Autolysis of phospholipids in homogenates of various plant tissues

The rates of autolysis of endogenous phospholipids were measured in homogenates of 17 cultivated plant tissues. For seven out of the 17 samples, the rates of autolysis of phosphatidylcholine (PC) were greater than 10% hydrolysis per hr (at pH 7.5 and 4°). The addition of dibucaine (2 mM) inhibited the autolytic rates in homogenates of 12 out of 17 samples. The only plant homogenates where dibucaine stimulated the autolytic rates were those of potato tubers. Although dibucaine inhibited the rate of autolysis of PC in potato leaf homogenates there was no advantage to adding it during homogenization and preparation of differential centrifugation fractions from potato leaves. Homogenization with different types and concentrations of osmotica affected the rates of autolysis of PC. Buffering the homogenates at pH 8 drastically inhibited the rates of autolysis in potato leaf homogenates but had little effect on bean leaf homogenates. Various strategies for controlling the rate of membrane lipid breakdown in different plant extracts are discussed.     

Regulatory effect of pig heart phospholipids on heart muscle AMP-deaminase

AMP-deaminase purified from pig heart has been found to be activated by liposomes prepared from phospholipids extracted from pig heart mitochondria, microsomes and cytoplasm, as well as by intact microsomes. The activation by phospholipids occurred only in the presence of ATP and after the enzyme had been preincubated with liposomes for 30 min. Liposomes prepared from cardiolipin displayed an inhibitory effect on pig heart AMP-deaminase.     

Subcellular distribution and some properties of alanine aminotransferase in striated muscles of the crayfish, trout, carp, frog, pigeon and rabbit.

Subcellular distribution and some physicochemical properties of alanine aminotransferase in striated muscles of the crayfish, trout, carp, frog, pigeon and rabbit were studied. It was established that: (1) Alanine aminotransferase activity in all mentioned animals occurred almost entirely in the cytosolic fraction of the muscles. Total activity and activity per mg protein were highest in crayfish and pigeon muscles and lowest in carp and trout muscles. (2) The pH optimum for the muscles of homoiotherms and poikilotherms ranged from 7.5 to 8, Km values for L-alanine were of the order 10(-3)--10(-2) M and those for alpha-ketoglutarate 10(-4) M. (3) A 10 degree C temperature increase of the incubation medium was accompanied by a 70--90% increase in activity. (4) The higher the alanine aminotransferase activity of the muscles, the relatively higher their alanine production during electrical stimulation. (5) From the above results it is concluded that alanine aminotransferase in striated muscles regulates the rate of glycolysis and energy production under conditions of anaerobiosis through the formation of alanine.     

Beta-actinin isoforms in various types of muscle and non-muscle tissues

We found that beta-actinin isoforms are present in various types of tissues in adult chicken by using immunoblotting after two dimensional gel electrophoresis; for this purpose, an antibody was raised against beta-actinin purified from adult chicken breast muscle (pectoralis major). One of the beta-actinin subunits, beta I, was present in all tissues we examined, i.e. skeletal (pectoralis major, semitendinosus, and anterior latissimus dorsi), cardiac, and smooth (gizzard) muscles, non-muscle (brain, liver, and kidney) tissues and blood, whereas another subunit, beta II, was present only in muscle tissues. A new subunit (designated beta III) that was found in the embryonic stages of skeletal muscle (Asami, Funatsu & Ishiwata (1988) J. Biochem. 103, 72-75) was present instead of beta II in non-muscle tissues and blood. In cardiac and smooth muscles, beta III coexisted with beta I and beta II. The antibody of beta-actinin did not cross-react to cytoplasmic beta-actinin (molecular weight, 80,000 daltons) found in kidney. It was suggested that the combination of beta I and beta III present in non-muscle tissues and blood is identical to the barbed end capping protein isolated from brain by Killiman and Isenberg (EMBO J. 1, 889-894 (1982)). It is likely that beta-actinin forms a genetic family whose constituents have an ability to cap either the pointed or barbed end of actin filaments.     

Multiple forms of phosphofructokinase in developing rabbit and guinea pig tissues.

Multiple forms of phosphofructokinase in striated muscle and cardiac muscle of developing rabbit (Oryctolagus cuniculus) undergo changes with development, but not in brain and liver. The cardiac muscle of the 1-day-old rabbit contains phosphofructokinase A₄ together with the four hybrid forms which were tentatively called A₃C, A₂C₂, AC₃, and C₄. In older animals, phosphofructokinase C₄ disappears first, followed by the hybrid forms, and only phosphofructokinase A₄ persists in the adult animal. Both phosphofructokinase A₄ and phosphofructokinase C₄, as well as their hybrid forms, are present in developing embryonic brain and also in the brains of adult animals. Developing rabbit liver contains a single form of phosphofructokinase, but two isoenzymes are consistently seen in guinea pig liver. In striated muscle from fetal and 1-day-old rabbit, two isoenzymes are found, tentatively identified as A₄ and the A₃C hybrid. The results suggest that fetal phosphofructokinase A₄ and phosphofructokinase C₄, and their hybrids, might be present in striated muscle. Guinea pig tissues show a pattern of phosphofructokinase isoenzymes different from that in rabbit tissues.     

Dietary fatty acid composition changes mitochondrial phospholipids and oxidative capacities in rainbow trout red muscle

Dietary conditioning of juvenile trout changed the acyl chain composition of mitochondrial phospholipids and the oxidative capacities of muscle mitochondria. Trout were fed three diets differing only in fatty acid (FA) composition. The highly unsaturated 22:6 n-3 (DHA) accounted for 0.4, 14, and 30% of fatty acids in Diets 1, 2 and 3. After 10 weeks of growth, the dietary groups differed markedly in FA composition of mitochondrial phospholipids, with significant dietary effects for virtually all FA. Mean mitochondrial DHA levels were 19, 40 and 33% in trout fed Diets 1, 2 and 3. Mitochondrial oxidative capacities changed with diet, while mitochondrial concentrations of cytochromes and of the adenylate nucleotide translocase (nmol mg¹ protein) did not. Mitochondria from fish fed Diet 1 had higher non-phosphorylating (state 4) rates at 5°C than those fed other diets. When phosphorylating (state 3) rates differed between dietary groups, rates at 5 and 15°C were higher for fish fed the more unsaturated diets. Stepwise multiple regressions indicated that FA composition could explain much (42–70%) of the variability of state 4 rates, particularly at 5°C. At 15°C, FA composition explained 16–42% of the variability of states 3 and 4 rates. Similar conclusions were obtained for the complete data set (trout fed diets 1, 2 and 3) and for the data from trout achieving similar growth rates (e.g. those fed Diets 1 and 2). Neither general characteristics of membrane FA, such as % saturates, unsaturation index, n-3, n-6 or n-3/n-6 nor levels of abundant unsaturated FA such as DHA or 18:1(n-9 + n-7), were systematically correlated with mitochondrial capacities even though they differed considerably between trout fed the different diets. Relatively minor FA (20:5n-3, 20:0, 18:2n-6, 18:3n-3, 18:0 and 15:0) showed better correlations with mitochondrial oxidative capacities. This supports the concept that acyl chain composition modulates mitochondrial capacities via interactions between membrane proteins and specific FA of particular phospholipid classes in their microenvironment.     

Comparative aspects of aminotransferases in the rat, pigeon and rainbow trout.

1. The activities of aminotransferases catalysing the transfer of amino groups from aspartate, alanine and leucine to 2-oxoglutarate in different tissues of the rat, pigeon and trout have been determined. 2. Alanine-2-oxoglutarate aminotransferase was high in the liver of the rat and trout and low in that of the pigeon. 3. Aspartate-2-oxoglutarate aminotransferase was usually the dominant aminotransferase in all tissues and was highest in oxidative tissues where the TCA cycle is active. Its activity in the various livers is not correlated with the function of aspartate in nitrogen excretion. 4. The activity of aspartate-2-oxoglutarate aminotransferase in oxidative tissues argues that aspartate in conjunction with this enzyme serves as a buffer of oxaloacetate to keep the TCA cycle running and/or to mediate the transfer of reducing equivalents across mitochondrial membranes.