The spontaneous incorporation of proteins into preformed bilayers.
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Adsorption↗
Biomedical subjects
D Zakim
Publications and source records attributed to D Zakim.
Reconstitution of membrane proteins: sequential incorporation of integral membrane proteins into preformed lipid bilayers.
Several integral membrane proteins can be inserted sequentially into preformed unilamellar vesicles (ULV's) composed of dimyristoylphosphatidylcholine (DMPC) and cholesterol in a gel phase. Thus, proteoliposomes of DMPC, cholesterol, and bacteriorhodopsin from Halobacterium halobium rapidly incorporate UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase from beef heart mitochondria, and additional bacteriorhodopsin, added sequentially. This process of spontaneous incorporation can be regulated to produce complex artificial membranes that contain phospholipids and proteins at ratios (mol/mol) equivalent to what is found in biological membranes. The ability of the lipid-protein bilayers to incorporate additional integral membrane proteins is not affected by annealing of the proteoliposomes at 37 degrees C nor by the order of addition of the proteins. Bacteriorhodopsin-containing vesicles formed by the sequential addition of integral membrane proteins demonstrate light-driven proton pumping. Therefore, they have retained a vesicular structure. Vesicles containing one or two different proteins will fuse with each other at 21 degrees C or with ULV's devoid of proteins. Incorporation of bacteriorhodopsin or UDPglucuronosyltransferase into proteoliposomes containing DMPC, with or without cholesterol as impurity, also occurs above the phase transition for DMPC. The presence of a protein in a liquid-crystalline bilayer provides the necessary condition for promoting the spontaneous incorporation of other membrane proteins into preformed bilayers.
Animals↗
Developmental alterations in hepatic UDP-glucuronosyltransferase. A comparison of the kinetic properties of enzymes from adult sheep and fetal lambs.
The kinetic properties of hepatic microsomal UDP-glucuronosyltransferase were studied in sheep in the perinatal period, using acetaminophen as the aglycone. Kinetic analyses indicated that activity at Vmax was significantly less in fetal microsomes (113, 135 or 141 days) as compared with the adult sheep. However, these differences between fetal and adult animals were not due simply to smaller amounts of UDP-glucuronosyltransferases catalyzing conjugation of acetaminophen in fetuses versus adults. Thus, the kinetic properties of UDP-glucuronosyltransferase(s) were different in fetus and adult. The "fetal" versus "adult" enzyme had a higher affinity for UDP-glucuronic acid, but a poorer affinity for acetaminophen. Furthermore, enzyme in fetal liver (113 days of gestation) was activated about 30% by the allosteric effector UDP-N-acetylglucosamine, whereas enzyme in adult liver was activated by 500%. These differences between fetal and adult enzymes diminished just prior to parturition (141-day fetus). Enzyme in microsomes from the 141-day fetus responded to UDP-N-acetylglucosamine-like enzyme in adult microsomes and had affinities for substrates that were similar to "adult" enzymes. These data indicate that maturation of the system that glucuronidates acetaminophen is a complex process. It may involve the expression in fetuses of a type of UDP-glucuronosyltransferase that is different from that expressed in the adult. An alternative but not mutually exclusive possibility is that maturation of the glucuronidation system involves modification of enzyme function by alteration of the phospholipids in the immediate environment of UDP-glucuronosyltransferase within the microsomal membrane.
Animals↗
Physical-chemical model for the entry of water-insoluble compounds into cells. Studies of fatty acid uptake by the liver.
The spontaneous transfer of water-insoluble substances from plasma to the interior of cells would involve a series of steps in which the substance of interest dissociates from albumin in plasma, enters the outer half of the plasma membrane of a cell, crosses the bilayer, and then dissociates from the inner half of the plasma membrane to enter cell cytosol and diffuses to sites of its metabolism. We have examined the behavior of long-chain fatty acids in the uptake process, assuming that none of these steps is facilitated by the cell during the entry of fatty acids into the liver. Comparison of the spontaneous rates for each individual step with rates of uptake of fatty acid by perfused liver leads to the conclusion that the uptake of fatty acids is not limited by kinetic factors but is determined instead by the equilibrium distribution (Keq) of fatty acids between albumin in plasma and the phospholipids of the plasma membrane. This idea was examined further by determining whether there was a relationship between the value for Keq and rates of uptake of a fatty acid and the pattern of kinetics for uptake. The data indicate that there is a linear relationship between Keq and the rate of uptake, that uptake rates can be predicted with a high degree of accuracy from thermodynamic data, and that the pattern of kinetics of uptake is compatible with the idea that the uptake rate is determined by the relative affinity of a fatty acid for albumin and membranes.
Animals↗
Reconstitution of membrane proteins: catalysis by cholesterol of insertion of integral membrane proteins into preformed lipid bilayers.
The presence of cholesterol in small unilamellar vesicles (ULV) of dimyristoylphosphatidylcholine (DMPC) catalyzes fusion of the vesicles at temperatures below the upper limit for the gel to liquid-crystalline phase transition of the DMPC. The extent to which ULV grow depends on the concentration of cholesterol in the vesicles and on temperature. Maximum growth occurs at 21 degrees C. It decreases as the temperature is lowered below 21 degrees C. Growth does not occur at temperatures above the phase transition. In addition, the presence of cholesterol in ULV of DMPC catalyzes the insertion of integral membrane proteins into the vesicles. Thus, bacteriorhodopsin from Halobacterium halobrium, UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, and cytochrome oxidase from beef heart mitochondria formed stable lipid-protein complexes spontaneously when added to ULV containing cholesterol at temperatures under which these vesicles would fuse. Incorporation of these proteins into the ULV of DMPC did not occur in the absence of cholesterol or in the presence of cholesterol when the temperature of the system was above that for the phase transition. It appears that cholesterol lowers the energy barrier for fusion of ULV of DMPC and for insertion of integral membrane proteins into these bilayers. Studies with bacteriorhodopsin suggest that the energy barrier for insertion of proteins into ULV containing cholesterol is smaller than the energy barrier for fusion of the ULV with each other.
Animals↗
Interface between membrane biology and clinical medicine.
Many enzymes that are embedded within membranes of cells are sensitive to the chemical and physical properties of the lipid components of the membrane. Because of this, the functions of these integral membrane-bound enzymes can be regulated to some extent by changes within the lipid portions of biologic membranes. That the functions of membrane-bound proteins can be manipulated by modifications of their intimate environment is not surprising. It is well known, for example, that the properties of the surrounding aqueous phase can modulate the function of proteins that are soluble in the cytosol of cells. In contrast, whereas significant changes in the chemical composition and physical properties of the aqueous portion of the cell (e.g., ionic strength and pH) are not allowed, normally tolerable fluctuations of diet appear to influence significantly the composition and properties of the lipid portions of intracellular membranes to the extent of altering the function of some membrane-bound enzymes. In addition, it appears that changes of this type can be induced by diseases that alter dietary intake and/or intermediary metabolism. In other words, it is likely that the functions of at least some integral membrane proteins can be manipulated in patients. Such manipulations may prove to be efficacious. Alternatively, the importance of diet and processes of intermediary metabolism for altering the course of certain diseases may not be fully appreciated. It is worthwhile to review, therefore, current ideas of how the lipid portion of a membrane interacts with integral proteins. The property of the lipids that appears to be most important in this regard is their viscosity. The types of manipulations of function of membrane-bound enzymes that can be achieved are illustrated by in vitro effects secondary to varying the lipids used to reconstitute pure, delipidated forms of these enzymes. The functions of pure delipidated enzymes are discussed for the hepatic drug-metabolizing enzyme uridine diphosphoglucuronyltransferase. In addition, data are presented to indicate that the function of this enzyme can be modified extensively in intact animals by changing the diet.
Cell Membrane↗
The role of membrane lipids in the regulation of membrane-bound enzymes.
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Animals↗
Reconstitution of membrane proteins. Spontaneous association of integral membrane proteins with preformed unilamellar lipid bilayers.
We have developed a simple method for reconstituting pure, integral membrane proteins into phospholipid-protein vesicles. The method does not depend on use of detergents or sonication. It has been used successfully with three different types of integral membrane proteins: UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase (EC 1.9.3.1) from pig heart, and bacteriorhodopsin from Halobacterium halobium. The method depends on preparing unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) that contain a small amount of myristate as fusogen. Under conditions that the vesicles of DMPC have the property of fusing, all of the above proteins incorporated into bilayers. Two events appear to be involved in forming the phospholipid-protein complexes. The first is a rapid insertion of all proteins into a small percentage of total vesicles. The second is slower but continued fusion of the remaining phospholipid-protein vesicles, or proteoliposomes, with small unilamellar vesicles of DMPC. This latter process was inhibited by conditions under which vesicles of DMPC themselves would not fuse. On the basis of proton pumping by bacteriorhodopsin and negative staining, the vesicles were unilamellar and large. The data suggest that insertion of the above integral membrane proteins into vesicles occurred independently of fusion between vesicles.
Animals↗
Fatty acids bound to unilamellar lipid vesicles as substrates for microsomal acyl-CoA ligase.
Palmitate incorporated into single-layered vesicles of phosphatidylcholine was used as a substrate for palmitoyl coenzyme A ligase (palmitoyl-CoA ligase) in microsomes from rat liver. This was done in order to avoid the use of detergents for dispersal of the water-insoluble palmitate and the possibility of precipitating palmitate added to the aqueous assay as a salt suspension. The activity of the ligase measured when palmitate was added to assays as a component of phospholipid vesicles was 10-40-fold greater vs. activities reported in the literature using other methods for adding fatty acids to the assay system. Phospholipids, however, had no direct effect on the activity of palmitoyl-CoA ligase. The data indicate, therefore, that the activity of this enzyme has been underestimated because of the manner in which fatty acid was added to the assay, which has a significant effect on the activity of the ligase. It is shown too that the rate of spontaneous transfer of palmitate from unilamellar vesicles of phosphatidylcholine to microsomes via a hydrated intermediate is far more rapid than the inherent catalytic activity of the fatty acyl-CoA ligase. The data also suggest that the membrane-associated pool of fatty acid and not fatty acid in the aqueous phase of the assay is the pool of substrate interacting with the ligase.
Animals↗
Rates of hydration of fatty acids bound to unilamellar vesicles of phosphatidylcholine or to albumin.
The rates of hydration of naturally occurring fatty acids bound to unilamellar vesicles of dimyristoylphosphatidylcholine were measured by following the rate of quenching of the inherent fluorescence of albumin. Rates of hydration of fatty acids bound to albumin could be estimated from the same data. The data show that these rates depend on the chain length and unsaturation of the fatty acid. Increasing chain length diminishes the rate of hydration whereas increasing unsaturation increases this rate. Rates of hydration of fatty acids bound to lipid vesicles appear to be rapid enough to account for intracellular movement between compartments in the absence of carrier proteins. It is uncertain whether this is true for hydration of fatty acids bound to albumin. Rates for this process are about 100-300 times slower vs. rates of hydration of fatty acids bound to lipid vesicles.
Animals↗
Substrate specificity of fatty-acyl-CoA ligase in liver microsomes.
The substrate specificity of fatty-acyl-CoA ligase in liver microsomes has been studied in a system in which fatty acids are present initially as complexes with unilamellar vesicles of phosphatidylcholine. The latter were prepared by cosonication of phospholipids and different fatty acids. As compared with previous studies of the enzyme the activity of acyl-CoA ligase is several-fold higher for assays carried out with fatty acid substrates added as components of a bilayer. This was true for all fatty acids studied. Also as compared with data reported previously in the literature there was a systematic relationship between the structure of fatty acids, activity at Vmax for synthesis of acyl-CoA and avidity of binding to the ligase. Activity at Vmax was greatest for lauric acid and decreased with increasing chain length. The apparent avidity of enzyme for fatty acids was greatest for octanoic acid and decreased as chain length increased.
Adenosine Triphosphate↗
Studies of the catalytic mechanism of microsomal UDP-glucuronyltransferase. Alpha-glucuronidase activity and its stimulation by phospholipids.
The rate at which a specific, purified form of microsomal UDP-glucuronyltransferase (designated as the GT2P type of this enzyme) catalyzes the hydrolysis of UDP-glucuronic acid was measured with pure, delipidated enzyme and enzyme reconstituted with different lysophosphatidylcholines. This activity of the GT2P type of UDP-glucuronyltransferase is referred to as alpha-glucuronidase activity. For delipidated enzyme, the rate of hydrolysis of UDP-glucuronic acid catalyzed by GT2P extrapolated to infinite concentrations of UDP-glucuronic acid was 1 X 10(-9) mol/min/mg of protein. This compares with a rate of glucuronidation of p-nitrophenol of 96 X 10(-9) mol/min/mg of enzyme, for delipidated enzyme. Addition of oleoyl- or myristoyllysophosphatidylcholine to GT2P did not affect the alpha-glucuronidase activity significantly. This activity was stimulated, however, in the presence of compounds that bind at the aglycone site but that do not undergo glucuronidation. alpha-Glucuronidase activity extrapolated to infinite concentration of UDP-glucuronic acid was 4.0 X 10(-9) mol/min/mg for delipidated enzyme assayed in the presence of less than saturating concentrations of p-nitrophenyl phenyl ether. Moreover, when the aglycone site of GT2P was occupied by ethers, the alpha-glucuronidase activity of this enzyme was enhanced by addition of phospholipids to delipidated enzyme. The extent of activation of the alpha-glucuronidase activity of GT2P, when the aglycone site was occupied, depended on the acyl chain of the lipid added to delipidated enzyme. These data indicate that the GT2P form of UDP-glucuronyltransferase catalyzes the hydrolysis of UDP-glucuronic acid at a significant rate and that lysophosphatidylcholines can influence this rate.
Animals↗
Evidence that UDP-glucuronyltransferase in liver microsomes at 37 degrees C is in a gel phase lipid environment.
UDP-glucuronyltransferase (EC 2.4.1.17) in intact, untreated microsomes from pig liver is activated by relatively low concentrations of UDP-N-acetylglucosamine. This property is absent after treatment of microsomes with detergents or phospholipases, and also is not a characteristic of pure, delipidated enzyme. Sensitivity to activation by UDP-N-acetylglucosamine was reconstituted, however, by incorporation of pure, delipidated enzyme into unilamellar bilayers of phosphatidylcholine that were in a gel phase. Warming of these bilayers to just above the temperature for the gel to liquid crystalline phase transition led to an abrupt loss of sensitivity of UDP-glucuronyltransferase to activation by UDP-N-acetylglucosamine. These experiments establish that sensitivity to activation by UDP-N-acetylglucosamine is an inherent property of UDP-glucuronyltransferase. The data also suggest that the lipid environment of UDP-glucuronyltransferase in intact, untreated microsomes can modulate the sensitivity of this enzyme to allosteric activation by UDP-N-acetylglucosamine, and that this lipid environment is in a gel phase in intact microsomes at 37 degrees C.
Animals↗
The effect of choline deficiency on the activity of a phosphatidylcholine-requiring enzyme: activity and properties of UDP-glucuronyltransferase in choline-deficient rats.
The effect of choline deficiency on the kinetic properties of the microsomal enzyme UDP-glucuronyltransferase (EC2.4.1.17) was investigated in rats. Animals fed choline-deficient diets, as compared with animals fed a choline-replete diet or standard laboratory chow, showed almost a three-fold increase in enzyme activity when the enzyme was assayed at physiological concentrations of UDP-glucuronic acid (0.25 mM). The increase in activity appeared to be due to an enhanced affinity of the enzyme for UDP-glucuronic acid rather than to an increase in the amount of enzyme. These data indicate that the kinetic properties of tightly bound membrane enzymes are altered by a dietary change that is known to cause liver disease in the rat.
Animals↗
Structural, functional and hybridization studies of the glutathione S-transferases of rat liver.
We have purified five forms of glutathione S-transferase from rat liver. One form was the glutathione S-transferase B (ligandin), which is composed of two non-identical subunits with molecular weights of 22,000 (Ya) and 25,000 (Yc). Two of the other transferases were Ya and Yc homodimers. The other two transferases were also homodimers, but their subunit, Yb, had a molecular weight of 24,000. The three proteins containing either Ya or Yc subunits had similar substrate specificities, and all three contained peroxidase activity. The greatest peroxidase activity was present in proteins containing the Yc subunit. Enzymes composed of Yb subunits had minimal peroxidase activity in addition to different substrate specificities. The Ya and Yc containing enzymes bound the ligands bilirubin, and indocyanine green with high affinity (KD less than 5 microM), although the KD values of the YcYc protein were consistently 4- to 12-fold greater than those of the other two transferases. Studies were performed to define the origins of the various isozymes. There was no evidence for conversion of Yc to either Ya or Yb during storage or under conditions favorable to proteolysis. Hybridization studies were performed under denaturing conditions (6 M guanidine-HCl), and a YaYc hybrid was formed from the YaYa and YcYc proteins. In addition, both YaYa and YcYc hybrids were formed from transferase B. The hybrids were functionally similar to the proteins isolated originally from the liver. Attempts to form a YaYb hybrid from the YbYb and YaYa transferases were unsuccessful. This result is consistent with the lack of this enzyme form in the liver. Glutathione S-transferase B and the Ya and Yc homodimers appeared to be hybrids of common subunits. These three transferases had very similar functional and structural characteristics and differed from the transferases that are composed of Yb subunits.
Animals↗
Evidence for an active site arginine in UDP-glucuronyltransferase.
2,3-Butanedione inactivates the pure form of UDP-glucuronyltransferase used in these experiments (GT2P) (EC 2.4.1.17) purified from pig liver microsomes. The kinetics of the reaction indicates that 2,3-butanedione reacts with two amino acids that affect activity. A rapid, partial inactivation is followed by a slower rate of inactivation that leads eventually to completely inactive enzyme. UDP-glucuronic acid and glucuronic acid, as compared with UDP, are effective as protectors against the slow, secondary phase of inactivation; no ligand tested protected against the rapid phase of inactivation. The lipid environment of GT2P was a determinant of the pseudo-first order rate constant for the slow phase of inactivation, but did not affect the rate of the rapid phase of inactivation. The data suggest that GT2P contains an active site arginine that interacts with the -COO- at C-6 of the glucuronic acid moiety of UDP-glucuronic acid.
Amino Acids↗
Modulation of the number of ligand binding sites of UDP-glucuronyltransferase by the gel to liquid-crystal phase transition of phosphatidylcholines.
The kinetics of a pure, delipidated form of microsomal UDP-glucuronyltransferase is non-Michaelis-Menten when the enzyme is reconstituted into unilamellar vesicles of phosphatidylcholine that are in a gel phase. Double reciprocal plots of velocity as a function of the concentration of UDP-glucuronic acid show a downward curvature under these conditions. Binding studies indicate that the basis for the kinetic pattern is the presence of one high affinity and one low affinity binding site for UDP-glucuronic acid. The two classes of binding sites seem to be generated by the presence of two subunits that bind UDP-glucuronic acid within a single molecule of UDP-glucuronyltransferase. Melting the phospholipids from the gel phase to the liquid-crystal phase is associated with a switch from non-Michaelis-Menten to Michaelis-Menten kinetics for UDP-glucuronyltransferase. Binding studies for interaction of UDP-glucuronic acid with enzyme present in a liquid-crystal lipid phase indicate that the two binding sites for UDP-glucuronic acid do not become identical in this setting. Instead, one of the sites becomes nonfunctional. Binding studies carried out with UDP as ligand lead to similar results. There is a high affinity and a low affinity site for UDP when enzyme is reconstituted into a phospholipid bilayer in a gel phase. There is only one UDP binding site per holoenzyme when enzyme is reconstituted into a phospholipid bilayer in a liquid-crystal phase. Delipidated enzyme or enzyme reconstituted with lysophosphatidylcholine displays Michaelis-Menten kinetics. Binding studies show that these forms have only one binding site for UDP-glucuronic acid per holoenzyme. However, they have two nonidentical binding sites for binding of UDP. Thus, the physical properties of its phospholipid milieu influence the number of functional binding sites of UDP-glucuronyltransferase.
Animals↗
A comparison of the kinetic properties of two different forms of microsomal UDPglucuronyltransferase.
Two forms of UDPglucuronyltransferase (EC 2.4.1.17) have been purified from microsomes of pig liver. One form is free of phospholipids and the other contains a small amount of residual phospholipids. Each form, however, is responsive to activation on addition of purified phospholipids. Comparison of kinetic properties of these enzymes, after reconstitution with identical phospholipid environments, indicate that these are unique functional forms of UDPglucuronyltransferase. The two differ by as much as 100-fold in their rates of conjugation at Vm of p-nitrophenol. Relative rates of glucuronidation of a variety of phenolic aglycones are different for the two enzymes, which suggests different reaction mechanisms. The energetic basis for binding of UDP-glucuronic acid to the active sites is different for the two forms of UDPglucuronyltransferase. Moreover, one form, but not the other, binds Mn2+, which leads to modulation of kinetic properties.
Animals↗