Are plant microsomal ATPases lipid-dependent enzymes?

Potato microsomes were delipidated by aqueous acetone solutions of increasing concentrations. Lipid extraction did not change the basal ATPase activity of these membranes (measured in the absence of added mineral ions), but affected the Mg²⁺-dependent ATPase activity. Low acetone concentrations (5–15%) moderately stimulated the Mg²⁺-ATPase; more concentrated solutions (20–50% acetone) dramatically decreased the activity of this enzyme, but 70 and 90% acetone solutions enhanced it again, as compared to the activity of the 50% acetone-treated fraction. This last stimulation could be explained by the selective extraction of an inhibitor of Mg²⁺-ATPase by concentrated acetone solutions. After lipid extraction with 50–90% acetone solutions, the initial Mg²⁺-dependent ATPase activity could not be restored by adding lipids to delipidated microsomes. These results strongly suggest that, in potato microsomes, the Mg²⁺-dependent ATPase was a lipid-dependent enzyme, but suitable relipidation conditions remain to be found to definitely prove this lipid dependence.     

Stimulation of microsomal N-demethylation by solubilized NADPH-cytochrome c reductase

The addition of solubilized NADPH-cytochrome c reductase to phenobarbital pretreated microsomes increases both the Vm value for the N-demethylation of S(+)-N, N-dimethylamphetamine and the total level of reductase activity sedimenting with microsomes. Preliminary data indicate that the increase in Vm is a nonlinear function of added reductase and demonstrates saturation at a N-demethylase level approximately five times greater than the endogenous activity. These results indicate that the added reductase is bound to microsomes and is capable of functionally coupling with the cytochrome P₄₅₀ monooxygenase system.     

Thyroxine stimulation of rate liver microsomal NADH-cytochrome c reductase in vitro

Rat liver microsomal NADH-cytochrome $\text{c}$ reductase activity is stimulated by 20 μM thyroxine $\text{in}$$\text{vitro}$. Thyroxine does not influence microsomal NADH-dichlorophenolindophenol reductase, NADPH-cytochrome $\text{c}$ reductase, or NADPH-dichlorophenolindophenol reductase activity. Stimulation of NADH-cytochrome $\text{c}$ reductase activity is not mediated by super-oxide and is likely due to enhanced reduction or oxidation of cytochrome $\text{b}$5.     

NADH-cytochrome c reductase activity in cultured human lymphocytes: Similarity to the liver microsomal NADH-cytochrome b5 reductase and cytochrome b5 enzyme system

A NADH-cytochrome c reductase activity was increased upon mitogen stimulation of human lymphocytes. The activity was not inhibited by antimycin A or rotenone but was specifically inhibited by antibodies elicited against rat liver NADH-cytochrome b₅ reductase or cytochrome b₅. The activity was linear with cellular homogenates up to 5.2 × 10⁶ cells/ml and had abroad pH optimum of 7.7. The presence of 3-methylcholanthrene in mitogen stimulation media had no effect on the NADH-cytochrome c reductase activity but differentially induced the benzo(a)pyrene hydroxylase (AHH) activity. The reductase activity was present in nonstimulated cells and appears not to be significantly increased in activity per cell upon mitogen-stimulation of the peripheral lymphocyte.     

Regulation of liver base-exchange activity by acidic phospholipids

Liver microsomes were enriched in liposomal acidic lipids by Ca²⁺-dependent fusion of liposomes at pH 7.0. The extent of fusion was monitored by the transfer of radioactive cholesteryl oleate. The enrichment of membranes in phosphatidylserine inhibited ethanolamine base-exchange, whereas the fusion with phosphatidylinositol inhibited both ethanolamine and serine base-exchange reactions. In contrast, these two phospholipids had scarce effects on choline base-exchange. Phosphatidic acid did not suppress any of the three base-exchange activities. Possible functional implications are discussed.Abbreviations DTT dithiothreitol - HEPES 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - SHB suerose-HEPES buffer (0.25M sucrose, 3mM HEPES, pH 7.4)     

Effect of acidic phospholipids on sphingosine kinase

Sphingosine-1-phosphate (SPP) is a unique sphingolipid metabolite involved in cell growth regulation and signal transduction. SPP is formed from sphingosine in cells by the action of sphingosine kinase, an enzyme whose activity can be stimulated by growth factors. Little is known of the mechanisms by which sphingosine kinase is regulated. We found that acidic phospholipids, particularly phosphatidylserine, induced a dose-dependent increase in sphingosine kinase activity due to an increase in the apparent Vmax of the enzyme. Other acidic phospholipids, such as phosphatidylinositol, phosphatidic acid, phosphatidylinositol bisphosphate, and cardiolipin stimulated sphingosine kinase activity to a lesser extent than phosphatidylserine, whereas neutral phospholipids had no effect. Diacylglycerol, a structurally similar molecule which differs from phosphatidic acid in the absence of the phosphate group, failed to induce any changes in sphingosine kinase activity. Our results suggest that the presence of negative charges on the lipid molecules is important for the potentiation of sphingosine kinase activity, but the effect does not directly correlate with the number of negative charges. These results also support the notion that the polar group confers specificity in the stimulation of sphingosine kinase by acidic glycerophospholipids. The presence of a fatty acid chain in position 2 of the glycerol backbone was not critical since lysophosphatidylserine also stimulated sphingosine kinase, although it was somewhat less potent. Dioleoylphosphatidylserine was the most potent species, including a fourfold stimulation, whereas distearoyl phosphatidylserine was completely inactive. Thus, the degree of saturation of the fatty acid chain of the phospholipids may also play a role in the activation of sphingosine kinase. © 1996 Wiley-Liss, Inc.     

Stimulation of plant plasma membrane Ca2+-ATPase activity by acidic phospholipids

The effect of phospholipids on the activity of the plasma membrane (PM) Ca²⁺-ATPase was evaluated in PM isolated from germinating radish ( Raphanus sativus L. cv. Tondo Rosso Quarantino) seeds after removal of endogenous calmodulin (CaM) by washing the PM vesicles with EDTA. Acidic phospholipids stimulated the basal Ca²⁺-ATPase activity in the following order of efficiency: phosphatidylinositol 4,5-diphosphate (PIP2)≈phosphatidylinositol 4-monophosphate>phosphatidylinositol≈phosphatidylserine≈phosphatidic acid. Neutral phospholipids as phosphatidylcholine and phosphatidylethanolamine were essentially ineffective. When the assays were performed in the presence of optimal free Ca²⁺ concentrations (10 μ M ) acidic phospholipids did not affect the Ca²⁺-ATPase activated by CaM or by a controlled trypsin treatment of the PM, which cleaved the CaM-binding domain of the enzyme. Analysis of the dependence of Ca²⁺-ATPase activity on free Ca²⁺ concentration showed that acidic phospholipids increased V_max and lowered the apparent K_m for free Ca²⁺ below the value measured upon tryptic cleavage of the CaM-binding domain; in particular, PIP2 was shown to lower the apparent K_m for free Ca²⁺ of the Ca²⁺-ATPase also in trypsin-treated PM. These results indicate that acidic phospholipids activate the plant PM Ca²⁺-ATPase through a mechanism only partially overlapping that of CaM, and thus involving a phospholipid-binding site in the Ca²⁺-ATPase distinct from the CaM-binding domain. The physiological implications of these results are discussed.     

Substrate specificity of potato lipoxygenase

The substrate specificity of potato lipoxygenase was examined using a partially purified enzyme preparation from tubers of a potato variety with low lipolytic acyl hydrolase activity. Potato lipoxygenase is fully active only on free linoleic acid or linolenic acid, and only acts directly on more complex glyceride moieties in the absence of any significant endogenous lipolytic acyl hydrolase activity.     

Conversion of p-coumaric acid to p-hydroxybenzoic acid by cell free extracts of potato tubers and Polyporus hispidus

The conversion of p-coumaric acid to p-hydroxybenzoic acid was demonstrated in vitro in both potato tuber and P. hispidus. The mechanism of the enzyme system is non-oxidative. This is the first report of a cell free system which is capable of converting a C₆-C₃ acid to the corresponding C₆-C₁ derivative from a fungus.     

Preparation of chaconines by enzymic hydrolysis of potato berry alkaloids

Endogenous enzyme activity in a blend of potato berries and blossoms converts the contained glycoalkaloids within 24 hr to a mixture of α-solanine an     

Stimulation by local anesthetics of the metabolism of acidic phospholipids in the rat pineal gland

The efficacy of five local anesthetics in causing stimulation of phospholipid metabolism in rat pineal gland $\text{in vitro}$ paralleled their anesthetic potency and decreased in the order: dibucaine, tetracaine, cocaine, procaine, lidocaine. When stimulation occurred, the patterns of labeling resembled that produced by propranolol, a β-adrenergic receptor blocking agent with local anesthetic activity. Isotope incorporation into phosphatidylglycerol and CDP-diglyceride was markedly enhanced and increases of labeling of phosphatidic acid and phosphatidylinositol were also seen. At concentrations of 1–10 mM, propranolol and local anesthetics inhibited labeling of phosphatidylcholine and phosphatidylethanolamine by more than 90% and incorporation of ³²P_i into other phospholipids to a smaller extent.     

Biosynthesis of cytokinins by potato cell cultures

Potato cells grown in liquid culture incorporated mevalonic acid lactone-[2-¹⁴C] into free cytokinin (zeatin riboside and zeatin and the cytokinin of RNA (zeatin riboside). The cytokinin liberated by catabolism of RNA can account for no more than 40% of the free cytokinins.     

Effects of bovine serum albumin and phospholipids on activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase in sweet potato roots

Sweet potato microsomal 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase preincubated at 30?C was inactivated 50to 60%. The inactivation depended on temperature and was muchless with preincubation below 20?C. High concentration (above0.6%, w/v) of bovine serum albumin not only prevented inactivationbut also increased the activity. Even after preincubation fora given time without bovine serum albumin, its addition at 1%(w/v) prevented inactivation during further incubation, althoughit was unable to restore the activity to the initial level. Microsomal lipids were hydrolyzed during preincubation at 30?C.There was a positive correlation between formation of fattyacids during the preincubation and loss of HMG-CoA reductaseactivity. The micelles prepared from sweet potato microsomalphospholipids also prevented enzyme inactivation. These resultssuggest that the hydrolysis of microsomal phospholipids inducesthe instability of microsomal HMG-CoA reductase by alteringmicrosomal membrane structures and that the enzyme requiresphospholipids for its activity. Besides bovine serum albumin and phospholipids, NADPH₂ and HMG-CoAadded together prevented inactivation of this enzyme but notwhen added separately. ¹ This paper constitutes Part 128 in the series "The PhytopathologicalChemistry of Sweet Potato with Black Rot and Injury." This workwas supported in part by a grant from the Ministry of Education. (Received October 28, 1976; )