Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.     

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.     

Phospholipid and fatty acid composition in mitochondria from spinach (Spinacia oleracea) leaves and petioles. A comparative study.

Essentially chlorophyll-free mitochondria from photosynthetic (leaf) and non-photosynthetic tissue (petiole) were isolated from spinach (Spinacia oleracea). Leaf mitochondria were found to contain more phosphatidylcholine than phosphatidylethanolamine compared with petiole mitochondria. Galactolipids were found in small and equal amounts (5 mol of galactolipids/100 mol of galactolipids and phospholipids) in both leaf and petiole mitochondria. Fatty acid composition showed a significant difference in the amounts of C18:2 and C18:3 acids. The C18:2/C18:3 ratio was more than twice as high in all of the phospholipids studied from petiole mitochondria compared with the ratio in leaf mitochondria. More than 50% (mol/100 mol) of the fatty acids in the major lipids (phosphatidylcholine, phosphatidylethanolamine and cardiolipin) in petiole mitochondria were C18:2. In the minor lipids (phosphatidylinositol and phosphatidylglycerol), C16:0 dominated in both leaf and petiole mitochondria.     

The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro.

The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases.     

Effects of the haem precursor 5-aminolaevulinate on tryptophan metabolism and disposition in the rat.

5-Aminolaevulinate administration to rats inhibits cerebral 5-hydroxytryptamine synthesis by decreasing tryptophan availability to the brain secondarily to activation of hepatic tryptophan pyrrolase. The results show that tryptophan metabolism and disposition can be influenced by changes in liver haem concentration, and are discussed briefly in relation to mood disorders in the hepatic porphyrias.     

Conformational changes induced in lens alpha- and gamma-crystallins by modification with glucose 6-phosphate. Implications for cataract.

There is good evidence that the non-enzymic chemical modification of proteins plays a role in the aetiology of cataract and diabetic sequelae. This paper presents new evidence that glycosylation of two major lens structural crystallins, alpha- and gamma-crystallins, by glucose 6-phosphate (G6P) induces conformational changes in the proteins. In addition the surface charge on the molecules is altered. These changes would affect protein-protein and protein-water interactions within the lens and could lead to disruption of the short-range order of the lens proteins which is essential for lens transparency. Conformational changes to lens proteins are known to occur in human cataractous lenses but their cause in vivo is not established. Cumulative chemical modification of proteins, over a period of decades, is a strong candidate as a causal agent.     

The interaction in vivo of transferrin and asialotransferrin with liver cells.

Rat transferrin or asialotransferrin doubly radiolabelled with 59Fe and 125I was injected into rats. A determination of extrahepatic and hepatic uptake indicated that asialotransferrin delivers a higher fraction of the injected 59Fe to the liver than does transferrin. In order to determine in vivo the intrahepatic recognition sites for transferrin and asialotransferrin, the liver was subfractionated into parenchymal, endothelial and Kupffer cells by a low-temperature cell isolation procedure. High-affinity recognition of transferrin (competed for by an excess of unlabelled transferrin) is exerted by parenchymal cells as well as endothelial and Kupffer cells with a 10-fold higher association (expressed per mg of cell protein) to the latter cell types. In all three cell types iron delivery occurs, as concluded from the increase in cellular 59Fe/125I ratio at prolonged circulation times of transferrin. It can be calculated that parenchymal cells are responsible for 50-60% of the interaction of transferrin with the liver, 20-30% is associated with endothelial cells and about 20% with Kupffer cells. For asialotransferrin a higher fraction of the injected dose becomes associated with parenchymal cells as well as with endothelial and Kupffer cells. Competition experiments in vivo with various sugars indicated that the increased interaction of asialotransferrin with parenchymal cells is specifically inhibited by N-acetylgalactosamine whereas mannan specifically inhibits the increased interaction of asialotransferrin with endothelial and Kupffer cells. Recognition of asialotransferrin by galactose receptors from parenchymal cells or mannose receptors from endothelial and Kupffer cells is coupled to active 59Fe delivery to the cells. It is concluded that, as well as parenchymal cells, liver endothelial and Kupffer cells are also quantitatively important intrahepatic sites for transferrin and asialotransferrin metabolism, an interaction exerted by multiple recognition sites on the various cell types.     

A carrier-mediated transport for folate in basolateral membrane vesicles of rat small intestine.

The mechanism of exit of folate from the enterocyte, i.e. transport across the basolateral membrane, is not known. In this study we examined, using basolateral membrane vesicles, the transport of folic acid across the basolateral membrane of rat intestine. Uptake of folic acid by these vesicles represents transport of the substrate into the intravesicular compartment and not binding to the membrane surface. The rate of folic acid transport was linear for the first 1 min of incubation but decreased thereafter, reaching equilibrium after 5 min of incubation. The transport of folic acid was: (1) saturable as a function of concentration with an apparent Km of 0.6 +/- 0.17 microM and Vmax. of 1.01 +/- 0.11 pmol/30 s per mg of protein; (2) inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate and methotrexate (Ki = 2 and 1.4 microM, respectively); (4) electroneutral; (5) Na+-independent; (6) sensitive to the effect of the anion exchange inhibitor 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS). These data indicate the existence of a carrier-mediated transport system for folic acid in rat intestinal basolateral membrane and demonstrate that the transport process is electroneutral, Na+-independent and sensitive to the effect of anion exchange inhibition.     

Protein phosphorylation in rat mammary acini and in cytosol preparations in vitro. Phosphorylation of acetyl-CoA carboxylase is unaffected by cyclic AMP.

Phosphorylation of soluble proteins in rat mammary acinar cells was investigated. When phosphorylation proceeded in intact cells, in the presence of [32P]Pi, the major non-casein phosphoproteins, including acetyl-CoA carboxylase, were unresponsive to incubation conditions that caused major increases in the intracellular concentration of cyclic AMP. The overall 32P specific radioactivity (c.p.m./microgram of protein) of acetyl-CoA carboxylase, assessed after affinity purification of the enzyme with avidin-Sepharose, was unchanged by incubation under such conditions. Furthermore, the distribution of 32P among tryptic phosphopeptides of the enzyme, resolved by reversed-phase h.p.l.c., was not altered by cyclic AMP-increasing treatments of the acinar cells. When cytosol fractions were incubated with [gamma-32P]ATP, some phosphoproteins responded to the addition of micromolar concentrations of dibutyryl cyclic AMP or cyclic AMP by undergoing an enhancement of phosphate incorporation. In these experiments in vitro, protein phosphatase activity did not make a major contribution to the net phosphorylation of individual phosphoproteins, and acetyl-CoA carboxylase was not prominent among the phosphoproteins identified after short (less than 1 min) incubations of cytosols with [gamma-32P]ATP. The resistance of protein phosphorylation to variations in the cyclic AMP concentration in intact mammary epithelial cells, demonstrated by this work, is one of several mechanisms that ensure the pleiotropic refractoriness of those cells to agents which normally cause a stimulation of adenylate cyclase activity in hormone-sensitive cells.