A Study on the Effect of Lysolecithin and Phospholipase A on Membrane-Bound Galactosyltransferase

1974 ◽  
Vol 52 (11) ◽  
pp. 1053-1066 ◽  
Author(s):  
Sailen Mookerjea ◽  
James W. M. Yung
Keyword(s):  
Liver Microsomes ◽  
Fatty Acyl ◽  
Phospholipase A ◽  
Synthetase Activity ◽  
Rat Liver Microsomes ◽  
Physiological Concentration ◽  
Activation Effect ◽  
Membrane Bound ◽  
Hydrolysis Of

Addition of lysolecithin caused very marked activation of UDP-galactose:glycoprotein galactosyltransferase in rat liver microsomes and in Golgi-rich membranes. Lysolecithin activated galactosyltransferase when the enzyme was assayed both with endogenous acceptor and with exogenous proteins or monosaccharides as acceptors. Lactose synthetase activity in presence of α-lactalbumin was also stimulated by lysolecithin. Lecithin, lysophosphatidylethanolamine, lysophosphatidic acid, and glycerophosphorylcholine did not activate the enzyme, suggesting that both fatty acyl and phosphorylcholine groups of the lysolecithin molecule are required for the observed activation. The degree of activation was about the same when myristoyl-, palmitoyl-, oleoyl-, or stearoyllysolecithin were tested. The activation by lysolecithin was observed well within the physiological concentration of the lipid in the liver cell. Saturating amounts of Triton masked the effect of lysolecithin.Brief preincubation with phospholipase A activated the enzyme and generated lysolecithin in the membranes. Triton and lysolecithin activated the enzyme without any lag time, whereas phospholipase A activation was dependent on preincubation and also on an alkaline pH favorable for the hydrolysis of phospholipid. EDTA blocked the activation effect of phospholipase A but had no effect on activation by lysolecithin. Albumin and cholesterol opposed the effects of lysolecithin and phospholipase A on the enzyme. Two successive incubations of the microsomes with lysolecithin caused considerable release of the enzyme into the soluble fraction. The role of lysolecithin in the activation of the enzyme is probably related to the solubilization of the membrane and consequent enhanced interaction of the enzyme with substrate. Lysolecithin also activated N-acetylglucosaminyl- and sialyltransferase activities in microsomes. A possible role of lysolecithin is indicated in the regulation of glycosylation reactions in mammalian system.

scholarly journals A STUDY OF THE NUCLEOSIDE TRI- AND DIPHOSPHATE ACTIVITIES OF RAT LIVER MICROSOMES

1962 ◽  
Vol 15 (3) ◽  
pp. 563-578 ◽  
Author(s):  
Lars Ernster ◽  
Lois C. Jones
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Physiological Role ◽  
Sodium Deoxycholate ◽  
Inorganic Pyrophosphatase ◽  
Rat Liver Microsomes ◽  
High Concentrations ◽  
Hydrolysis Of ◽  
No Activation

Rat liver microsomes catalyze the hydrolysis of the triphosphates of adenosine, guanosine, uridine, cytidine, and inosine into the corresponding diphosphates and inorganic orthophosphate. The activities are stimulated by Na2S2O4, and inhibited by atebrin, chlorpromazine, sodium azide, and deaminothyroxine. Sodium deoxycholate inhibits the ATPase activity in a progressive manner; the release of orthophosphate from GTP and UTP is stimulated by low, and inhibited by high, concentrations of deoxycholate, and that from CTP and ITP is unaffected by low, and inhibited by high, concentrations of deoxycholate. Subfractionation of microsomes with deoxycholate into ribosomal, membrane, and soluble fractions reveals a concentration of the triphosphatase activity in the membrane fraction. Rat liver microsomes also catalyze the hydrolysis of the diphosphates of the above nucleosides into the corresponding monophosphates and inorganic orthophosphate. Deoxycholate strongly enhances the GDPase, UDPase, and IDPase activities while causing no activation or even inhibition of the ADPase and CDPase activities. The diphosphatase is unaffected by Na2S2O4 and is inhibited by azide and deaminothyroxine but not by atebrin or chlorpromazine. Upon fractionation of the microsomes with deoxycholate, a large part of the GDPase, UDPase, and IDPase activities is recovered in the soluble fraction. Mechanical disruption of the microsomes with an Ultra Turrax Blender both activates and releases the GDPase, UDPase, and IDPase activities, and the former effect occurs more readily than the latter. The GDPase, UDPase, and IDPase activities of the rat liver cell reside almost exclusively in the microsomal fraction, as revealed by comparative assays of the mitochondrial, microsomal, and final supernatant fractions of the homogenate. The microsomes exhibit relatively low nucleoside monophosphatase and inorganic pyrophosphatase activities, and these are unaffected by deoxycholate or mechanical treatment. Different approaches toward the function of the liver microsomal nucleoside tri- and diphosphatases are reported, and the possible physiological role of the two enzymes is discussed.

Effect of nucleotides on UDP-N-acetylglucosamine pyrophosphatase and N-acetylglucosaminyltransferase activities in microsomal membranes

1979 ◽  
Vol 57 (6) ◽  
pp. 557-565 ◽  
Author(s):  
Sailen Mookerjea
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Inhibitory Effect ◽  
Rat Liver Microsomes ◽  
Glycoprotein Synthesis ◽  
Microsomal Membranes ◽  
Pyrophosphatase Activity ◽  
Triton X ◽  
Hydrolysis Of

Rat liver microsomes solubilized by incubating with lysolecithin or Triton X-100 showed very active UDP-N-acetylglucosamine pyrophosphatase activity leading to the hydrolysis of the substrate into N-acetylglucosamine-1-P and N-acetylglucosamine. ATP, GTP, CDPcholine, and CDPglucose exerted a considerable inhibitory effect on the solubilized membrane pyrophosphatase activity. CDPcholine and CDPglucose, in addition, appeared to stimulate the transfer of N-acetylglucosamine into endogenous and exogenous acceptor proteins. Evidence is also presented of an inhibitory effect of ATP (and to some extent GTP) on N-acetylglucosaminyltransferase activity. This inhibitory effect of ATP and GTP became clearly evident when the pyrophosphatase activity in the membranes was virtually eliminated in the presence of CDPcholine and CDPglucose. The effect of ATP and GTP on the solubilized membrane enzymes indicated that the inhibition of pyrophosphatase activity alone did not determine the rate of transfer of sugar to protein. The results also suggested that the UDP-N-acetylglucosamine pyrophosphatase and N-acetyiglucosaminyltransferase activities were controlled independently and the effect of each nucleotide on these enzymes should, therefore, be carefully evaluated to understand its role in glycopolymer biosynthesis. Also, a possible role of choline and its derivatives in glycoprotein synthesis is discussed.

scholarly journals Acyl-CoA binding and acylation of UDP-glucuronosyltransferase isoforms of rat liver: their effect on enzyme activity

1995 ◽  
Vol 312 (1) ◽  
pp. 301-308 ◽  
Author(s):  
A Yamashita ◽  
M Watanabe ◽  
T Tonegawa ◽  
T Sugiura ◽  
K Waku
Keyword(s):  
Fatty Acid ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Fatty Acyl ◽  
Binding Activity ◽  
Rat Liver Microsomes ◽  
Sequencing Analysis ◽  
Terminal Amino ◽  
Udp Glucuronosyltransferase ◽  
Hydrolysis Of

When [14C]arachidonoyl-CoA was incubated with crude extracts of rat liver microsomes, [14C]arachidonic acid was incorporated into many proteins, suggesting that modification of these proteins with fatty acid, i.e. acylation, occurred. Using a [14C]arachidonyl-CoA labelling assay, 50 and 53 kDa proteins were purified from rat liver microsomes to near homogeneity by sequential chromatography on Red-Toyopearl, hydroxyapatite, heparin-Toyopearl, Blue-Toyopearl and UDP-hexanolamine-agarose. Acylation of the 50 and 53 kDa proteins occurred in the absence of any other protein, suggesting that these molecules catalyse autoacylation. The acylation was dependent on the length of the incubation period and the concentration of [14C]arachidonoyl-CoA. The 50 and 53 kDa proteins also had acyl-CoA-binding activity; initial rates of acyl-CoA binding and acylation were 0.25 and 0.004 min-1 respectively. The proteins also had weak but distinct acyl-CoA-hydrolysing activity (0.006 min-1). These results suggest that the proteins catalysed the sequential reactions of binding to acyl-CoA, autoacylation, and hydrolysis of fatty acid. N-terminal amino acid sequencing analysis showed these proteins to be UDP-glucuronosyltransferase (UDPGT) isoforms. UDPGT activity was inhibited by arachidonoyl-CoA. These results suggest that binding of acyl-CoA and acylation of UDPGT isoforms regulate the enzyme activities, implying a possible novel function for fatty acyl-CoA in glucuronidation, which is involved in the metabolism of drugs, steroids and bilirubin.

Autoregulation of 3, 3′,5′-triiodothyronine production by rat liver microsomes

1981 ◽  
Vol 98 (2) ◽  
pp. 240-245 ◽  
Author(s):  
T. Kaminski ◽  
J. Köhrle ◽  
R. Ködding ◽  
R.-D. Hesch
Keyword(s):  
Rat Liver ◽  
Incubation Medium ◽  
Liver Microsomes ◽  
Inhibitory Effect ◽  
Incubation Mixture ◽  
Rat Liver Microsomes ◽  
Experimental Conditions ◽  
Physiological Concentration ◽  
Enzyme Binding ◽  
Ph Dependent

Abstract. Conversion of thyroxine (T4) to 3,3′,5′-triiodothyronine (rT3) was studied in rat liver microsomes. Addition of rT3 at a physiological concentration to the incubation medium inhibited the deiodination of thyroxine to rT3. With a concentration of rT3 greater than 37.6 nM no net rT3 production at pH 8.0 was observed. Further increases in rT3 concentration resulted only in degradation of added rT3 and no net synthesis of rT3 from T4 could be detected. The inhibitory effect of rT3 upon its own production from T4 was pH dependent, 5 fold lower amounts of hormone being required to inhibit completely rT3 production at pH 7.4 than at pH 8.0. With the same experimental conditions no significant effect of rT3 on the conversion of T4 to 3,5,3′-triiodothyronine (T3) could be observed at pH 8.0 with all concentrations of added iodothyronine. A linear production of 3,3′-T2 from added rT3 was determined over the whole range of rT3 concentration, suggesting a lack of saturation of deiodinating enzyme. Binding of rT3 by anti-rT3 antibody added to the incubation mixture enhanced rT3 production from T4 by protecting rT3 from being degraded and/or diminishing the inhibitory effect of this iodothyronine on its own production. It was concluded that rT3 influenced its own production and that this effect may represent an important autoregulatory process in the iodothyronine metabolism.

scholarly journals Fatty acyl-CoA esters inhibit glucose-6-phosphatase in rat liver microsomes

1995 ◽  
Vol 307 (2) ◽  
pp. 391-397 ◽  
Author(s):  
R Fulceri ◽  
A Gamberucci ◽  
H M Scott ◽  
R Giunti ◽  
A Burchell ◽  
...  
Keyword(s):  
Palmitic Acid ◽  
Phosphatase Activity ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Fatty Acyl ◽  
Rat Liver Microsomes ◽  
Control Value ◽  
Scattering Measurements ◽  
Induced Changes ◽  
Enzymic Assay

In native rat liver microsomes glucose 6-phosphatase activity is dependent not only on the activity of the glucose-6-phosphatase enzyme (which is lumenal) but also on the transport of glucose-6-phosphate, phosphate and glucose through the respective translocases T1, T2 and T3. By using enzymic assay techniques, palmitoyl-CoA or CoA was found to inhibit glucose-6-phosphatase activity in intact microsomes. The effect of CoA required ATP and fatty acids to form fatty acyl esters. Increasing concentrations (2-50 microM) of CoA (plus ATP and 20 microM added palmitic acid) or of palmitoyl-CoA progressively decreased glucose-6-phosphatase activity to 50% of the control value. The inhibition lowered the Vmax without significantly changing the Km. A non-hydrolysable analogue of palmitoyl-CoA also inhibited, demonstrating that binding of palmitoyl-CoA rather than hydrolysis produces the inhibition. Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer demonstrated that palmitoyl-CoA alone or CoA plus ATP and palmitic acid altered the microsomal permeability to glucose 6-phosphate, but not to glucose or phosphate, indicating that T1 is the site of palmitoyl-CoA binding and inhibition of glucose-6-phosphatase activity in native microsomes. The type of inhibition found suggests that liver microsomes may comprise vesicles heterogeneous with respect to glucose-6-phosphate translocase(s), i.e. sensitive or insensitive to fatty acid ester inhibition.

scholarly journals The conversion of exogenous retinol and related compounds into retinyl phosphate mannose by adult Brugia pahangi in vitro

1983 ◽  
Vol 214 (2) ◽  
pp. 367-376 ◽  
Author(s):  
J C W Comley ◽  
J J Jaffe
Keyword(s):  
Liver Microsomes ◽  
Beta Carotene ◽  
Synthetase Activity ◽  
Rat Liver Microsomes ◽  
Brugia Pahangi ◽  
Glycoprotein Synthesis ◽  
Absolute Requirement ◽  
Endogenous Lipid ◽  
Related Compounds

Adult Brugia pahangi took up and incorporated beta-carotene and free retinol in vitro. The uptake of retinol was 50 times greater than that of beta-carotene under similar incubation conditions. beta-Carotene was almost entirely metabolized, primarily to retinol. The metabolism of retinol by B. pahangi in vitro was less extensive, with a variety of retinoids tentatively identified, including retinyl phosphate (Ret-P), retinyl phosphate mannose (Ret-P-Man) and anhydroretinol as minor metabolites. B. pahangi microsomes were also shown to biosynthesize Ret-P-Man from exogenous Ret-P and GDP-mannose, but not from endogenous lipid acceptors alone. In this circumstance an unidentified lipid appeared to be mannosylated by B. pahangi. The rate of mannose transfer to exogenous Ret-P by B. pahangi microsomes was 150 pmol X min -1. (mg of protein) -1. Ret-P-Man synthetase activity from both B. pahangi and rat liver microsomes had an absolute requirement for bovine serum albumin and MnCl2, and occurred in the absence of detergent. The results suggest a biochemical role for vitamin A in B. pahangi, possibly in filarial glycoprotein synthesis.

Role of the Sarcoplasmic Reticulum Ca2+-ATPase on Heat Production and Thermogenesis

2001 ◽  
Vol 21 (2) ◽  
pp. 113-137 ◽  
Author(s):  
Leopoldo de Meis
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atp Hydrolysis ◽  
Surrounding Medium ◽  
Chemical Energy ◽  
The Past ◽  
Sarcoplasmic Reticulum Membrane ◽  
Membrane Bound ◽  
Hydrolysis Of ◽  
Heat Released

The sarcoplasmic reticulum of skeletal muscle retains a membrane bound Ca2+-ATPase which is able to interconvert different forms of energy. A part of the chemical energy released during ATP hydrolysis is converted into heat and in the bibliography it is assumed that the amount of heat produced during the hydrolysis of an ATP molecule is always the same, as if the energy released during ATP cleavage were divided in two non-interchangeable parts: one would be converted into heat, and the other used for Ca2+ transport. Data obtained in our laboratory during the past three years indicate that the amount of heat released during the hydrolysis of ATP may vary between 7 and 32 kcal/mol depending on whether or not a transmembrane Ca2+ gradient is formed across the sarcoplasmic reticulum membrane. Drugs such as heparin and dimethyl sulfoxide are able to modify the fraction of the chemical energy released during ATP hydrolysis which is used for Ca2+ transport and the fraction which is dissipated in the surrounding medium as heat.

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