酸が生成され,添加したクエン酸は消費されず麹中にクエン酸が蓄積されることが示された.しかし,A. sojaeを使用すると同条件で,添加したクエン酸がわずかに消費されておりA. oryzaeと若干異なっていた.一方,180%散水,33°C培養では両菌株ともに添加したクエン酸は消費,減少した. (4) クエン酸代謝関遵酵素活性はA. oryzae,A. sojaeともに,100%散水,23°C培養麹では180%散水33°C培養麹に比較してクエン酸シンターゼ活性が高く,アコニターゼ活性が低い傾向にありクエン酸の生成,消費と関連していた.