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Biomedical subjects

R M Dawson

Publications and source records attributed to R M Dawson.

At least 55 records · Page 3Linked to original sources
PubMed1983

The precision of results from nonlinear regression analysis of data following a three-parameter one-exponential equation.

Simulated experimental data were generated from error-free data following the equation y = A - Be-kt where A, B, and k are constants and were analyzed by iterative nonlinear regression using one of two basic published computer programs. The effect of the simulated experimental error in y on the precision of the computed constants A, B, and k was evaluated. The errors were either independent of y (simple errors) or proportional to y (relative errors) and outliers were sometimes introduced. Other factors investigated were the number of data points per regression, the range of values of y, and the effect of weighting the data. The results show that the errors in the computed constants, and particularly the rate constant k, may be considerably magnified with respect to the errors in the experimental data. The quantitative relationships that are presented are useful aids in the design of biochemical experiments in which the above equation is applicable.

Computers↗
PubMed1982

Proteolytic activation can produce a phosphatidylinositol phosphodiesterase highly sensitive to Ca2+.

The phosphatidylinositol phosphodiesterase of rat brain shows little activity under conditions likely to pertain in vivo (neutral pH and micromolar Ca(2+) concentrations). A short incubation of a brain supernatant with trypsin, or a longer pre-incubation of the supernatant alone, produce new forms of the enzyme, which are active under such conditions. A possible role of receptor-linked proteinases in initiating phosphatidylinositol catabolism is discussed.

Animals↗
PubMed1982

Heterogeneity of the calcium-dependent phosphatidylinositol phosphodiesterase in rat brain.

1. The Ca(2+)-dependent phosphatidylinositol phosphodiesterase (phospholipase C-type) from the cytosolic supernatant of rat brain was active against exogenous [(32)P]-phosphatidylinositol from pH5.0 to pH8.5. However, the activity in the range pH7.0-8.5 could not be recovered after precipitation with (NH(4))(2)SO(4); most of the enzyme activity was recovered in the 30-50% fraction and showed a single sharp pH optimum at 5.5. 2. The cytosolic supernatant was analysed by isoelectric focusing on acrylamide gels, and assay at pH5.5. Four peaks of phosphodiesterase activity were found at pI ranges 7.4-7.2, 6.0-5.8, 4.8-4.4 and 4.2-3.8. 3. The cytosolic supernatant was also applied to a chromatofocusing column, and again assayed at pH5.5. Four peaks were eluted: minor, but consistent, activity at the beginning of the elution with a pI of near 7.2 or above; a second peak at pH6.0-5.85; a third broad peak with a wide range pH5.3-4.2; and a fourth peak, which was eluted by washing the column with 1m-NaCl, suggesting an isoenzyme with a pI below 4.0 (supported by the result of the isoelectric focusing). 4. If all the chromatofocusing fractions were assayed at pH7.0 or 8.0 (at 1mm-Ca(2+)), only a single sharp peak was detected, with a pI of 4.6-4.8. This peak disappeared on (NH(4))(2)SO(4) fractionation (30-50%) of the cytosolic supernatant, whereas the four peaks with activity at pH5.5 were virtually unaffected. 5. The four activities (assayed at pH5.5) separated by chromatofocusing produced inositol 1:2-cyclic monophosphate, inositol 1-monophosphate and diacylglycerol as enzymic products. 6. We conclude that the Ca(2+)-dependent phosphatidylinositol phosphodiesterase exhibits considerable heterogeneity, both with respect to pH optima of activity, and its isoelectric properties.

Ammonium Sulfate↗
PubMed1982

Phosphatidylinositol-hydrolysing enzymes in blowfly salivary glands.

The salivary glands of adult blowflies (Calliphora erythrocephala) contain enzymes that hydrolyse phosphatidylinositol, predominantly by a Ca2+-independent deacylation, though a Ca2+-dependent phosphodiesterase (phospholipase C) activity could be detected. The deacylating enzymes could also hydrolyse phosphatidylcholine and phosphatidylethanolamine, and were secreted in the saliva. Homogenization of salivary glands prelabelled with [3H]inositol resulted in a rapid deacylation of the endogenous 3H-labelled phosphatidylinositol; this hydrolysis was unaffected by addition of 5-hydroxytryptamine to the homogenate.

Animals↗
PubMed1982

Hydrolysis of phosphatidylinositol by pancreas and pancreatic secretion.

1. The secretion from sheep pancreas and a supernatant fraction prepared from the gland contained an EDTA-insensitive acid phospholipase A1 which readily deacylated phosphatidylinositol (pH optimum, 5.3), 1-acylglycerophosphoinositol and phosphatidic acid, but had limited action of phosphatidylcholine and phosphatidylethanolamine even with deoxycholate present. The enzyme was not a triacylglycerol lipase. 2. The action of the phospholipase A1 on phosphatidylinositol was inhibited effectively by Ca2+ and Mg2+, probably by interaction of these ions with the substrate. 3. In the presence of calcium the decomposition of phosphatidylinositol and lysophosphatidylinositol by the supernatant fraction was overwhelmingly by phosphodiesterase action (EC 3.1.4.10), producing inositol monophosphate and its cyclic derivative. Its pH optimum was about 6.0 but with considerable activity extending to pH 8.5. 4. The phosphodiesterase was not secreted in the pancreatic juice.

Animals↗
PubMed1982

The stimulation of the brain alkaline phospholipase A1 attacking phosphatidylethanolamine by various salts and metal chelators.

Rat brain contains a soluble, high molecular weight phospholipase A1 of alkaline pH optimum which shows a preference for phosphatidylethanolamine as substrate. There is evidence that the same enzyme exists in liver and kidney. At low osmotic concentrations of buffer the enzyme is markedly stimulated by CaCl2. However, MgCl2 and MnCl2 are equally as effective although at concentrations above 2 mM the activation falls away with MnCl2. The phospholipase A1 is stimulated by divalent metal ion chelators (EDTA, EGTA, CDTA) and by sodium phosphate and sodium sulphate. The activity is inhibited by hexanol, benzyl alcohol, diethylether and detergents. Although the activity can be inhibited by saturated and unsaturated fatty acids, no evidence could be obtained that the activators function by counteracting the inhibitory action of fatty acids liberated at the interface of the substrate and incubation medium. It is suggested that to achieve good enzymic hydrolysis a certain type of organised hydrated phosphatidylethanolamine structure is required in which the negative zeta potential has been reduced by metallic cations in the incubation medium.

Animals↗
PubMed1981

On the insensitivity of sheep to the almost complete microbial destruction of dietary choline before alimentary-tract absorption.

1. Injection of [Me-14C]choline into sheep indicated that the small amount of phosphatidylcholine present in abomasal digesta was largely (69%) of non-dietary or ruminal origin. 2. Long-term feeding of [Me-3H]choline to sheep produced insignificant labelling of plasma phosphatidylcholine, indicating that more than 99% of the choline body pool was of non-dietary origin. 3. In contrast, when rats were fed with [Me-3H]choline for similar periods, 18-54% of the tissue phosphatidylcholine was derived from dietary choline. 4. The loss of [14C]choline and 32P from the plasma phosphatidylcholine after a single injection of these isotopes indicated a markedly slower turnover of choline in the sheep compared with the rat. This observation, coupled with a lack of liver glycerophosphocholine diesterase, provides an explanation for the insensitivity of the sheep to an almost complete microbial destruction of dietary choline before alimentary-tract absorption.

Abomasum↗
PubMed1981

Alkaline O leads to N-transacylation. A new method for the quantitative deacylation of phospholipids.

1. Quantitative O-deacylation of phospholipids has been achieved by incubation with a reagent containing monomethylamine, methanol and water. The reaction is primarily an O leads to N-transacylation with N-methyl fatty acid amides being formed. 2. The reagent can be removed easily by volatilization and under defined conditions no secondary decomposition of the phosphorus-containing deacylation products occurs. 3. The water-soluble phosphorus compounds derived by deacylation of mammalian tissue O-diacylated phospholipids have been completely separated by a single-dimensional paper ionophoresis with a volatile pH9 buffer. 4. The O-deacylated alkyl and alkenyl phospholipids have been examined by t.l.c. before and after catalytic hydrolysis with Hg2+. 5. A complete analysis of rat brain phospholipids by the above methods agrees closely with that obtained by other procedures.

Acylation↗
PubMed1981

The hydrolysis of phosphatidylinositol monolayers at an air/water interface by the calcium-ion-dependent phosphatidylinositol phosphodiesterase of pig brain.

1. The activity of Ca2+-dependent phosphatidylinositol phosphodiesterase (EC 3.1.4.10) of pig brain against [32P]phosphatidylinositol monolayers at an air/water interface has been measured. As the monolayer pressure was increased a sharp cut-off of enzymic hydrolysis occurred at 33 X 10(-3) N/m. 2. The addition of either phosphatidic acid, phosphatidylglycerol or oleyl alcohol increased the film pressure at which cut off occurred, as well as increasing the rate of hydrolysis at lower pressures. 3. The rate of hydrolysis, but not the cut-off pressure, was markedly increased by oleic acid and slightly increased by phosphatidylethanolamine. 4. Phosphatidylcholine, palmitoylcholine and octadecylamine decreased the cut-off pressure, as well as the enzymic activity below this pressure. 5. Stearic acid and stearyl alcohol had no effect on either the cut-off pressure or the activity. 6. All activators decreased the length of the lag phase before enzyme activity began, and phosphatidylcholine increased it. 7. These results are compared with the stimulatory and inhibitory effects of various amphiphiles observed previously with phosphatidylinositol dispersions [Irvine, Hemington & Dawson (1979) Eur. J. Biochem. 99, 525-530], and their possible relevance to the control of the phosphatidylinositol phosphodiesterase in vivo are discussed.

Amines↗
PubMed1981

Diabolic acids: occurrence and identification in natural products and their metabolism by simple-stomached and ruminant animals.

1. A method for gas-liquid chromatographic analysis of the dimethyl esters of a homologous series of diabolic acids is described. 2. Only when combined with low-resolution mass spectrometry could the method be used to show unequivocally the existence of diabolic acids in natural products. 3. Diabolic acids occurred naturally in rumen and faecal lipids of all ruminants examined but could not be detected in a number of ruminant tissue samples. 4. Infused 14C-labelled (C16:0)2 diabolic acid was poorly absorbed by both simple-stomached and ruminant animals. Oxidation was more extensive in the ruminant, but there was no evidence of deposition of diabolic acid in the tissues of either animal.

Animals↗
PubMed1981

The catabolism of phosphatidylinisitol by an EDTA-insensitive phospholipase A1 and calcium-dependent phosphatidylinositol phosphodiesterase in rat brain.

1. A rat brain supernatant and microsomal fraction contained a phospholipase A1 enzyme which hydrolysed phosphatidylinositol at pH 8 in the absence of calcium. Triolein and phosphatidylcholine were not attacked under the same incubation conditions. 2. No evidence could be obtained for a phospholipase A2 in the microsomal preparation, and in the presence of Ca2+ the release of fatty acid observed was due to phosphatidylinositol phosphodiesterase followed by diacylglycerol lipase action. 3. Brain phosphatidylinositol phosphodiesterase showed extensive activity in the alkaline range (7-8.5) as well as at pH 5-5.5. The activity at higher pH values required higher calcium concentrations and disappeared on purification of the soluble enzyme by ammonium sulphate fractionation. 4. In general the ratio between inositol 1,2-(cyclic)phosphate and inositol 1-phosphate produced by phosphodiesterase action decreased with increasing pH.

Animals↗
PubMed1980

Rabbit small intestinal brush border membrane preparation and lipid composition.

1. Rabbit intestinal brush border membrane vesicles were prepared either from frozen or fresh tissue and the lipid composition was analysed. 2. Lipids extracted from membranes prepared by the Ca2+-precipitation method (Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J. and Crane, R.K. (1973) Biochim. Biophys. Acta 323, 98--112; Kessler, M., Acuto, O., Storelli, C., Murer, H., Müller, M. and Semenza, G. (1978) Biochim. Biophys. Acta 506, 136--154) had exceptionally high levels of lysophospholipids and free fatty acids. An intrinsic, Ca2+-activated phospholipase A is responsible for the lipid decomposition. 3. It was necessary to modify the preparation of brush border membranes. Essentially, EGTA is used to keep the free Ca2+ concentration low so that phospholipases are inactivated, and Mg2+ instead of Ca2+ is employed to aggregate selectively contaminating membranes. The modified procedure gives membranes suitable for lipid analysis. 4. The molar ratio of neutral, phospholipid and glycolipid is about 1 : 1 : 1. The major neutral lipids are free cholesterol and fatty acids. The major phospholipids are phosphocholine-containing (approx. 45%) and phosphatidyl-ethanolamine (approx. 40%). The remainder (15--20%) is made up ppf acidic phospholipids, mainly phosphatidylserine and phosphatidylinositol. The major glycolipid is ceramide monohexodise. 5. The major fatty acids of total lipids and phospholipids are palmitic, stearic, oleic and linoleic acids. Individual phospholipids are characterised by distinct fatty acid compositions and differ markedly in the ratio of unsaturated-to-saturated fatty acid. X

Animals↗
PubMed1980

Complex lipids of a lipolytic and general-fatty-acid-requiring Butyrivibrio sp. isolated from the ovine rumen.

The complex lipids of the naturally-occurring general-fatty-acid-auxotroph Butyrivibrio S2 [Hazlewood & Dawson (1979) J. Gen. Microbiol. 112, 15-27] grown with palmitic acid as sole fatty-acid supplement have been investigated and some have been isolated in a state of purity and analysed. The majority are phospholipids (84%) and many contain galactose. They typically possess few esterified long-chain fatty-acid residues (C16:0), but are rich in esterified butyric acid and C16-alkenyl groups. Most of the phosphorus-containing lipids, including the two major lipids of the organism, contain esterified diabolic acid, a long-chain vicinal dimethyl-substituted dicarboxylic acid [Klein, Hazlewood, Kemp & Dawson (1979) Biochem. J. 183, 691-700] in definite stoichiometric relationship to phosphorus. No phosphatidylglycerol was present, but its monobutyroyl ester was detected as a minor component. Galactofuranosyldiacylglycerol (plasmalogen) and its monobutyroyl ester, cetyl alcohol and diacylglycerol were also identified.

Chemical Phenomena↗
PubMed1980

Structure of diabolic acid-containing phospholipids isolated from Butyrivibrio sp.

The structures of the diabolic acid-containing phospholipids of Buryrivibrio S2 grown in the presence of palmitic acid have been investigated. Generally they consist of two conventional bacterial phospholipid or galactolipid structures linked by esterification through a single diabolic acid residue. The main lipid consists of the butyroyl ester of sn-1-alkenylglycero-3-phospho-1'-sn-glycerol joined in this way to the butyroyl ester of sn-1-alkenyl-3-galactosylglycerol by esterification of the vacant 2-hydroxy groups of the alkenyl-substituted glycerol molecules. A lipid that possess a palmitoyl group rather than one of the butyroyl groups of the latter structure has also been detected. The lipid occurring in the second highest concentration consists of two molecules of sn-1-alkenylglycero-3-phospho-sn-1'-glycerol butyroyl ester linked through diabolic acid in a similar manner to the main lipid. Other lipids with the latter structure either minus a butyroyl group or with palmitoyl group, instead of one of the buryroyl groups, exist as minor components.

Chemical Phenomena↗
PubMed1980

Phosphatidylinositol phosphodiesterase in higher plants.

1. The lower regions of the stem of celery (Apium graveolens L.) contain a soluble enzyme that hydrolyses phosphatidylinositol. 2. The lipoidal product of hydrolysis is diacylglycerol, and the water-soluble products are 1:2-cyclic phosphoinositol and phosphoinositol in the approximate proportions of 60% and 40% respectively: this indicates that a phosphodiesterase (phospholipase C-like) activity is cleaving the phosphatidylinositol. 3. The enzyme requires a bivalent cation, Ca2+ being the most effective activator. 4. The enzyme has a pH optimum, depending on conditions of assay, of pH 5.9-6.6 and in this pH range shows no detectable activity against phosphatidylcholine or phosphatidylethanolamine. 5. The activity is stimulated by phosphatidic acid and slightly inhibited (30% at concentrations equimolar with phosphatidylinositol) by phosphatidylcholine. 6. The phosphodiesterase was also detected (but not quantified) in the tips of the flowers in cauliflowers, in outer leaves of onion and in the elongating stem of daffodils. 7. The enzyme's properties are compared with equivalent mammalian enzymes, and its possible role in the catabolism of phosphatidylinositol in higher plants is discussed.

Cations↗