Functional lysosomal hydrolase size as determined by radiation inactivation analysis.

Electron inactivation analysis with 16 MeV electrons was used to determine the functional target size of a number of commonly studied lysosomal hydrolases. Observed values ranged from a low of 62 000 +/- 4000 Da for beta-galactosidase to a high of 200 000 +/- 17 500 Da (mouse beta-glucuronidase). One group of lysosomal hydrolases (N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, alpha-galactosidase, beta-mannosidase, beta-glucosidase, arylsulphatase A and sphingomyelinase) had target sizes in the range 100 000-120 000 Da, whereas alpha-glucosidase and alpha-fucosidase exist as complex multimers in the 150 000-160 000 Da range. Analysis of freeze-dried cell material showed little evidence of species (mouse versus human) variation in the functional size of most lysosomal hydrolases with the exception of beta-glucuronidase. Our findings suggest the potential usefulness of lysosomal hydrolases as endogenous marker enzymes in studies where the target size of proteins of unknown molecular mass is to be determined.     

Regulation of hepatic cholesterol ester hydrolase and acyl-coenzyme A:cholesterol acyltransferase in the rat

Cholesterol exists within the hepatocyte as free cholesterol and cholesteryl ester. The proportion of intrahepatic cholesterol in the free or ester forms is governed in part by the rate of cholesteryl ester formation by acyl-coenzyme A:cholesterol acyltransferase (ACAT) and cholesteryl ester hydrolysis by neutral cholesterol ester (CE) hydrolase. In other cell types both ACAT and CE hydrolase activities are regulated in response to changes in the need for cellular free cholesterol. In rats, we performed a variety of experimental manipulations in order to vary the need for hepatic free cholesterol and to examine what effect, if any, this had on the enzymes that govern cholesteryl ester metabolism. Administration of a 20-mg bolus of lipoprotein cholesterol or a diet supplemented with 2% cholesterol resulted in an increase in microsomal cholesteryl ester content with little change in microsomal free cholesterol. This was accomplished by an increase in cholesteryl esterification as measured by ACAT but no change in CE hydrolase activity. An increased need for hepatic free cholesterol was experimentally induced by intravenous bile salt infusion or cholestyramine (3%) added to the diet. ACAT activity was decreased with both experimental manipulations compared to controls, while CE hydrolase activity did not change. Microsomal cholesteryl ester content decreased significantly with little change in microsomal free cholesterol content. Addition of exogenous liposomal cholesterol to liver microsomes from cholestyramine-fed and control rats resulted in a 784 +/- 38% increase in ACAT activity. Nevertheless, the decrease in ACAT activity with cholestyramine feeding was maintained. These studies allowed us to conclude that changes in hepatic free cholesterol needs are met in part by regulation of the rate of cholesterol esterification by ACAT without a change in the rate of cholesteryl ester hydrolysis by CE hydrolase.     

The functional size of ferrochelatase determined in situ by radiation inactivation.

Ferrochelatase (EC 4.99.1.1) catalyzes the final step of heme biosynthesis, the insertion of iron(II) into protoporphyrin. It is an integral protein of the inner mitochondrial membrane. The functional size of bovine hepatic ferrochelatase has been studied in situ using radiation inactivation analysis. The functional unit required for enzymic activity in intact mitochondria was found to have a mass of 82 +/- 13 kDa. In contrast, the structural unit (evaluated in immunoblots following sodium dodecyl sulfate-polyacrylamide gel electrophoresis) has a mass of 40 +/- 10 kDa. Similar results were obtained when irradiation was performed on sodium cholate-solubilized mitochondria. The presence or absence of dithiothreitol during irradiation had no effect on target sizes obtained from either intact or solubilized mitochondria. Pairwise comparison of the functional and structural target sizes from each set of irradiated samples yielded a ratio of 2.0 +/- 0.4. Previous studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography have shown that a Mr 40,000 peptide is associated with ferrochelatase activity. This study shows that the functional size of bovine ferrochelatase is approximately 80 kDa; the data are most consistent with a model for active ferrochelatase composed of two structural subunits of about 40 kDa each.     

Lipoprotein lipase: size of the functional unit determined by radiation inactivation

Radiation inactivation was used to determine the functional molecular weight of lipoprotein lipase (LPL) in rat heart and adipose tissues. This technique reveals the size of the smallest unit required to carry out the enzyme function. Supernatant fractions of the tissue homogenates were exposed to high energy electrons at -135 degrees C. LPL activity showed a simple exponential decay in all samples tested. Because changes in nutritional state shift the distribution of LPL between the capillary endothelial and parenchymal cells within heart and adipose tissues, fasted and refed rats were used for the radiation studies. The functional molecular weight was calculated to be 127,000 +/- 15,000 (mean +/- SD) daltons for heart and adipose. Thus, the smallest unit required for enzyme function was the same in both of these tissues and did not vary with nutritional state. The data suggest that, compared with LPL monomer sizes reported in the range 55,000 to 72,000, this active unit constitutes a dimer.     

Functional size of acyl coenzyme A:diacylglycerol acyltransferase by radiation inactivation

Rat liver acyl coenzyme A:diacylglycerol acyltransferase, an intrinsic membrane activity associated with the endoplasmic reticulum, catalyzes the terminal and rate-limiting step in triglyceride synthesis. This enzyme has never been purified nor has its gene been isolated. Inactivation by ionizing radiation and target analysis were used to determine its functional size in situ. Monoexponential radiation inactivation curves were obtained which indicated that a single-sized unit of 72 +/- 4 kDa is required for expression of activity. The size corresponds only to the protein portion of the target and may represent one or several polypeptides.     

Molecular size of bovine lipoprotein lipase as determined by radiation inactivation

We have determined the size of the functional unit of bovine lipoprotein lipase by radiation inactivation. This was done in five different situations: 1) in a buffer with high salt concentration. In this situation the enzyme is relatively soluble and stable. 2) For an enzyme-heparin complex. This may reflect the physiological state of the enzyme at the vascular endothelium, where it is believed to be bound to a heparin-like molecule. 3) In the presence of lipid substrate and 4) with lipid substrate and activator protein. Here most of the enzyme is adsorbed to the substrate droplets. 5) For an enzyme-detergent complex; another model for enzyme-lipid interaction. In all five situations the enzyme activity decayed as an exponential function of radiation dose, and the target sizes were similar. The target size did not vary with the concentration of lipase protein. The combined data for bovine lipoprotein lipase yield a functional size of 72 kDa which is close to that expected for a dimer, 77 kDa.     

Functional molecular size of trypsin inhibitors as determined by radiation inactivation analysis

The molecular sizes of trypsin inhibitors obtained by radiation inactivation were investigated in relation to the functional domain size. The molecular weights obtained were 10,200 +/- 700 for ovomucoid, 17,800 +/- 400 for ovoinhibitor and 16,400 +/- 500 for soybean trypsin inhibitor. These values mostly agreed with the size of domain which has trypsin inhibitory activity, suggesting that the radiation inactivation analysis indicates the minimum functional domain size.     

Target size of neurotoxic esterase and acetylcholinesterase as determined by radiation inactivation.

The target size of neurotoxic esterase (NTE), the putative target site for the initiation of organophosphorus-compound-induced delayed neurotoxicity, and acetylcholinesterase (AChE) from hen brain were examined by determining the rate at which the activities of the esterases were destroyed by ionizing irradiation. Samples of hen brain were prepared by slowly drying a microsomal preparation under vacuum. The dried samples were then irradiated with electrons from a 1 MeV Van de Graaff generator. The doses ranged from 0 to 28 Mrad. The radiation doses were calibrated by the rate of inactivation of T1-bacteriophage plaque induction. Following the irradiation procedure, the samples were resuspended in buffer and enzymic activity was measured. The target size of NTE from hen brain was determined to be about 105 kDa, whereas hen brain AChE was found to have a target size of about 53 kDa. The target size of NTE was found to be similar in experiments with rat brain and cat brain. In addition, commercial preparations of electric-eel electric-organ AChE and horse serum butyrylcholinesterase were found to have target sizes that were identical with each other, and also were very similar to that of AChE from hen brain.     

Glibenclamide acts as an inhibitor of acyl-CoA:cholesterol acyltransferase enzyme

Sulfonylureas are used in the treatment of non-insulin-dependent diabetes mellitus. Little is known, however, about their effects on cholesterol metabolism. We tested in the present study the effects of glibenclamide (GB) on cholesterol esterification (CE) in macrophage-derived cells. GB inhibited intracellular accumulation of CE induced by acetylated LDL or oxidized LDL in J774 cells, but no such effect on total cholesterol, suggesting that the target of GB was acyl-CoA:cholesterol acyltransferase (ACAT). In the cell-free reconstitution ACAT assay, GB inhibited the ACAT activity with an IC(50) value of 20 microM. Furthermore, GB effectively inhibited the ACAT activity of PMA-stimulated THP-1 cells to the undifferentiated level of THP-1. In the whole-cell ACAT assay using CHO cells overexpressed with ACAT-1 or ACAT-2, GB inhibited the activity of both isozymes with similar potency. Our in vitro data suggest that sulfonylurea could be a potential seed for a new generation of ACAT inhibitors.     

Functional size of Photosystem II determined by radiation inactivation

The functional size of Photosystem II (PS II) was investigated by radiation inactivation. The technique provides an estimate of the functional mass required for a specific reaction and depends on irradiating samples with high energy -rays and assaying the remaining activity. The analysis is based on target theory that has been modified to take into account the temperature dependence of radiation inactivation of proteins. Using PS II enriched membranes isolated from spinach we determined the functional size of primary charge separation coupled to water oxidation and quinone reduction at the Q_B site: H₂O (Mn)₄ Y_z P680 Pheophytin Q phenyl-p-benzoquinone. Radiation inactivation analysis indicates a functional mass of 88 ± 12 kDa for electron transfer from water to phenyl-p-benzoquinone. It is likely that the reaction center heterodimer polypeptides, D1 and D2, contribute approximately 70 kDa to the functional mass, in which case polypeptides adding up to approximately 20 kDa remain to be identified. Likely candidates are the and subunits of cytochrome b ₅₅₉and the 4.5 kDa psbI gene product.Abbreviations Cyt cytochrome - PS Photosystem - P680 primary electron donor of Photosystem II - Q_A primary quinone acceptor of Photosystem II - Q_B secondary quinone acceptor of Photosystem II - Y_z tyrosine donor to P680     

Effect of albumin on acyl-CoA: lysolecithin acyltransferase,lysolecithin : lysolecithin acyltransferase and acyl-CoA hydrolase from rabbit lung

Acyl-CoA : lysolecithin and lysolecithin : lysolecithin acyltransferases, as well as acyl-CoA hydrolase are important enzymes in lung lipid metabolism. They use amphiphylic lipids as substrates and differ in subcellular localization. In this sense, lipid-protein interactions can be an essential factor in their activity. We have studied the effect of albumin, as lipid-binding protein model, in the activities of these enzymes. Acyl-CoA hydrolase was inhibited in the presence of albumin, whereas acyl-CoA : lysolecithin acyltransferase showed a complex effect of activation depending on both albumin concentration and palmitoyl-CoA/lysolecithin molar ratio. Lysolecithin : lysolecithin acyltransferase was affected differentially on its two activities. Hydrolysis remained unaffected and transacylation was inhibited by albumin. These results are consequence of the interaction of albumin with both lipidic substrates that changes their critical micellar concentration.Abbreviations TNS 6-(p-toluidino)-2-naphthalene-sulfonic acid - CMC Critical Micellar Concentration - LP Lysolecithin (1-acyl-sn-glycero-3-phosphocholine) - PalmCoA palmitoyl-CoA     

Molecular size of opiate (enkephalin) receptors in neuroblastoma-glioma hybrid cells as determined by radiation inactivation analysis

Opiate receptor binding decayed exponentially in mouse neuroblastoma-rat glioma (NG108-15) hybrid cell preparations following exposure to increasing doses of ionizing radiation (0.2 to 7.0 Mrads; 2.0 Mrads/min). Target size analysis revealed that [3H][D-Ala2, D-Leu5]enkephalin (agonist) and [3H]naloxone (antagonist) bound specifically to a component with an apparent molecular size of 200,000 +/- 20,000. Lyophilization of cells for the irradiation procedure did not significantly alter receptor affinity or binding capacity for these ligands. Furthermore, the loss of opiate receptor binding in irradiated cell samples could not be attributed to reduced receptor affinity since increasing concentrations of radiolabeled ligand failed to reverse the inhibition; nonspecific binding decreased only slightly under identical experimental conditions. The value of determining molecular size by radiation inactivation analysis was confirmed by showing that apparent target sizes for two representative lysosomal enzymes (beta-galactosidase and alpha-mannosidase) were consistent with results obtained previously using conventional methods. Thus, the data suggest that the ligand binding component of delta-opiate (enkephalin) receptors in NG108-15 cells has a minimum functional size of approximately 200,000.     

A 30 kDa functional size for the erythrocyte water channel determined in situ by radiation inactivation.

The functional unit size of the water channel in rabbit erythrocytes was assessed using target size analysis following radiation inactivation. Using Radiochromic nylon dosimetry, accurate values of accumulated dose yielded an absolute target analysis, leading to direct determination of molecular size. The erythrocyte water channel functional size was shown to be 30 kDa, and is identical to the size found in rat renal proximal tubule brush border membranes (1), suggesting close homology of these two water channels. The result suggests that the 28 kDa channel-like intrinsic protein (CHIP28) recently isolated from human erythrocytes and proximal tubule (2), which is believed to form water channels of oligomeric construction may have a functional water channel activity in monomeric form.     

The presence of acyl-CoA: cholesterol acyltransferase in Tetrahymena pyriformis W

1. The esterification of cholesterol was studied in Tetrahymena pyriformis an organism which does not synthesize sterols nor are sterols required for growth. 2. Microsomes catalyzed the esterification of cholesterol in the presence of oleoyl-CoA but not oleic acid or lecithin. 3. The enzyme has a similar sterol substrate specificity to that of mammalian acyl-CoA: cholesterol acyltransferase (ACAT) and was inhibited by the specific ACAT inhibitor 58-035. 4. The enzyme is constitutive since activity was observed in cells grown in sterol-free medium when cholesterol was added to the in vitro assay.     

The disulfide linkage and the free sulfhydryl accessibility of acyl-coenzyme A:cholesterol acyltransferase 1 as studied by using mPEG5000-maleimide

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a membrane protein located in the endoplasmic reticulum (ER). It plays important roles in cellular cholesterol homeostasis. Human ACAT1 (hACAT1) contains nine cysteines (C). To quantify and map its disulfide linkage, we performed thiol-specific modifications by mPEG(5000)-maleimide (PEG-mal) and iodoacetamide (IA) under denatured condition, using extracts that contain wild-type or various single C to A mutant hACAT1s. With the wild-type enzyme, seven Cs could be modified before dithiothreitol (DTT) treatment; nine Cs could be modified after DTT treatment. With the C528A or the C546A enzyme, all eight Cs could be modified before or after DTT treatment. With all other remaining single C to A mutant enzymes, six Cs could be modified before DTT treatment, and eight Cs could be modified after DTT treatment. We next performed Lys-C protease digestion on hACAT1 with a hemagglutinin (HA) tag at the C-terminus. The digests were treated with or without DTT and analyzed by SDS-PAGE and Western blotting. The two predicted C-terminal fragments (K496-K531 and N532-F550-HA tag) were trapped as a single peptide band, but only when the digests were treated without DTT. Thus, C528 and C546 near the enzyme's C-terminus form a disulfide. PEG-mal is impermeable to ER membranes. We used PEG-mal to map the localizations of the seven free sulfhydryls and the disulfide bond of hACAT1 present in microsomal vesicles. The results show that C92 is located on the cytoplasmic side of the ER membrane and the disulfide is located in the ER lumen, while all other free Cs are located within the hydrophobic region(s) of the enzyme.     

Chlamydia pneumoniae induces macrophage-derived foam cell formation by up-regulating acyl-coenzyme A: cholesterol acyltransferase 1

In macrophages, the accumulation of cholesteryl esters synthesized by acyl-coenzyme A: cholesterol acyltransferase 1(ACAT1) plays a crucial role in foam cell formation, a hallmark of early atherosclerotic lesions. It is suggested that Chlamydia pneumoniae (C. pneumoniae) induces foam cell formation. However, the mechanism of foam cell formation induced by C. pneumoniae has not been fully elucidated. In this study, we found that C. pneumoniae increased the expression of acyl-coenzyme A: cholesterol acyltransferase 1(ACAT1) mRNA and protein in a dose-dependent manner in THP-1-derived macrophages exposed to low density lipoprotein (LDL). In addition, C. pneumoniae dose-dependently suppressed the expression of peroxisome proliferator-activated receptor gamma (PPAR γ) mRNA and protein. Rosiglitazone, a specific PPAR γ agonist, not only dose-dependently alleviated the down-regulation of PPAR γ expression by C. pneumoniae infection, but also dose-dependently inhibited the C. pneumoniae-induced ACAT1 expression. Furthermore, higher doses of rosiglitazone (10 and 20 μM) suppressed the C. pneumoniae-induced foam cell formation from morphological (Oil red O staining) and biochemical (zymochemistry method) criteria. These results first demonstrate that C. pneumoniae induces macrophage-derived foam cell formation by up-regulating ACAT1 expression via PPAR γ-dependent pathway, which may contribute to its pro-atherogenic properties.     

Human Acyl-CoA：cholesterol Acyltransferase （ACAT） and its Potential as a Target for Pharmaceutical Intervention against Atherosclerosis

Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes the formation of cholesteryl estersfrom cholesterol and long-chain fatty-acyl-coenzyme A.At the single-cell level,ACAT serves as a regulatorof intracellular cholesterol homeostasis.In addition,ACAT supplies cholesteryl esters for lipoproteinassembly in the liver and small intestine.Under pathological conditions,the accumulation of cholesterylesters produced by ACAT in macrophages contributes to foam cell formation,a hallmark of the earlystage of atherosclerosis.Several reviews addressing various aspects of ACAT and ACAT inhibitors areavailable [1-8].This review briefly outlines the current knowledge on the biochemical properties of humanACATs,and then focuses on discussing the merit of ACAT as a drug target for pharmaceutical interventionsagainst atherosclerosis.     

Effects of high-density lipoprotein(2) on cholesterol transport and acyl-coenzyme A:cholesterol acyltransferase activity in P388D1 macrophages

High-density lipoproteins are the putative vehicles for cholesterol removal from monocyte-derived macrophages, which are an important cell type in all stages of atherosclerosis. The role of HDL(2), an HDL subclass that accounts for most variation in plasma HDL-cholesterol concentration, in cholesterol metabolism in monocyte-derived macrophages is not known. In this study, the dose-dependent effects of HDL(2) on cellular cholesterol mass, efflux, and esterification, and on cellular cholesteryl ester (CE) hydrolysis using the mouse macrophage P388D1 cell line was investigated. HDL(2) at low concentrations (40 microg protein/ml) decreased CE content without affecting cellular free cholesterol content (FC), CE hydrolysis, or cholesterol biosynthesis. In addition, HDL(2) at low concentrations reduced cellular acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and increased FC efflux from macrophages. Thus, HDL(2) has two potential roles in reverse cholesterol transport. In one, HDL(2) is an acceptor of macrophage FC. In the other, more novel role, HDL(2) increases the availability of macrophage FC through the inhibition of ACAT. Elucidation of the mechanism by which HDL(2) inhibits ACAT could identify new therapeutic targets that enhance the transfer of cholesterol from macrophages to the liver.     

Functional size of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase as determined by radiation inactivation

The functional molecular weight of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase was determined by radiation inactivation. Both isolated hepatic microsomes and primary hepatocytes were irradiated with high energy electrons at -135 degrees C, and the residual microsomal enzyme activity was subsequently determined. The loss of enzyme activity in both irradiated microsomes and microsomes isolated from irradiated hepatocytes followed a single exponential decay which corresponded to a molecular mass of 200 kDa. This minimal molecular size of the functional enzyme was unaffected by either addition of cholestyramine to the rat diet or addition of 25-hydroxycholesterol plus mevalonate to the isolated rat hepatocytes. In addition, surviving enzyme protein was determined by immunoprecipitation of radiolabeled enzyme from hepatocytes that had been incubated with [35S]methionine before irradiation. The target size for loss of the monomer subunits was 98 kDa. The simplest interpretation of these results is that rat liver 3-hydroxy-3-methylglutaryl-CoA reductase in situ is a noncovalently linked dimer of the Mr = 97,200 enzyme subunit.