Biomedical subjects

P Proulx

Publications and source records attributed to P Proulx.

At least 37 records · Page 2Linked to original sources

PubMed1985

Studies on the uptake of fatty acids by brush border membranes of the rabbit intestine.

Initial studies revealed that the uptake of palmitic acid and oleic acid into brush border membranes was similar when these were isolated from either whole small intestine, jejunum, or ileum. The uptake of these fatty acids was somewhat lower with membranes obtained from duodenum. Subsequent studies, all with membranes obtained from whole intestine, indicated an increase in binding with chain length of fatty acid of up to 16 carbons. Unsaturation decreased this uptake somewhat. Taurocholate and 1-palmitoyl lysolecithin had a moderate stimulatory effect on the binding of oleic acid and palmitic acid at concentrations of 10 and 0.5 mM, respectively, and inhibited at higher concentrations. Addition of 1.4 mM egg lecithin to the fatty acid - bile salt micelles, such that the lecithin - bile salt ratio was 0.2, decreased the uptake of fatty acids generally, but did not significantly affect the pattern of binding by membrane fractions isolated from different segments nor did it change the pattern of labelling when fatty acid chain length and unsaturation were varied. At lower concentrations, egg lecithin had little effect on the uptake of oleic acid, whereas dipalmitoyl phosphatidylcholine stimulated binding of both palmitic acid and oleic acid over the entire range of concentrations tested. Preincubation of the membranes with this saturated phospholipid stimulated the uptake of oleic acid, and addition of this choline lipid to the oleic acid - bile salt containing micelles did not substantially enhance fatty acid uptake in lipid-treated membranes. The binding of fatty acid was very rapid either in the presence or the absence of Ca2+, such that even in zero-time controls essentially equilibrium bindings were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Interaction of lipid vesicles with an heptoseless strain of Escherichia coli.

The conditions for uptake of lipid vesicles by the deep rough mutant of Escherichia coli, strain D21F2, and the parent strain, K12 were studied. A variety of lipids including phosphatidylcholine, phosphatidylethanolamine, diglyceride, cholesterol and palmitic acid were taken up much more readily by the deep rough mutant than the K12 strain. The uptake of lipid in the mutant strain was enhanced by Ca2+ at an optimal concentration of 2 mM, by alkaline pH and by growth of the cells up to the late exponential phase. With K12 cells, cholesterol and phosphatidylethanolamine were taken up from equimolar mixtures at similar rates thus suggesting the involvement of a fusion process. With D21F2 cells, the endogenous lipids of which had been labelled by growth in [3H] acetate, the uptake of exogenous [32P] phosphatidylethanolamine and [32P] lysophosphatidylethanolamine was not accompanied by a loss of endogenous lipid to the incubation medium. This precluded the involvement of an exchange mechanism. The absorbed exogenous lipid and the endogenous lipids of D21F2 cells displayed the same susceptibility to hydrolysis by added phospholipase C. This indicated that the exogenous lipid is inserted into lipid core of the membrane rather than adsorbed at the cell surface.

Calcium↗

PubMed1984

Studies on the mechanism of cholesterol uptake and on the effects of bile salts on this uptake by brush-border membranes isolated from rabbit small intestine.

The effect of bile salts and other surfactants on the rate of incorporation of cholesterol into isolated brush-border membranes was tested. At constant cholesterol concentration, a stimulatory effect of taurocholate was noticed which increased as the bile salt concentration was raised to 20 mM. Taurodeoxycholate was as effective as taurocholate at concentrations of up to 5 mM and inhibited at higher concentrations. Glycocholate was only moderately stimulatory whereas cholate was nearly as effective as taurocholate at concentrations above 5 mM. Other surfactants such as sodium lauryl sulfate and Triton X-100 were very inhibitory at all concentrations tried whereas cetyltrimethyl ammonium chloride was stimulatory only at a very low range of concentrations. These micellizing agents all caused some disruption of the membranes and the greater effectiveness of taurocholate in stimulating sterol uptake was partly relatable to the weaker membrane solubilizing action of this bile salt. Preincubation of membranes with 20 mM taurocholate followed by washing and exposure to cholesterol-containing lipid suspensions lacking bile salt, did not enhance the incorporation of the sterol. In the absence of bile salt the incorporation of cholesterol was unaffected by stirring of the incubation mixtures. Increasing the cholesterol concentration in the mixed micelle while keeping the concentration of bile salt constant caused an increase in rate of sterol incorporation. This increased rate was seen whether the cholesterol suspension was turbid, i.e., contained non-micellized cholesterol, or whether it was optically-clear and contained only monomers and micelles. When the concentration of taurocholate and cholesterol were increased simultaneously such that the concentration ratio of these two components was kept constant, there resulted a corresponding increase in rate of cholesterol uptake. The initial rates of cholesterol incorporation from suspensions containing micellar and monomer forms of cholesterol were much larger than from solutions containing only monomers of the same concentration. The rates of incorporation of cholesterol and phosphatidylethanolamine from mixed micelles containing these lipids in equimolar concentrations were very different. The results as a whole suggest at least for those experimental conditions specified in this study, that uptake of cholesterol by isolated brush-border membranes involves both the monomer and micellar phases of the bulk lipid and that the interaction of the micelles with membrane does not likely involve a fusion process.

Animals↗

PubMed1984

The effect of phosphoglycerides on the incorporation of cholesterol into isolated brush-border membranes from rabbit small intestine.

The incorporation of cholesterol from bile salt micelles into brush-border membranes was found to be similar whether these originated from jejunum, ileum or whole small intestine. This incorporation, however, was appreciably lower in membranes obtained from duodenum. Studies pursued with membranes from whole small intestine revealed that dipalmitoyl or dioleoylphosphatidylcholine, when micellized together with the sterol and taurocholate markedly inhibited the incorporation. The didecanoyl and dilauroyl analogues of this lipid class were without significant effect. Preincubation of the membranes for 30 min at 37 degrees C with or without dipalmitoylphosphatidylcholine had no effect on cholesterol incorporation. Again in this case, suppression of sterol uptake could be seen only when phosphatidylcholine was admixed with the sterol. In contrast, dipalmitoyl and dilauroylphosphatidylethanolamines were found to be stimulatory rather than inhibitory. Addition of palmitic acid to the sterol-bile salt micelles had no effect on the uptake of cholesterol; however, this fatty acid could completely reverse the inhibition of cholesterol uptake by dipalmitoylphosphatidylcholine. The present study supports the conclusion that cholesterol incorporation into isolated brush-border membranes is governed largely by factors which affect the partitioning of the sterol out of the bile salt micelle.

Animals↗

PubMed1982

Interaction of lipids with intestinal brush border membrane preparations.

Conditions for uptake of lipids by rabbit intestinal brush border membrane preparations were investigated. A variety of lipids were found to be incorporated, including choline and ethanolamine phosphatides as well as cholesterol, diglyceride, and fatty acid. The incorporation of those lipids tested was enhanced by Ca2+ and other divalent cations but not by monovalent cations. The optimal Ca2+ concentration was approximately 10 mM. The uptake varied with lipid and membrane protein concentration and proceeded at rates which were too rapid to measure under several assay conditions tried. Incorporations were decreased substantially outside the pH range of 6.5-8.0. The effect of one lipid, phosphatidylcholine, on the structural appearance of the membrane fraction was examined by electron microscopy. No free or surface-bound lipid structures could be detected and the membrane fractions appeared to be unchanged after uptake.

Animals↗

PubMed1982

Studies on the uptake of exogenous phosphoglycerides by Escherichia coli B cells.

The uptake of exogenous lipids by Escherichia coli B was studied under various conditions. Lysophosphoglycerides were absorbed more readily than diacyl analogues when sonicated dispersions of a single lipid were used. When these same lipids were admixed with coliform lipids and dispersed as vesicles, the uptake of lysophosphoglyceride diminished and was approximately equal to that of diacyl analogues. Neutral lipids such as diglyceride, fatty acid, and cholesterol were also absorbed when admixed and dispersed as vesicles with coliform lipids. The uptake of lysophosphoglyceride was stimulated slightly by all divalent cations tested except Mg2+; monovalent cations were ineffective. Uptake was accompanied by conversion of lysophosphoglyceride to diacyl analogues. In the presence of Ca2+, lysophosphatidylethanolamine also formed a more polar lipid product yet unidentified. The uptake of lipid did not cause release of 3H label into the medium from cells that had been grown in [3H]acetate-containing medium. Also, the [3H]phosphoglyceride content of such labelled cells remained constant. Thus the uptake process did not involve exchange of membrane lipid with the medium or an enhanced hydrolysis of endogenous lipids, but it represented a net gain of lipid by the cell. The uptake did not seem to involve a stable adsorption of lipid at the surface of the cell as could be judged from electron microscopic examination of the cells after incubation; the cell surfaces were devoid of adsorbed vesicle or liposomal types of structures and did not display evidence of expansion. [3H]Cholesterol-[32P]phospholipid mixtures were taken up without a change in isotopic ratio. This result together with the other evidence presented indicate a net uptake of exogenous lipid by a process likely involving fusion.

Acetates↗

PubMed1980

The formation of phosphatidylethanolamine from diglyceride by extracts of Escherichia coli.

Extensively dialyzed cell-free homogenates or washed particulate fractions, of Escherichia coli in the presence of added CTP, Mg2+, serine, and rac-glycerol 3-phosphate, incorporated [32P]phosphatidic acid into phosphatidylglycerol and cardiolipin but not into phosphatidylethanolamine. [14C]Phosphatidic acid could be incorporated by these preparations into phosphatidylethanolamine in the absence of added CTP and Mg2+. CDP-diglyceride did not significantly affect the formation of [14C]phosphatidylethanolamine from [14C]phosphatidic acid. 14C-labeled diglyceride was also readily incorporated into phosphatidylethanolamine in the absence of added cofactors by both homogenate and particulate fraction. Serine stimulated the incorporation somewhat whereas CTP + Mg2+ diminished this conversion slightly because of concurrent [14C]phosphatidic acid and [14C]phosphatidylglycerol formation. CDP-diglyceride did not significantly affect the conversion of 14C-labeled diglyceride to phosphatidylethanolamine. Dialyzed 14C-labeled cytosol fractions of E. coli, obtained from cells grown in medium containing [14C]serine, transferred some of their label to phosphatidylethanolamine in the presence of fresh particulate fraction. This transfer was stimulated by added diglyceride. The results indicate that phosphatidylethanolamine can be synthesized from exogenous diglyceride by a pathway which does not require CDP-diglyceride as an intermediate but which likely makes use of an endogenous cofactor supplying phosphorylethanolamine and (or) phosphorylserine.

Carbon Radioisotopes↗

PubMed1980

The uptake and acylation of exogenous lysophosphatidylethanolamine by Escherichia coli cells.

Escherichia coli envelopes were fractionated to yield inner and outer membrane fractions. Both these fractions were found to convert [14C]lysophosphatidylethanolamine to its diacyl analogue. Intact Escherichia coli cells were capable of absorbing exogenous labelled lysophosphatidylethanolamine and converting it to phosphatidylethanolamine. When the 14C- and 32P-labelled lyso analogue was used, both the absorption process and the conversion to diacyl analogue proceeded without a significant change in isotope ratio either in the presence or in the absence of added inorganic phosphate. The absorption process was not markedly stimulated by Ca2+ in the medium; it proceeded to an amount representing 25--30% of the endogenous membrane lipid and was accompanied by some degradation to water-soluble products which accumulated in the cell mainly, but also in the incubation medium. The absorbed lipid was recovered in both the inner and outer membrane fractions of the cell envelope. The results indicate that Escherichia coli inner and outer membranes are capable of absorbing exogenous lysophosphoglyceride and converting it into structurally useful diacyl analogue.

Calcium↗

PubMed1978

Partial purification of a lipolytic enzyme from Escherichia coli.

An enzyme with phospholipase Al activity was purified some 500-fold from Escherichia coli cell homogenates. Lipase, phospholipase A2, and lysophospholipase copurified with phospholipase A1 and the four activities displayed similar susceptibility to heat treatment. The phospholipase A and lipase activities were recovered in a single band when partially purified preparations were subjected to SDS gel electrophoresis. Phospholipase, lysophospholipase, and lipase all required Ca2+ for activity. Phosphatidylcholine, phosphatidylethanolamine, and their lyso analogues were all hydrolysed at equivalent rates and these were substantially greater than the rate of methylpalmitate or tripalmitoylglycerol hydrolyses under similar incubation conditions. Evidence for a direct but slow hydrolysis of the ester at position 2 of phosphoglyceride was obtained; however, release of fatty acid from this position is mostly indirect involving acyl migration to position 1 and subsequent release of the translocated fatty acid. Escherichia coli, therefore, appears to possess a lipolytic enzyme of broad substrate specificity acting mainly at position 1 but also at position 2 of phosphoglycerides and on triacylglycerols and methyl fatty-acid esters.

Escherichia coli↗

PubMed1978

The lipase activity of a partially purified lipolytic enzyme of Escherichia coli.

Escherichia coli lipase was found to have a broad pH optimum between pH 8 and 10. Long-chain acyl triacylglycerols such as trioleolglycerol were hydrolysed at a relatively slow rate, whereas, the shorter-chain acyl derivative tracapryloylglycerol was not. Triacylglycerols and diacylglycerols were broken down at a rate 10 to 15 fold greater than that for monoacylglycerol. Simple esters such as methyloleate and cetylpalmitate were hydrolysed at rates greater than that of triacyglycerol. Water-soluble esters such as p-nitrophenylacetate were not attacked. Hydrolysis of lipase substrate occurred more readily in the presence of an anionic detergent such as taurocholate. The enzyme had no marked preference for the 1- or 3-position of triacylglycerols but attacked these positions much more readily than position 2. The enzyme also catalyzed transacylation reaction with simple alcohols such as methanol or ethanol.

Escherichia coli↗

PubMed1978

Uptake of lysolecithin and other lysins by rat erythrocytes.

Lysis of rat erythrocytes by lysolecithin, palmitoyl carnitine and palmitoyl choline is inhibited by albumin and by certain lipids. Albumin rapidly forms a complex with the lysins thereby decreasing their reactivity with erythrocytes. Lecithin and cholesterol are lysis inhibitors that do not prevent the uptake of lysins but decrease their interactions with structural elements of the red cell membrane.

Animals↗

PubMed1977

Further studies on the cardiolipin phosphodiesterase of Escherichia coli.

The cardiolipin phosphodiesterase of Escherichia coli was further characterized. This enzyme has a pH optimum of 7.0 and is Mg2+ dependent. Mn2+ and Co2+ could replace Mg2+ but other divalent cations were inhibitory or without effect. The enzyme is not periplasmic and does not appear to be associated with membrane fractions prepared by different methods. It is recovered as a soluble protein in the cytosol fraction but could not be readily purified because of its instability. With cell-free systems, a requirement for ATP or ADP could be shown under certain defined conditions. Other nucleotides were less effective or ineffective in stimulating the phosphodiesterase. The cells displayed the highest activity during the middle to late exponential stage but no marked requirement for ATP was apparent when the phosphodiesterase was obtained from such freshly grown cells. If, however, cells were starved for several hours in saline medium, the cardiolipin phosphodiesterase level fell and a requirement for added ATP could be shown. The cardiolipin phosphodiesterase is an enzyme distinct from cardiolipin synthase. The assay conditions are quite different from each of these enzymes as are their subcellular distributions.

Adenosine Diphosphate↗

PubMed1975

Polyglycerophosphatide metabolism in Escherichia coli.

When Escherichia coli B cells were labelled with [14-C] glycerol and chased, there was a marked sparing of the phosphatidyl moiety compared to the nonacylated glycerol moiety of phosphatidylglycerol. When energy-depleted cells were restored to an energy-rich medium there resulted a conversion of 32-P-labelled cardiolipin to phosphatidylglycerol, a lack of phosphatidic acid accumulation and no loss in total polyglycerophosphatide counts. In cell-free extracts, phosphatidic acid produced from 32-P-labelled cardiolipin by the action of Escherichia coli phosphalipase D, was readily recycled to form poly-glycerophosphatide. In the presence of glycerol, such extracts displayed traansphosphatidylase activity by degrading cardiolipin to phosphatidyglycerol mainly. The results as a whole indicate that the enzyme synthesizing cardiolipin together with cardiolipin-hydrolyzing phospholipase D constitute a cycle which is normally involved in the turnover of polyglycerophosphatides in Escherichia coli.

Adenosine Triphosphate↗

PubMed1975

Phospholipase D activity of gram-negative bacteria.

A phospholipase hydrolyzing cardiolipin to phosphatidic acid and phosphatidyl glycerol was characterized in gram-negative bacteria but was absent in preparations of gram-positive bacteria, Saccharomyces cerevisiae, and rat liver mitochondria. In cell-free extracts of Escherichia coli, Salmonella typhimurium, Proteus vulgaris, and Pseudomonase aeruginosa, this cardiolipin-hydrolyzing enzyme had similar pH and Mg2+ requirements and displayed a specificity which excluded phosphatidyl glycerol and phosphatidyl ethanolamine as substrates.

Animals↗

PubMed1974