Intermediates and enzymes between alpha-ketoarginine and gamma-guanidinobutyrate in the L-arginine catabolic pathway of Pseudomonas putida.

In Pseudomonas putida P2 grown on L-arginine as the sole source of carbon and nitrogen, catabolism of L-arginine forms of alpha-ketoarginine, gamma-guanidinobutyrate, and gamma-aminobutyrate. A previously undetected intermediate, gamma-guanidinobutyraldehyde, is identified as the product of alpha-ketoarginine decarboxylase. An 86-fold purification of this enzyme is described. Activity is thiamine pyrophosphate-dependent and cofactor reassociation is facilitated by divalent cations. The order of effectiveness is Mn-2+ greater than Mg-2+, Co-2+ greater than Ca-2+ greater than Ni-2+ greater than Zn-2+. An inducible enzyme that catalyzes conversion of gamma-guanidinobutyraldehyde to gamma-guanidinobutyrate has been studied in cell-free extracts. NAD-+, but no other cofactors, is required. By differential nutritional growth experiments, 4 regulatory units for the L-arginine pathway are proposed and inducers of 2 units are identified.     

Properties and subcellular localization of elastase-like activities of arterial smooth muscle cells in culture

The interaction of cardiolipin with Ca2+ was assessed by measuring the cardiolipin-mediated extraction of 45Ca2+ from an aqueous to an organic (methylene chloride) phase. Cardiolipin binds Ca2+ with high affinity [Kd(apparent) = 0.70 +/- 0.17 microM (S.D.)]. Cation-cardiolipin interactions are selective. Interaction of cardiolipin with Ca2+ is insensitive to Na+, but is inhibited by divalent cations with Mn2+ greater than Zn2+ greater than Mg2+. In addition La3+ and Ruthenium red are particularly potent inhibitors of Ca2+ binding by cardiolipin. Cardiolipin-mediated extraction of Ca2+ into an aqueous phase is also inhibited by phosphatidylcholine. Inhibition of Ca2+-cardiolipin interaction by phosphatidylcholine (a phospholipid known to stabilize the bilayer conformation) may implicate inverted, non-bilayer lipid structures in the binding.     

Participation of the microsomal CDP-diglycerides in the mitochondrial biosynthesis of phosphatidylglycerol

Participation of microsomal CDP-diglycerides in mitochondrial biosynthesis of phosphatidylglycerol was studied by [3H]palmitoyl, [14C]linoleoyl, and [14C]arachidonoyl CDP-diglycerides and [3H]CDP-diglycerides which were bound to microsomal membranes, incubated with unlabelled mitochondrial membranes, and further incubated in the presence of radioactive sn-glycero-3-phosphate under conditions required for mitochondrial phosphatidylglycerol biosynthesis. Ten to 15% of microsomal radioactive CDP-diglycerides was transferred to mitochondrial membranes and incorporated into mitochondrial radioactive lipids identified as phosphatidylglycerol, phosphatidylglycerophosphate, and, when [14C]linoleoyl CDP-diglycerides were used, diphosphatidylglycerol (cardiolipin).     

Mechanism and localization of cardiolipin biosynthesis revisited: evidence for the identical mechanism and different localization in mitochondrial and submitochondrial membranes isolated from guinea pig and rat liver

The mechanism of cardiolipin (diphosphatidylglycerol) biosynthesis was examined in mitochondria and outer and inner mitochondrial membranes prepared from guinea pig and rat livers to determine whether this formation from phosphatidylglycerol was absolutely dependent on cytidinediphosphodiglyceride, as previously reported for intact mitochondria. Experimental results confirmed that the biosynthesis of cardiolipin, from the membrane-bound radioactive phosphatidylglycerol in intact mitochondria isolated from guinea pig and rat liver, was absolutely dependent on CDP-diglycerides and required the addition of divalent cations. Furthermore, the same mechanism for the biosynthesis of cardiolipin was operational in the outer and inner mitochondrial membranes. This biosynthesis was associated with both the outer and inner mitochondrial membranes prepared from guinea pig liver, but only with the inner mitochondrial membranes prepared from rat liver. The release of radioactive glycerol was also measured, but the amount obtained did not satisfy the stoichiometric requirement for CDP-diglyceride-independent biosynthesis of cardiolipin from 2 mol of phosphatidylglycerol with the liberation of 1 mol of glycerol. Therefore, it was concluded that this mechanism is not involved in the biosynthesis of cardiolipin in mitochondrial and submitochondrial membranes prepared from guinea pig and rat liver.     

Phosphatidylglycerol in lung surfactant. II. Subcellular distribution and mechanism of biosynthesis in vitro.

Lamellar inclusion bodies, apparent precursors for alveolar surfactant lining, have remarkably similar phospholipid composition to surfactant from alveolar lavage, but distinctly different from other fractions studied: mitochondria, microsomal fraction containing endoplasmic reticulum membranes, plasma membranes and nuclei. Surfactant contained (as % of total phospholipid phosphate): 75.5-77.0% lecithin, 11.0-11.2% phosphatidylglycerol, 4.2-4.6% phosphatidylethanolamine, 3.0-3.2% phosphatidylinositol, 1.5-1.7% bis-(monoacylglycerol) phosphate, 1.2-1.9% phosphatidylserine, and 0.7-1.5% sphingomyelin. Fatty acids of phosphatidylglycerol from lamellar bodies were similar to those from microsomes but different from those in mitochondria. Lung homogenate in continuous sucrose density gradient displayed two major activity peaks of phosphatidylglycerol synthesis: the heavier from mitochondria; the lighter from endoplasmic reticulum. Studies on mechanism of phosphatidylglycerol synthesis in vitro revealed (in these two fractions) CDP-diglyceride and sn-glycerol phosphate precursors to phosphatidylglycerol phosphate, that hydrolysed to phosphatidylglycerol. In microsomes disaturated CDP-diglycerides were 1.6-1.9 times more active substrates than in mitochondria, whereas CDP-diglycerides from egg lecithin were almost equally active. In contrast to lung mitochondria no cardiolipin synthesis was detected in microsomes. The highest specific activities for phosphatidate cytidyltransferase, CDP-diglyceride-inositol phosphatidyltransferase, choline phosphotransferase, and phosphatidylethanolamine methyltransferase were all found in microsomes. The present in vitro studies and additional evidence (M. Hallman and L. Gluck, (1975) Fed. Proc. 34, 274) support the hypothesis that de novo synthesis of surfactant lecithin phosphatidylinositol and phosphatidylglycerol takes place in the endoplasmic reticulum of alveolar cells.     

Modification of the biosynthesis and composition of polyglycerophosphatides in outer and inner mitochondrial membranes by cytidine liponucleotides

The biosynthesis of [3H]polyglycerophosphatides ([3H]phosphatidylglycerophosphate and [3H]phosphatidylglycerol) in mitochondrial and submitochondrial (outer and inner) membranes isolated from guinea pig liver was examined. Experimental results have established that the amount of biosynthesized [3H]polyglycerophosphatides and the relative amounts of biosynthesized [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate can be influenced by varying the composition of fatty acids in CDP-diglycerides and by altering the incubation time of the mixture containing CDP-diglycerides (obligatory precursor), sn-[2-3H]glycerol-3-phosphate and mitochondria or submitochondrial membranes. The changes thus obtained in respect to the amount and composition of biosynthesized [3H]polyglycerophosphatides are different in mitochondria and submitochondrial membranes. The highest amount of biosynthesized [3H]polyglycerophosphatides was obtained with CDP-didecanoin and inner mitochondrial membranes. The greatest accumulation of [3H]phosphatidylglycerol with CDP-didecanoin was obtained in mitochondria and outer mitochondrial membranes, while in inner mitochondrial membranes the amounts of [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate accumulated were approximately the same. In general, prolongation of the incubation time decreased the relative amounts of [3H]phosphatidylglycerolphosphate and increased the amount of accumulated [3H]phosphatidylglycerol, but the absolute amounts of these [3H]polyglycerophosphatides were more dependent on fatty acid composition of CDP-diglycerides tested. The following cytidine liponucleotides were tested: CDP-didecanoin, CDP-dipalmitin, CDP-diolein, and CDP-diglycerides containing saturated and unsaturated fatty acids similar to those in egg yolk lecithin. The formation of [3H]cardiolipin from [3H]phosphatidylglycerol in the presence of CDP-didecanoin and Mn2+ was found in both the outer and inner mitochondrial membranes.     

Phosphatidyltransferase activity in Bacillus megaterium.

Phosphatidyl transfer between phosphatidylethanolamine, phosphatidylglycerol or phosphatidylserine as donors and primary hydroxyl acceptors including ethanolamine, glycerol, serine and Triton X-100 has been shown to be catalysed by membrane particles derived from Bacillus megaterium strains ATCC 13632 and ATCC 14581. The rate of cardiolipin synthesis from phosphatidylglycerol in the presence of ethanolamine was an order of magnitude greater than that of phosphatidylethanolamine formation. Cardiolipin synthesis from phosphatidylethanolamine in the presence of glycerol was also observed, and was 1.5-fold greater than the formation of phosphatidylglycerol. Similar heat lability, effects of pH and of Triton X-100 for phosphatidyl transfer and cardiolipin synthesis indicate that both reactions were catalysed by cardiolipin synthase.     

Partial purification of diphosphatidylglycerol synthetase from liver mitochondrial membranes

The enzyme responsible for the conversion of phosphatidylglycerol to diphosphatidylglycerol (cardiolipin) in the presence of cytidine diphosphate diacylglycerol is firmly associated with mitochondrial membranes and is not extracted with hypotonic or hypertonic media or with nonionic detergents. Some solubilization was obtained with bile salt solutions, but the zwitter-ionic detergent. Miranol H2M, was most effective in extracting the enzyme. The Miranol extracts were fractionated by column chromatography on Bio-Gel A-1.5 m. The solubilized enzyme is considerably more active in converting unsaturated than saturated phosphatidyl-glycerols, but shows little preference for the cytidine diphosphate diacylglycerols with different fatty acyl substituents. There is an absolute dependence upon divalent cations with the order of effectiveness: Co2+ much greater than Mn2+ greater than Mg2+. In the presence of optimal levels of Co2+ other divalent cations are inhibitory with the order of inhibition: Cd2+ greater than Zn2+ greater than Ca2+ greater than Ba2+ greater than Cu2+ greater than Hg2+ greater than Ni2+. The solubilized enzyme exhibited no requirement for added phospholipids and several phospholipids inhibited the reaction in the order: diphosphatidylglycerol greater than phosphatidylethanolamine greater than phosphatidylserine greater than phosphatidylinositol.     

Biosynthesis of Cardiolipin in Plant Mitochondria

The properties of cardiolipin synthase were investigated in mitochondria and submitochondrial fractions from etiolated mung bean (Vigna radiata L.) seedlings. Direct evidence is presented that the enzyme utilizes CDP-diacylglycerol in addition to phosphatidylglycerol for the synthesis of cardiolipin. Cardiolipin synthase had an alkaline pH optimum of about 9 and required divalent cations for activity. Maximal activity was obtained in the presence of 16 mM MnCl2. The apparent Km values for CDP-diacylglycerol and phosphatidylglycerol were 0.8 and 50 [mu]M, respectively. Cardiolipin synthase was localized predominantly in the inner membrane of mung bean mitochondria and displayed a substrate species specificity. Highest activities were measured with the dioleoyl species of both CDP-diacylglycerol and phosphatidylglycerol, and somewhat lower activities were measured with mixed species of the two substrates containing a palmitoyl and an oleoyl group. On the other hand, the cardiolipin synthase hardly used the dipalmitoyl species and strongly discriminated against CDP-dipalmitoylglycerol from a mixture with CDP-dioleoylglycerol.     

Calcium-induced insulin release in monolayer culture of the endocrine pancreas. Studies with ionophore A23187.

The role of Ca2+ on insulin release has been studied by the use of ionophore A23187. The ionophore complexes divalent cations and permits Ca2+ entry into cells by acting as a carrier in the plasma membranes. Cultured cells obtained by enzymatic digestion of pancreases from newborn rats were studied on the 3rd day of culture. With Ca2+ in the incubation medium the ionophore induced sustained insulin release even in the absence of glucose. Optimal effects of the ionophore were observed at 3 and 10 mug per ml in the presence of 0.3 to 1.0 mM Ca-2+. Under these conditions the insulin release was greater than that caused by 16.7 mM glucose. A graded response was observed to changes in Ca-2+ concentration from 0.1 to 1.0 mM Ca-2+. Higher Ca-2+ concentrations caused a large amount of insulin to be released promptly, but the release was not sustained. Mg-2+ and Sr-2+ were not found to substitute for Ca-2+. Ba-2+ at 0.3 mM stimulated insulin release even in the absence of ionophore. Cyclic adenosine 3':5'-monophosphate was able to increase ionophore-induced insulin release. The alpha-adrenergic effect of epinephrine to inhibit insulin release was not observed in the presence of Ca-2+ and the ionophore, and a stimulatory effect of epinephrine was seen. This unusual stimulatory effect of epinephrine was blocked by propranolol indicating a beta-adrenergic mechanism for epinephrine. It is concluded that Ca-2+, which plays an essential role in the stimulus-secretion coupling, can alone initiate and cause sustained insulin release.     

Activation of the action potential Na+ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin.

The activation of the action potential Na+ ionophore by veratridine and batrachotoxin is time- and concentration-dependent and completely reversible. Batrachotoxin acts more slowly than veratridine. The concentration dependence of activation at equilibrium suggests reversible interaction of each toxin with a single class of independent sites having dissociation constants at physiologic ion concentrations of 80 plus or minus 13 muM for veratridine and 0.4 plus or minus muM for batrachotoxin. The maximum velocity of Na+ uptake at 50 mM Na+ is 128 plus or minus 12 nmol/min/mg in the presence of batrachotoxin compared to 48 plus or minus 4 nmol/min/mg in the presence of veratridine. Treatment of cells with excess veratridine in addition to batrachotoxin inhibits batrachotoxin-dependent 22-Na+ uptake. The concentration dependence of this inhibition suggests that it reflects competitive displacement of batrachotoxin from its binding site by veratridine. The activation by veratridine and batrachotoxin is inhibited in a competitive manner by divalent cations. The inhibition by divalent cations exhibits significant ion specificity with Mn-2+ greater than Co-2+ greater than Ni-2+ greater than Ca-2+ greater than Mg-2+ greater than Sr-2+. The inhibition constants (KI) for Ca-2+ are 0.84 mM for veratridine-dependent 22-Na+ uptake and 1.2 mM for batrachotoxin-dependent 22-Na+ uptake. The activation by veratridine and batrachotoxin is inhibited in a noncompetitive manner by tetrodotoxin. The apparent KD for tetrodotoxin as 11 plus or minus 1 nM in the presence of 150 mM Na+ and approximately 8.5 nM in 50 mM Na+. Divalent cations do not affect the apparent KD for tetrodotoxin. A hypothesis is presented which suggests that batrachotoxin, veratridine, and divalent cations interact with an activation site associated with the action potential Na+ ionophore, whereas tetrodotoxin interacts with a physically and functionally independent site involved in the transport of monovalent cations by the ionophore.     

Sequence and inactivation of the pss gene of Escherichia coli. Phosphatidylethanolamine may not be essential for cell viability

Phosphatidylethanolamine is the only zwitterionic phospholipid in Escherichia coli and accounts for 70-80% of the total glycerophospholipids of this organism. To investigate the function of phosphatidylethanolamine in E. coli, we constructed an inactivated allele (pss93::kan) of the gene encoding the phosphatidylserine synthase which catalyzes the committed step to the synthesis of phosphatidylethanolamine. Growth of this mutant was dependent on a plasmid-borne copy of the wild type gene. After curing the mutant of the wild type gene, growth stopped when the content of phosphatidylethanolamine reached 30% of the total phospholipid. Divalent metal ions at millimolar concentrations suppressed the growth phenotype of the mutant in the following order of efficiency: Ca2+ greater than Mg2+ greater than Sr2+. Although phosphatidylserine synthase activity was not detectable, phosphatidylethanolamine was still present at 0.007% of the total phospholipid after growth for many generations in rich medium containing 20 mM Mg2+. The remainder of the phospholipid was primarily phosphatidylglycerol and cardiolipin with no other unique phosphate-containing chloroform-soluble material present. The phospholipid to protein ratio and the fatty acid composition were very similar to the parental strain. The broad divalent metal ion auxotrophy brought about by the lack of phosphatidylethanolamine suggests a primarily structural role for this phospholipid in E. coli.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

Activation of three types of membrane currents by various divalent cations in identified molluscan pacemaker neurons

We investigated membrane currents activated by intracellular divalent cations in two types of molluscan pacemaker neurons. A fast and quantitative pressure injection technique was used to apply Ca2+ and other divalent cations. Ca2+ was most effective in activating a nonspecific cation current and two types of K+ currents found in these cells. One type of outward current was quickly activated following injections with increasing effectiveness for divalent cations of ionic radii that were closer to the radius of Ca2+ (Ca2+ greater than Cd2+ greater than Hg2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than Pb2+ greater than Sr2+ greater than Mg2+ greater than Ba2+). The other type of outward current was activated with a delay by Ca2+ greater than Sr2+ greater than Hg2+ greater than Pb2+. Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, and Ni2+ were ineffective in concentrations up to 5 mM. Comparison with properties of Ca2(+)-sensitive proteins related to the binding of divalent cations suggests that a Ca2(+)-binding protein of the calmodulin/troponin C type is involved in Ca2(+)-dependent activation of the fast-activated type of K+ current. Th sequence obtained for the slowly activated type is compatible with the effectiveness of different divalent cations in activating protein kinase C. The nonspecific cation current was activated by Ca2+ greater than Hg2+ greater than Ba2+ greater than Pb2+ greater than Sr2+, a sequence unlike sequences for known Ca2(+)-binding proteins.     

Solubilization, purification, and characterization of cardiolipin synthase from rat liver mitochondria. Demonstration of its phospholipid requirement.

Cardiolipin is a specific and functionally important phospholipid of mitochondria, and its biosynthesis is considered to be crucial for the assembly of this organelle. However, little information is available about the enzyme cardiolipin synthase, largely because it has not yet been isolated. We solubilized cardiolipin synthase from rat liver mitochondrial membranes with Zwittergent 3-14 and purified it by Mono Q anion exchange chromatography, Superose 12 gel filtration, and Mono P chromatofocusing. Cardiolipin synthase is one of the most acidic mitochondrial proteins (isoelectric point, pH 4-5) and appears as a 50-kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme requires CO2+ for activity, has an alkaline pH optimum (pH 8-9), and exhibits Km values of 45 and 1.6 microM for phosphatidylglycerol and CDP-diacylglycerol, respectively. Cardiolipin synthase loses activity during purification, and the activity can be partially reconstituted by the addition of phospholipids. The most effective phospholipid is phosphatidylethanolamine which reactivates in a cooperative manner. Cardiolipin reactivates hyperbolically at low concentrations but inhibits the enzyme at higher concentrations. In addition, cardiolipin shifts the sigmoidal reactivation curve of phosphatidylethanolamine toward lower concentrations. It is suggested that cardiolipin synthase requires interaction with several molecules of phosphatidylethanolamine and at least one molecule of cardiolipin for full enzymatic activity.     

Synthesis of a new phosphatidylserine spin-label and calcium-induced lateral phase separation in phosphatidylserine-phosphatidylcholine membranes.

A new phosphatidylserine spin label with nitroxide stearate attached at the 2 position has been synthesized by the reaction of spin-labeled CDP-diglyceride with L-serine under the catalytic action of phosphatidylserine synthetase. Some structural properties of pure phosphatidylserine (PS) and binary PS-phosphatidylcholine (PC) membranes were studied with the spin label. PS membrane became solidified on lowering solution pH, 50% solidification being attained at pH 3.5. The membrane was also solidified by addition of Ca-2+. The effect of Ba-2+,Sr-2+, and Mg-2+ was smaller than that of Ca-2+. The calcium-induced lateral phase separation in the binary membrane was studied from the side of the calcium-receiving lipid. The results confirmed and extended our previous conclusion drawn with PC spin label. The phase diagram of the binary membrane in the presence of Ca-2+ was determined. Not all PS molecules were aggregated to form the solid patches but some remained dissolved in the fluid PC matrix. The fluid PS fraction was larger for the membranes containing more PC. The membrane with 10% PS still had a significant fraction of solid phase. The rate of calcium-induced aggregation was greatly dependent on the PS content. The aggregation was almost complete within 5 min in the membrane containing 67% PS, while it was still proceeding after several hours in the membrane with 20% PS. The rate-limiting step was suggested to be in the formation of "stable" nuclei consisting of larger aggregates. The possible biological significance of the ionotropic phase separation was discussed whereby a transient density fluctuation was emphasized.     

Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.     

Membrane microenvironment regulation of carnitine palmitoyltranferases I and II

CPT (carnitine palmitoyltransferase) 1 and CPT2 regulate fatty acid oxidation. Recombinant rat CPT2 was isolated from the soluble fractions of bacterial extracts and expressed in Escherichia coli. The acyl-CoA chain-length-specificity of the recombinant CPT2 was identical with that of the purified enzyme from rat liver mitochondrial inner membranes. The Km for carnitine for both the mitochondrial preparation and the recombinant enzyme was identical. In isolated mitochondrial outer membranes, cardiolipin (diphosphatidylglycerol) increased CPT1 activity 4-fold and the Km for carnitine 6-fold. It decreased the Ki for malonyl-CoA inhibition 60-fold, but had no effect on the apparent Km for myristoyl-CoA. Cardiolipin also activated recombinant CPT2 almost 4-fold, whereas phosphatidylglycerol, phosphatidylserine and phosphatidylcholine activated the enzyme 3-, 2- and 2-fold respectively. Most of the recombinant CPT2 was found to have substantial interaction with cardiolipin. A model is proposed whereby cardiolipin may hold the fatty-acid-oxidizing enzymes in the active functional conformation between the mitochondrial inner and outer membranes in conjunction with the translocase and the acyl-CoA synthetase, thus combining all four enzymes into a functional unit.     

Characterization of the cardiolipin synthetase activity of Escherichia coli envelopes

The synthesis of cardiolipin from phosphatidylglycerol catalyzed by isolated envelopes of Escherichia coli occurs without the utilization of endogenous CDP-diglyceride as a substrate. The synthesis of cardiolipin has been assayed distinct from the synthesis of bis-phosphatidic acid. Envelope fractions isolated from cultures exposed to treatments which increase the relative rate of cardiolipin synthesis in vivo were found not to have increased amounts of cardiolipin synthetase activity in vitro. We suggest that the relative increase of cardiolipin synthesis observed during these treatments stems from the lack of an energy requirement for the cardiolipin synthetase reaction and the presence of large amounts of cellular phosphatidylglycerol.     

Calcium-sensitive univalent cation channel formed by lysotriphosphoinositide in bilayer lipid membranes.

A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to univalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb+ greater than Cs+ greater than Na+ greater than K+ greater than Li+. The conductance of the membrane was increased up to a value of about 10-2 ohm-1/cm2 with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn+ greater than Cd2+ greater than Ca2+ greater than Sr2+ greater than Mg2+.