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D R Janero

Publications and source records attributed to D R Janero.

At least 55 records · Page 3Linked to original sources
PubMed1989

Thiobarbituric acid-reactive malondialdehyde formation during superoxide-dependent, iron-catalyzed lipid peroxidation: influence of peroxidation conditions.

A systematic study of the influence of biological lipid peroxidation conditions on lipid hydroperoxide decomposition to thiobarbituric acid-reactive malondialdehyde is presented. A superoxide-dependent, iron-catalyzed peroxidation system was employed with xanthine oxidase plus hypoxanthine plus ferric iron-adenosine diphosphate complex as free radical generator. Purified cardiac membrane phospholipid (as liposomes) was the peroxidative target, and 15-hydroperoxy-eicosatetraenoic acid was used as a standard lipid hydroperoxide. Exposure of myocardial phospholipid to free radical generator at physiological pH (7.4) and temperature (37 degrees C) was found to support not only phospholipid peroxidation, but also rapid lipid hydroperoxide breakdown and consequent malondialdehyde formation during peroxidation. Under lipid peroxidation conditions, oxidative injury to the phospholipid polyunsaturated fatty acids required superoxide radical and ferric iron-adenosine diphosphate complex, whereas 37 degrees C temperature and trace iron were sufficient for lipid hydroperoxide decomposition to malondialdehyde. Harsh thiobarbituric acid-test conditions following peroxidation were not mandatory for either lipid hydroperoxide breakdown or thiobarbituric acid-reactive malondialdehyde formation. However, hydroperoxide decomposition that had begun in the peroxidation reaction could be completed during a subsequent thiobarbituric acid test in which no lipid autoxidation took place. Iron was more critical than heat in promoting the observed hydroperoxide decomposition to malondialdehyde during the lipid peroxidation reaction at 37 degrees C and pH 7.4. These data demonstrate that the radical generator, at physiological pH and temperature, serves a dual role as both initiator of membrane phospholipid peroxidation and promotor of lipid peroxide breakdown and thiobarbituric acid-reactive malondialdehyde formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Cardiac membrane vitamin E and malondialdehyde levels in heart muscle of normotensive and spontaneously-hypertensive rats.

The vitamin E (alpha-tocopherol) and free and bound malondialdehyde (MDA) in ventricular heart muscle and myocardial membrane from Wistar-Kyoto (W/K) normotensive and spontaneously hypertensive (SH) rats have been measured directly by high performance liquid chromatography (HPLC). Thiobarbituric acid-reactive substance (TBA-RS) in the myocardium and heart-muscle membrane of the two strains was also quantified by a colorimetric TBA test. It was found that SH-rat myocardium and myocardial membrane contained more than 3-fold less alpha-tocopherol than did heart muscle and cardiac membrane of the normotensive rat. Coincident with this relative vitamin E deficiency were several-fold greater amounts of MDA and TBA-RS in SH-rat myocardium and myocardial membrane. Most (87%) of the MDA in SH-rat heart muscle, but only 40% in W/K-rat heart muscle, was free (i.e., unbound). These results offer direct evidence that SH-rat myocardium is vitamin E-deficient and highly peroxidative, relative to cardiac muscle of the normotensive W/K parent strain. The lower vitamin E content of SH-rat myocardium is particularly striking, because SH-rat myocardial membrane was found to contain approximately 35% more phospholipid than myocardial membrane in the W/K rat. Although the amounts of myocardial TBA-RS are greater in the SH strain, they do not reflect the actual MDA profiles of the heart muscles or the heart membranes and cannot be used as a quantitative index of cardiac oxidative-injury status due to non-MDA TBA-RS in both strains.

Animals↗
PubMed1989

Oxidative injury to myocardial membrane: direct modulation by endogenous alpha-tocopherol.

Peroxidation of myocardial-membrane phospholipid is considered an important pathogenic component of heart muscle damage in ischemia and reperfusion. The extent to which membrane alpha-tocopherol (vitamin E) in the heart can modulate such damage and protect against it is a matter of controversy. The relative alpha-tocopherol deficit of spontaneously-hypertensive (SH) rat myocardium as compared to the myocardium of the Wistar-Kyoto (W/K) normotensive parent strain prompted use of these animals to identify and characterize any protective antiperoxidant role of endogenous, myocardial-membrane alpha-tocopherol. With exposure to a superoxide- and iron-containing initiator of peroxidation, the membrane complements from the ventricular myocardia of the SH rat and the W/K parent strain were found to have very different peroxidative-injury profiles. SH-rat myocardial membrane demonstrated a marked sensitivity to peroxidation as reflected in the acute onset and rapid progression of phospholipid damage. The greater susceptibility of SH-rat myocardial membrane to free-radical attack could not be explained by inter-strain compositional differences in membrane polyunsaturated fatty acids or fatty aldehydes. Rather, the basis for the enhanced peroxidation was identified as the 3-fold lower alpha-tocopherol content of SH-rat myocardial membrane with respect to the heart-muscle membrane from the normotensive animal. The relative alpha-tocopherol deficit not only increased the susceptibility of SH-rat cardiac membrane to damage under pro-oxidant conditions, but also reduced the efficacy of exogenously supplied antioxidant intervention. These findings demonstrate that membrane alpha-tocopherol tone is a critical protectant of myocardial phospholipid against oxidative injury and acts as a determinant of the course of heart-membrane peroxidative damage.

Animals↗
PubMed1989

Propranolol as xanthine oxidase inhibitor: implications for antioxidant activity.

Propranolol is the beta-blocker most widely used in the management of cardiovascular disorders. It has been proposed that propranolol may act as a "chain-breaking" antioxidant. We have directly examined the ability of propranolol to inhibit superoxide-dependent, iron-promoted cardiac membrane phospholipid peroxidation, with xanthine oxidase (XOD) as a physiologically-recognized, enzymatic superoxide generator. Our results demonstrate that propranolol not only protects cardiac-membrane lipid from peroxidative damage, but also acts as a simple, reversible XOD inhibitor, noncompetitive with xanthine substrate. Propranolol, at effective antiperoxidant and XOD-inhibitory concentrations, cannot scavenge superoxide radical. The antiperoxidative profile of propranolol resembles that of the known XOD inhibitor allopurinol, although allopurinol, a tight-binding substrate-analog competitive with xanthine, inhibits XOD in a manner mechanistically very different from that of propranolol. Furthermore, the antiperoxidative profiles of both propranolol and allopurinol do not resemble those of chain-breaking antioxidants such as alpha-tocopherol. These data, along with the tendency of propranolol to concentrate in myocardial membranes and cytosol, suggest that the observed antioxidant action of propranolol, as a consequence of XOD inhibition, could play a pharmacologic role in propranolol's cardioprotective effects.

Allopurinol↗
PubMed1989

Prevention of oxidative injury to cardiac phospholipid by membrane-active "stabilizing agents".

Three lipophilic, membrane-active "stabilizing agents," cepharanthine, chlorpromazine, and trifluoperazine, were found to protect myocardial membrane phospholipid from peroxidative injury. The compounds prevented, in a concentration-dependent manner, the cardiac phospholipid peroxidation which resulted from lipid exposure to superoxide-dependent, iron-promoted oxygen-radical chemistry of the type thought to be a causative factor in ischemic-reperfusion tissue damage. Chlorpromazine's antiperoxidant IC50 (i.e., concentration at which peroxidation was inhibited by 50%) was 180 microM; the antiperoxidant potencies of cepharanthine (IC50 = 90 microM) and trifluoperazine (IC 50 = 100 microM) were some two-fold greater. These agents, at effective antiperoxidant concentrations, did not inhibit the enzymatic superoxide source, xanthine oxidase, scavenge superoxide radical, or act like a chain-breaking antioxidant. The data raise a possibility that the these three membrane-active compounds, as lipophilic anesthetics, may exert antiperoxidant effects by inducing structural changes in the lipid-rich (membrane or liposome) target of free radical attack.

Alkaloids↗
PubMed1988

Protection of cardiac membrane phospholipid against oxidative injury by calcium antagonists.

Calcium antagonists representative of the four major chemical classes were assessed for their abilities to prevent peroxidation of rat heart membrane lipids through xanthine oxidase-dependent, superoxide-driven, iron-promoted oxygen radical chemistry. The dihydropyridines nifedipine and nitrendipine did not affect peroxidation, even at a concentration (500 microM) approaching their solubility limit. The benzothiazepine diltiazem did protect the cardiac lipids against oxidative injury, but at high micromolar concentrations: 50% inhibition of peroxidation (antiperoxidant IC50) required 510 microM diltiazem. The phenylalkylamines verapamil and gallopamil (D-600) were likewise weak antiperoxidants (approximately 35% inhibition of peroxidation at 500 microM). In contrast, two other alkylamines, bepridil and prenylamine, were very effective membrane lipid protectants with respective antiperoxidant IC50 values of 55 and 75 microM. The diphenylpiperazines flunarizine (IC50 = 190 microM) and cinnarizine (IC50 = 180 microM) displayed moderate antiperoxidant activity. No Ca2+ antagonist inhibited xanthine oxidase under conditions whereby 10 microM allopurinol inhibited enzyme activity by 50%. The effects of the Ca2+ antagonist-antiperoxidants on the kinetics of cardiac membrane lipid peroxidation indicate that they inhibit peroxidation by intercepting oxy- and/or lipid free radical intermediates. These data raise the possibility that antiperoxidant action may contribute to the spectrum of pharmacologic and therapeutic activities of certain Ca2+ antagonists.

Animals↗
PubMed1988

Protection of rat myocardial phospholipid against peroxidative injury through superoxide-(xanthine oxidase)-dependent, iron-promoted Fenton chemistry by the male contraceptive gossypol.

Metal-promoted oxygen free-radical chemistry is a cause of tissue damage in many disease states, such as myocardial ischemia. The effect of gossypol, a polyphenolic plant pigment and male contraceptive, on the peroxidation of myocardial membrane phospholipid was studied and quantitatively characterized. As a result of exposure to xanthine oxidase (superoxide)-dependent, iron-promoted Fenton chemistry, cardiac phospholipid was readily peroxidized with defined kinetics. The peroxidation could be blocked by substances which interdict at specific points in the Fenton chemistry: superoxide dismutase, alpha-tocopherol, the iron chelator desferrioxamine, and the xanthine oxidase substrate-analogs allopurinol and oxypurinol. The oxidative-injury system displayed a characteristic antiperoxidant response to each type of inhibitor. Gossypol, at low micromolar concentrations, profoundly altered the rate and extent of myocardial phospholipid peroxidation. Gossypol was ineffective as a xanthine oxidase inhibitor and as a superoxide scavenger at concentrations that abolished myocardial lipid peroxidation. Since metal chelation was an effective means of preventing lipid peroxidation in this system only when the iron therein was completely chelated, the low anti-peroxidant IC50 for gossypol, 1.1. microM, relative to the concentration of iron (100 microM) did not support a functionally significant antiperoxidant role for gossypol as an iron chelator. Rather, it appears that, at low micromolar gossypol concentrations which approximate the peak plasma concentrations in humans, the antiperoxidant effects of gossypol against superoxide-mediated, iron-promoted lipid damage rest with the ability of gossypol to intercept lipid radical intermediates as a "chain-breaking" aromatic phenol.

Allopurinol↗
PubMed1988

Specific binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) to the intact canine platelet.

Binding of 3H-labeled 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor; PAF) to the intact, washed canine platelet has been defined and characterized as being specific and receptor-mediated. Under the conditions described, specific binding to 2 X 10(7) canine platelets reached saturation within 10 min at a [3H]PAF concentration of approximately 0.4 nM. Non-specific binding was accountable for, at most, some 30% of the total PAF bound at equilibrium. Above approximately 0.4 nM [3H]PAF, total binding and non-specific binding increased in parallel. Since no involvement of PAF ligand in dog platelet intermediary metabolism during the binding incubation could be demonstrated, non-specific PAF binding may reflect a partitioning of the molecule into a cellular compartment (perhaps the platelet membranes). Equilibrium analysis revealed that the canine platelet has one class of specific binding sites with a Kd of 0.63 +/- 0.02 nM PAF, a Bmax of 222 +/- 10 fmol/10(7) platelets, and, at most, 1.33 +/- 0.06 X 10(3) binding sites/platelet. [3H]PAF specific binding to the canine platelet is ligand-selective and stereo-selective, as demonstrated by the relative abilities of non-labeled PAF and various PAF analogs/metabolites to inhibit [3H]PAF specific binding in a concentration-dependent manner. The extents to which PAF and PAF analogs were able to displace specifically-bound [3H]PAF from the canine platelet correlated well with their physiological (i. e., pro-aggregatory) effects. These data offer the first quantitative description of canine platelet high-affinity PAF binding sites/receptors and link receptor-mediated PAF binding to canine platelet physiology.

Animals↗
PubMed1988

Amphiphile-induced heart muscle-cell (myocyte) injury: effects of intracellular fatty acid overload.

Lipid amphiphile toxicity may be an important contributor to myocardial injury, especially during ischemia/reperfusion. In order to investigate directly the potential biochemical and metabolic effects of amphiphile overload on the functioning heart muscle cell (myocyte), a novel model of nonesterified fatty acid (NEFA)-induced myocyte damage has been defined. The model uses intact, beating neonatal rat myocytes in primary monolayer culture as a study object and 5-(tetradecyloxy)-2-furoic acid (TOFA) as a nonmetabolizable fatty acid. Myocytes incubated with TOFA accumulated it as NEFA, and the consequent NEFA amphiphile overload elicited a variety of cellular defects (including decreased beating rate, depletion of high-energy stores and glycogen pools, and breakdown of myocyte membrane phospholipid) and culminated in cell death. The amphiphile-induced cellular pathology could be reversed by removing TOFA from the culture medium, which resulted in intracellular TOFA "wash-out." Although the development and severity of amphiphile-induced myocyte injury could be correlated with both the intracellular TOFA/NEFA content (i.e., the level of TOFA to which the cells were exposed) and the duration of this exposure, removal of amphiphile overload did not inevitably lead to myocyte recovery. TOFA had adverse effects on myocyte mitochondrial function in situ (decoupling of oxidative phosphorylation, impairing respiratory control) and on myocyte oxidative catabolism (transiently increasing fatty acid beta oxidation, citric acid cycle flux, and glucose oxidation). The amphiphile-induced bioenergetic abnormalities appeared to constitute a state of "metabolic anoxia" underlying the progression of myocyte injury to cell death. This anoxic state could be ameliorated to some extent, but not prevented, by carbohydrate catabolism.

Adenosine Triphosphate↗
PubMed1988

Analysis of cardiac membrane phospholipid peroxidation kinetics as malondialdehyde: nonspecificity of thiobarbituric acid-reactivity.

When exposed to xanthine oxidase (superoxide)-dependent, iron-promoted Fenton chemistry, purified cardiac membranes evidenced, by the thiobarbituric acid (TBA) test, a virtually instantaneous peroxidative response with a maximal linear rate of 5.8 nmol malondialdehyde (MDA)-equivalents/mEquivalents lipid ester reacted/min. Yet when the lipids purified from these same membranes and reconstituted into liposomes were peroxidized under identical reaction conditions, the TBA test indicated that a pronounced (approximately 20-min) lag period preceded a maximal peroxidation rate of only 2.1 nmol MDA-equivalents/mEquivalents lipid ester reacted/min. After 120 min of peroxidation, the cardiac membranes yielded some 300 nmol TBA-reactive MDA-equivalents/mEquivalent ester, whereas the isolated membrane lipids evidenced approximately 40% less TBA-reactivity. To verify that these quantitative and kinetic differences in membrane (phospho)-lipid peroxidation occurred with removal of the lipids from their membrane milieu, the MDA produced during both cardiac membrane peroxidation and the peroxidation of the lipids derived therefrom was isolated as its free anion by ion-pair high-pressure liquid chromatography. As quantified spectrophotometrically, true MDA production during myocardial membrane peroxidation was identical in kinetics and in amount to the production of TBA-reactive substance from the peroxidized isolated membrane lipids. These results demonstrate that significant non-MDA, TBA-reactive species are generated during the peroxidation of cardiac membranes, especially before the maximal rates of bona fide MDA production. As a direct consequence, artifactual levels and kinetics of membrane lipid peroxidation do result.

Animals↗
PubMed1988

Radioligand competitive binding methodology for the evaluation of platelet-activating factor (PAF) and PAF-receptor antagonism using intact canine platelets.

High-affinity, stereoselective, and ligand-selective specific binding of 3H-labeled platelet-activating factor (PAF) to its receptor on the dog platelet is reproducible over wide mass and concentration ranges of [3H]PAF. The [3H]PAF specific binding can be competitively inhibited by low picogram amounts of nonlabeled PAF. These observations have led to the formulation of radioligand competitive binding methodology for the detection and estimation of PAF in a biological lipid sample and the quantitative evaluation of PAF-receptor antagonism. The methodology is predicated upon correlation between the ability of a PAF analog/biological lipid sample/(synthetic) substance to inhibit [3H]PAF specific binding to the washed canine platelet and the known inhibition of [3H]PAF specific binding by standard, nonradioactive PAF. Application of this methodology to lipid extracts of human saliva has uncovered the finding that subjects with upper respiratory infection and chronic allergies have high saliva PAF contents. Pharmacologically active antiallergy agents known to inhibit PAF-induced pathology in animal models of disease were demonstrated, with the methology advanced, to act as PAF-receptor antagonists, and their potencies were quantified. These investigations indicate that the system proposed, in its ease, economy, sensitivity, specificity and capacity, has practical value for detecting and estimating PAF in biological lipid extracts and for evaluating PAF-receptor antagonism.

Animals↗
PubMed1988

Myocardial vitamin E (alpha-tocopherol) contents of spontaneously hypertensive and Wistar-Kyoto normotensive rats.

As a membrane-associated antioxidant, tissue vitamin E (alpha-tocopherol) may help protect heart muscle from irreversible necrosis during myocardial ischemia. The vitamin E content of heart-muscle membrane from the spontaneously hypertensive (SH) rat, an animal model of cardiovascular disease, has been determined and compared to that of the myocardial membranes of the Wistar-Kyoto normotensive (W/K) parent strain. It was found that SH-rat myocardial membranes contained some 3-fold less vitamin E than did cardiac membranes in the normotensive strain. These data demonstrate that SH-rat myocardium is deficient in membrane alpha-tocopherol and, hence, in its lipid-soluble antioxidant capacity with respect to heart muscle of the normotensive animal.

Animals↗
PubMed1984

Synthesis, processing, and secretion of hepatic very low density lipoprotein.

Very low density lipoprotein (VLDL) is the major vehicle in the plasma which carries triacylglycerol synthesized in the liver to peripheral tissues for utilization. Estrogen-induced chick parenchymal liver cells (hepatocytes) synthesize and secrete large amounts of VLDL. These cells, in a primary monolayer culture system developed in this laboratory, have been employed to study the operative and regulatory aspects of VLDL synthesis, assembly, and secretion. Some 10 min are required for the translation of the principle VLDL protein constituent, apolipoprotein B, and 30-35 min are required for the two newly translated chick VLDL apolipoproteins, apolipoprotein B and apolipoprotein II, to be secreted. Apolipoprotein B is synthesized on membrane-bound polysomes as a contiguous polypeptide chain of 350K molecular weight (MW) and is not assembled posttranslationally from smaller-peptide precursors. Translocation of puromycin-discharged apolipoprotein B nascent chains into the endoplasmic reticulum lumen and their subsequent secretion are independent of both ongoing protein synthesis and the attachment of the nascent peptides to ribosomes. Apolipoprotein B nascent chains discharged by puromycin assemble with glycerolipid (mainly triacylglycerol) and are secreted as immunoprecipitable VLDL. Core oligosaccharides are added to the apolipoprotein B nascent chain co-translationally in at least two stages, at molecular weights of approximately 120K and approximately 280K . Inhibition of N-linked glycosylation of apolipoprotein B with tunicamycin affects neither the assembly of glycerolipids into VLDL nor the secretion of the VLDL particle, indicating that aglyco -apolipoprotein B can serve as a functional component for VLDL assembly and secretion. Active synthesis of the VLDL apolipoproteins is required, however, for glycerolipid assembly into VLDL and secretion from the hepatocyte. The differential kinetics with which newly synthesized apolipoproteins and glycerolipids are secreted as VLDL and the timing of the effects of protein-synthesis inhibitors on their secretion indicate that VLDL constituents are assembled sequentially in the intact liver cell. The bulk of the VLDL triacylglycerol and some VLDL phosphoglyceride is introduced early in the secretory pathway proximal, yet subsequent to apopeptide synthesis, while a significant fraction of VLDL phosphoglyceride associates with the resulting triacylglycerol-rich lipid-protein complexes just prior to their secretion as mature VLDL. Within the context of current models for VLDL structure, the late assembly of phosphoglyceride into VLDL is taken to represent a surface maturation of the nascent VLDL particle.

Animals↗
PubMed1983

Sequential assembly of very low density lipoprotein apolipoproteins, triacylglycerol, and phosphoglycerides by the intact liver cell.

Primary cultures of estrogen-induced chick parenchymal liver cells have been used to study the assembly of the apolipoprotein and glycerolipid constituents of hepatic very low density lipoprotein (VLDL). Liver cell monolayers in serum-free medium were pulse-labeled for 2.5 min with either [3H]leucine or [3H]glycerol and were then chased for 4 h. The kinetics of secretion of VLDL 3H-apolipoproteins (labeled from [3H]leucine) and [3H]triacylglycerol and 3H-phosphoglycerides (labeled from [3H]glycerol) was determined through the chase. Maximal rates of VLDL [3H]triacylglycerol secretion were reached by 20-25 min into the chase, preceding the initial appearance of 3H-apolipoproteins in the medium at 30-35 min of chase time. The secretion of VLDL 3H-phosphoglycerides was maximal much earlier, between 5 and 15 min into the chase, plateaued for 15 min, and then began again at 30 min, thereby displaying a distinctive biphasic pattern. In the presence of cycloheximide or puromycin, such that apopeptide synthesis was halted from the start of the chase, the secretion of VLDL 3H-glycerolipid was depressed after 30 min of chase, without having influenced the temporal pattern of the newly synthesized VLDL 3H-apolipoprotein and 3H-glycerolipid secretion. As compared to the cycloheximide-treated cells in which apolipoprotein B nascent chains were arrested intracellularly, the puromycin-treated cells secreted discharged apolipoprotein B nascent chains as VLDL constituents assembled with triacylglycerol. The differential kinetics of 3H-apolipoprotein, [3H]triacylglycerol, and 3H-phosphoglyceride secretion as VLDL and the timing of the effects of protein synthesis inhibitors on their secretion indicate that VLDL constituents are assembled sequentially in the intact liver cell. Some VLDL phosphoglyceride and the VLDL triacylglycerol are assembled with apolipoprotein early in the secretory pathway, forming a triacylglycerol-rich lipid-protein particle into which further phosphoglyceride is introduced just prior to its secretion as mature VLDL.

Animals↗
PubMed1982

Association and assembly of triglyceride and phospholipid with glycosylated and unglycosylated apoproteins of very low density lipoprotein in the intact liver cell.

Using estrogen-induced chick liver cells which synthesize and secrete large amounts of very low density lipoprotein (VLDL), we have previously shown (Siuta-Mangano, P., Howard, S., Lennarz, W. J., and Lane, M. D. (1982) J. Biol. Chem. 257, 4292-4300) that the major protein constituent of VLDL, the 350,000 molecular weight apoprotein (apoprotein B), is synthesized as a single polypeptide to which core oligosaccharides are added co-translationally. This system has now been employed to study the assembly of the apoproteins of VLDL with their glycerolipid (triglyceride and phospholipid) components and the secretion of the VLDL glycerolipids. In the presence of cycloheximide such that VLDL apoprotein synthesis is inhibited 98%, the secretion of lipids labeled from a [3H]palmitate pulse by hepatocyte monolayers was halted only after completed apoprotein chains had cleared the cell. Under conditions whereby tunicamycin inhibited [3H]glucosamine incorporation into apoprotein B by 98% and [3H]leucine incorporation into the VLDL apoproteins minimally, the unglycosylated form of apoprotein B assembled with the usual complement of triglyceride and phospholipid as did glycosylated apoprotein B to form a VLDL which was readily secreted by the hepatocyte. Taken together, these findings demonstrate that whereas apoprotein synthesis is necessary for the secretion of the major lipid components of VLDL, glycosylation of apoprotein B is not required for either the assembly of VLDL glycerolipids or for the secretion of the VLDL particle.

Animals↗
PubMed1982

Thylakoid membrane biogenesis in Chlamydomonas reinhardtii 137+. II. Cell-cycle variations in the synthesis and assembly of pigment.

Synthesis of the chlorophyll and the major carotenoid pigments and their assembly into thylakoid membrane have been studied throughout the 12-h light/12-h dark vegetative cell cycle of synchronous Chlamydomonas reinhardtii 137+ (wild-type). Pulse exposure of cells to radioactive acetate under conditions in which labeling accurately reflects lipogenesis, followed by cellular fractionation to purify thylakoid membrane, allowed direct analysis of the pigment synthesis and assembly attendant to thylakoid biogenesis. All pigments are synthesized and assembled into thylakoids continuously, but differentially, with respect to cell-cycle time. Highest synthesis and assembly rates are confined to the photoperiod (mid-to-late G1) and support chlorophyll and carotenoid accretion before M-phase. The lower levels at which these processes take place during the dark period (S, M, and early-to-mid G1) have been ascribed to pigment turnover. Within this general periodic pattern, pigment synthesis and assembly occur in a "multi-step" manner, i.e., by a temporally-ordered, stepwise integration of the various pigments into the thylakoid membrane matrix. The cell-cycle kinetics of pigment assembly at the subcellular level mirror the kinetics of pigment synthesis at the cellular level, indicating that pigment synthesis not only provides chlorophyll and carotenoid for thylakoid biogenesis but may also serve as a critical rate-determinant to pigment assembly.

Carotenoids↗
PubMed1982

Membrane lipid metabolism in Chlamydomonas reinhardtii 137+ and Y-1: I. Biochemical localization and characterization of acyltransferase activities.

The acyltransferases involved in the synthesis of the chloroplast membrane glycerolipids were analysed biochemically in dark-grown and greening Chlamydomonas reinhardtii y-1 as well as in the synchronous wild-type algae (strain 137+) and wild-type membranes. Using oleoyl-CoA as a substrate, three acyltransferase enzyme activities were detected. Glycerol-3-phosphate (glycerol-3-P) acyltransferase exhibited a pH optimum of 8.0 and was inhibited by addition of N-ethylmaleimide (MalNEt). Lysophosphatidate (PtdLys) acyltransferase exhibited a pH optimum of 7.0 and was not affected by the addition of MalNEt. From preliminary analyses, the activity at pH 5.5 appeared to be associated with dihydroxyacetone phosphate acyltransferase activity. Both glycerol-3-P and PtdLys acyltransferases were analysed further and found to be present in dark-grown and light-induced y-1 cells as well as in synchronous 137+ cells and their photosynthetic membranes. Both enzyme activities were enriched at least 10-fold in the photosynthetic membranes of 137+ chloroplasts relative to the activities present in the whole cells. This enrichment is indicative of their intrinsic localization in the thylakoids, suggesting that the photosynthetic membranes exhibit a greater degree of autonomy with respect to the synthesis of their membrane lipids than previously reported. A role for glycerol-3-P and PtdLys acyltransferases in the synthesis of the chloroplast membrane lipids is suggested further by the increases in both enzyme activities coincident with and preceding thylakoid biogenesis following light induction of dark-grown y-1 cells. Increased acyltransferase activity preceded the increase in the chlorophyll content of greening y-1 cells, which is a generally accepted marker for thylakoid synthesis. The increase in the PtdLys acyltransferase activity upon light-induction of the y-1 cells was both more immediate and more dramatic than the increase in glycerol-3-P acyltransferase activity. PtdLys acyltransferase activity was negligible in dark-grown cells and the dramatic increase upon light induction may be important in the subsequent initiation of chloroplast membrane lipid synthesis. On the basis of the localization of acyltransferase enzyme activities to the photosynthetic membranes of 137+ cells and the increase in acyltransferase activity both preceding and occurring in concert with thylakoid synthesis, we propose a direct role for the photosynthetic membranes in the synthesis of their membrane lipid components.

Acyl Coenzyme A↗