Amino acid composition and terminal residues of aspartate aminotransferase from ox heart

1. The amino acid composition of highly purified aspartate aminotransferase from ox heart was determined. 2. Alanine is the only N-terminal residue. 3. Leucine was identified as the only C-terminal residue. 4. No disulphide bridges are present in the enzyme molecule. 5. The thiol groups are not equally accessible, the accessibility being comparatively easier in the apoenzyme molecule.     

Fatty acid composition of mitochondrial phospholipids of the ischemic heart

Gas liquid chromatography was used to study fatty acid (FA) composition of lysophosphatidylcholine (lyso-PC) and phosphatidylcholine in isolated rabbit heart mitochondria. In control, lyso-PC and PC were found to contain 95 and 66% of unsaturated FA, respectively. At 1 hour of ischemia (autolysis at 37 degrees C) the percentage of saturated FA in lyso-PC noticeably increased whereas FA composition remained unchanged. It is concluded that changes in FA composition of lyso-PC are caused by phospholipase A2 action.     

The isotope-exchange reactions of ox heart phosphofructokinase

1. Ox heart phosphofructokinase catalyses isotope-exchange reactions at pH6.7 between ADP and ATP, and between fructose 6-phosphate and fructose 1,6-diphosphate, the latter reaction being absolutely dependent on the presence of the magnesium complex of ADP. 2. The reaction kinetics are hyperbolic with respect to substrate concentration for both exchange reactions (within the experimental error). 3. The influence of pH, AMP and citrate suggests that the fructose 6-phosphate-fructose 1,6-diphosphate exchange is subject to effector control, and is abolished by dissociation of the enzyme. 4. These results are discussed in relation to the reaction mechanism of the enzyme.     

Effect of sex and dietary fat intake on the fatty acid composition of phospholipids and triacylglycerol in rat heart

Variations in the fatty acid composition of lipids in the heart alter its function and susceptibility to ischaemic injury. We investigated the effect of sex and dietary fat intake on the fatty acid composition of phospholipids and triacylglycerol in rat heart. Rats were fed either 40 or 100 g/kg fat (9:1 lard:soybean oil) from weaning until day 105. There were significant interactive effects of sex and fat intake on the proportions of fatty acids in heart phospholipids, dependent on phospholipid classes. 20:4n-6, but not 22:6n-3, was higher in phospholipids in females than males fed a low, but not a high, fat diet. There was no effect of sex on the composition of triacylglycerol. These findings suggest that sex is an important factor in determining the incorporation of dietary fatty acids into cardiac lipids. This may have implications for sex differences in susceptibility to heart disease.     

Purification and general properties of aspartate aminotransferase of ox heart

1. A five-step procedure for preparing highly purified aspartate aminotransferase from ox heart is described. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis in starch gel and in polyacrylamide gel. 3. The pure enzyme has an isoelectric point of about pH5, and E(1%) (1cm.) 14.40 at 278mmu. 4. The molecular weight of the pure enzyme was determined as 96000 by sedimentation equilibrium. 5. The pH optimum for the pure enzyme was about 8. It was determined by a new assay technique. 6. A difference in the electrophoretic migration rate between the enzyme from ox heart and brain and the enzyme from pig heart and brain suggests a species specificity rather than an organ specificity. 7. A new effect of deionization on the visible-absorption spectrum of the enzyme was observed.     

Separation of phosphoinositides and other phospholipids by two-dimensional thin-layer chromatography

A simple, rapid, two-dimensional TLC system is presented which resolves the four phosphoinositide cycle phospholipids as well as all commonly encountered major and minor phospholipids. Ca2+-free lipid samples are loaded onto silica gel HL plates and developed first in 48:40:7:5 chloroform:methanol:water:concentrated ammonia, and then in 55:25:5 chloroform:methanol:formic acid. The method was applied successfully to human erythrocytes, human platelets, and BL/VL3 murine lymphoma cells.     

Effect of exogenous N-stearoylethanolamine on fatty acid composition of individual phospholipids in the isolated rat heart under postischemic reperfusion

Changes of the individual phospholipid fatty acid composition under the normothermal short-time ischemia with following reperfusion were investigated. Modification of the phospholipid fatty acid (FA) composition under ischemia-reperfusion didn't bear total character and was more manifested in cardiolipin (CL) and phosphatidylethanolamine (PE). The decrease of short chain FA in these phospholipids (more than by 50%) was observed. The amount of unsaturated FA included in CL increased and whole the saturated ones decreased. This caused the rise of the unsaturation index. The selective type of the changes suggested that they had an adaptive character. The addition of the N-stearoilethanolamine (NSE) into the perfusion solution caused a normalization of saturated and unsaturated FA relative amount, as well as of omega-3 and omega-9 FA level in CL. The modification of the FA composition of phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI) was also found. The quantity of arachidonic acid in PC increased by 26% and the amount of stearinic acid enhanced in PS. The labeled N-([1-(14)C]-palmitoil)-ethanolamine was found in different lipid classes of the rat organs immediately 5 min following intraperitoneal injection. Approximately 1/3 of all incorporated label accumulated in the phospholipid fraction, and more than 50% of the labels were found in CL.     

Separation and quantitation of phospholipids and lysophospholipids by high-performance liquid chromatography

We describe a comprehensive approach to the separation, quantitation, and characterization of phospholipids and lysophospholipids present in complex biological samples. The central feature is a normal-phase HPLC separation of individual phospholipid and lysophospholipid classes. In this single chromatographic step, phospholipids and lysophospholipids are separated and recovered for quantitation by organic phosphate assay and characterization by acyl-group composition. Recovery of phospholipids and lysophospholipids from HPLC averages 80-90%. Isolated phospholipid and lysophospholipid fractions are available for separation of individual molecular species by second-dimension reverse-phase HPLC and characterization of individual molecular species by mass spectrometry.