Single session exercise stimulates formation of pre beta 1-HDL in leg muscle

Physical activity can raise the level of circulating HDL cholesterol. Pre beta 1-HDL is thought to be either the initial acceptor of cellular cholesterol or virtually the first particle in the pathway of the formation of HDL from apolipoprotein A-I and cellular lipids. We have therefore sought to identify pre beta 1-HDL in arterial and venous circulations of exercising legs in healthy individuals and in subjects with stable Type 2 diabetes mellitus. Blood samples were taken simultaneously from the femoral artery and vein before and after 25 min cycling exercise. The major findings were, first, that exercise significantly increased plasma concentration of pre beta 1-HDL (20% increase, P < 0.05) and second, that the pre beta 1-HDL concentration was significantly higher in the venous compared with the arterial blood both before and after exercise in both diabetics and controls. In the combined population, formation of pre beta 1-HDL at rest was 9.9 +/- 5.2 mg/min and exercise enhanced pre beta 1-HDL formation 6.6-fold in both groups.     

Analytical performance of a sandwich enzyme immunoassay for pre beta 1-HDL in stabilized plasma

We have established an immunoassay for pre beta 1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because pre beta 1-HDL is unstable during storage, fresh plasma must be used for pre beta 1-HDL measurements. In this study, we describe a method of stabilizing pre beta 1-HDL, and evaluate the analytical performance of the immunoassay for pre beta 1-HDL. Fresh plasma was stored under various conditions with or without a pretreatment consisting of a 21-fold dilution into 50% (v/v) sucrose. Pre beta 1-HDL concentration was measured by immunoassay. In nonpretreated samples, pre beta 1-HDL decreased significantly from the baseline after 6 h at room temperature. Although pre beta 1-HDL was more stable at 0 degrees C than at room temperature, it increased from 30.2 +/- 8.5 (SE) to 56.5 +/- 5.5 mg/l apolipoprotein A-I (apoA-I) (P < 0.001) in hyperlipidemics, and from 18.4 +/- 1.2 to 37.9 +/- 3.3 mg/l apoA-I (P < 0.001) in normolipidemics after 5-day storage. After 30-day storage at -80 degrees C, pre beta 1-HDL increased from 29.0 +/- 4.0 to 38.0 +/- 5.7 mg/l apoA-I (P < 0.001) in hyperlipidemics, whereas it did not change in normolipidemics. In pretreated samples, pre beta 1-HDL concentration did not change significantly under any of the above conditions. Moreover, pre beta 1-HDL concentrations determined by immunoassay correlated with those determined by native two-dimensional gel electrophoresis (n = 24, r = 0.833, P < 0.05). An immunoassay using MAb55201 with pretreated plasma is useful for clinical measurement of pre beta 1-HDL.     

Biochemical and molecular characterization of a lipase produced by Rhizopus oryzae

A novel strain of Rhizopus oryzae WPG secretes a noninduced lipase (ROLw) in the culture medium; purified ROLw is a protein of 29 kDa, the 45 N-terminal amino acid residues were sequenced, this sequence is very homologous to Rhizopus delemar lipase (RDL), Rhizopus niveus lipase (RNL) and R. oryzae lipase (ROL29) sequences; the cloning and sequencing of the part of the gene encoding the mature ROLw, shows two nucleotides differences with RDL, RNL and ROL29 sequences corresponding to the change of the residues 134 and 200; ROLw does not present the interfacial activation phenomenon when using tripropionin or vinyl propionate as substrates; the lipase activity is maximal at pH 8 and at 37 degrees C, specific activities of 3500 or 900 U mg(-1) were measured at 37 degrees C and at pH 8, using olive oil emulsion or tributyrin as substrates, respectively; ROLw is unable to hydrolyse triacylglycerols in the presence of high concentration of bile salts; it is a serine enzyme as it is inhibited by tetrahydrolipstatin and was stable between pH 5 and pH 8.     

Biochemical and physiological properties of an extracellular protease produced by Bacillus megaterium

A protease, excreted by a sporogeneous strain of B. megaterium, growing exponentially in a minimum glucose ammonium medium, was isolated. It is a neutral endopeptidase, stabilized by Ca⁺⁺, inhibited by o-phenanthroline, but not by di-isopropylfluorophosphate. The specificity, studied on insulin B-chain, glucagon, cytochrome c, and dipeptides substrates, indicated the need for a dipeptide backbone with both substituted amino and carboxyl groups. A requirement was observed for a nonpolar lateral chain in the amino acid whose amino group was involved in the peptide bond (Leu, Phe, Ala, He, Val). Rates of hydrolysis varied also with the amino acid whose carboxyl group was involved (e.g., His > Ser > Ala > Gly). In complex medium, supplemented with Yeast Extract, the biosynthesis of the protease was repressed during growth, but the same enzyme was excreted during sporulation. The repression was apparently of the same nature as that controlling sporulation during and after growth (e.g., repression by a mixture of amino acids or high concentration of glucose). An asporogeneous mutant showed a normal product ion of protease under all conditions, and a low intracellular protease turnover after growth. A mutant unable to produce protease showed a normal sporulation and a high protein turnover. This protease, here termed megapeptidase, seems to be a typical growth enzyme, not related to either the sporulation process or to the protein turnover after growth.     

Biochemical properties of the alkaline lipase of Bacillus flexus XJU-1 and its detergent compatibility

The possibility of using Bacillus flexus XJU-1 lipase in detergent preparations was studied. The enzyme was monomeric protein as confirmed by liquid chromatography-mass spectrometry and its molecular weight was 15.95 kDa. The lipase showed optimum activity at pH 10.0 and was 100% stable for 24 h at pH 10.0 and 11.0. It exhibited maximum activity at 70°C and retained more than 70% of the initial activity at 60, 70 and 80°C for 24 h. The activity was stimulated by Ca²⁺, Ba²⁺, Mg²⁺ and Co²⁺, whereas 50% of the initial activity was lost with Fe³⁺ and Hg²⁺. The activity was inhibited by 10 mM N-bromosuccinimide and tosyl-L-lysylchloromethylketone, while N-ethylmaleimide, phenylmethylsulphonylfluoride and urea did not show any effect. The enzyme significantly hydrolysed olive, cottonseed, sunflower, groundnut, and gingelly oils. With p-nitrophenyl palmitate, V_max and K_m were 62.5 U/mL and 2.25 mM, respectively. The lipase maintained its stability in Tween-80, Triton-100 and H₂O₂ at 1%, but an activation of 10% and a reduction of 15% in relative activity were observed with NaClO and sodium dodecyl sulphate, respectively. The enzyme retained maximum storage stability for 20 days at ?20, 4 and 30°C. In the presence of 0.7% (w/v) Ariel, Henko, Super wheel, Tide plus and Rin, a retention of more than 84.90% initial activity was recorded after 24 h at 60°C. The supplementation of the lipase to the detergents improved the olive oil stain removal. These properties suggested the present enzyme as a potential additive for detergent preparations.     

Hydrolytic properties of lipase produced by entomopathogenic fungusZoophthora (Erynia) ovispora

Summary Crude lipase produced by fungusZoophthora (Erynia) ovispora demonstrates activity high enough to be used as hydrolytic reagent in organic syntheses. Its effectiveness and enantioselectivity is comparable with those of commercial lipases.     

Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.

Chimeric molecules between human lipoprotein lipase (LPL) and rat hepatic lipase (HL) were used to identify structural elements responsible for functional differences. Based on the close sequence homology with pancreatic lipase, both LPL and HL are believed to have a two-domain structure composed of an amino-terminal (NH2-terminal) domain containing the catalytic Ser-His-Asp triad and a smaller carboxyl-terminal (COOH-terminal) domain. Experiments with chimeric lipases containing the HL NH2-terminal domain and the LPL COOH-terminal domain (HL/LPL) or the reverse chimera (LPL/HL) showed that the NH2-terminal domain is responsible for the catalytic efficiency (Vmax/Km) of these enzymes. Furthermore, it was demonstrated that the stimulation of LPL activity by apolipoprotein C-II and the inhibition of activity by 1 M NaCl originate in structural features within the NH2-terminal domain. HL and LPL bind to vascular endothelium, presumably by interaction with cell surface heparan sulfate proteoglycans. However, the two enzymes differ significantly in their heparin affinity. Experiments with the chimeric lipases indicated that heparin binding avidity was primarily associated with the COOH-terminal domain. Specifically, both HL and the LPL/HL chimera were eluted from immobilized heparin by 0.75 M NaCl, whereas 1.1 M NaCl was required to elute LPL and the HL/LPL chimera. Finally, HL is more active than LPL in the hydrolysis of phospholipid substrates. However, the ratio of phospholipase to neutral lipase activity in both chimeric lipases was enhanced by the presence of the heterologous COOH-terminal domain, demonstrating that this domain strongly influences substrate specificity. The NH2-terminal domain thus controls the kinetic parameters of these lipases, whereas the COOH-terminal domain modulates substrate specificity and heparin binding.     

Lipoprotein electrostatic properties regulate hepatic lipase association and activity.

The effect of lipoprotein electrostatic properties on the catalytic regulation of hepatic lipase (HL) was investigated. Enrichment of serum or very low density lipoprotein (VLDL) with oleic acid increased lipoprotein negative charge and stimulated lipid hydrolysis by HL. Similarly, enrichment of serum or isolated lipoproteins with the anionic phospholipids phosphatidylinositol (PI), phosphatidic acid, or phosphatidylserine also increased lipoprotein negative charge and stimulated hydrolysis by HL. Anionic lipids had a small effect on phospholipid hydrolysis, but significantly stimulated triacylglyceride (TG) hydrolysis. High density lipoprotein (HDL) charge appears to have a specific effect on lipolysis. Enrichment of HDL with PI significantly stimulated VLDL-TG hydrolysis by HL. To determine whether HDL charge affects the association of HL with HDL and VLDL, HL-lipoprotein interactions were probed immunochemically. Under normal circumstances, HL associates with HDL particles, and only small amounts bind to VLDL. PI enrichment of HDL blocked the binding of HL with HDL. These data indicate that increasing the negative charge of HDL stimulates VLDL-TG hydrolysis by reducing the association of HL with HDL. Therefore, HDL controls the hydrolysis of VLDL by affecting the interlipoprotein association of HL. Lipoprotein electrostatic properties regulate lipase association and are an important regulator of the binding and activity of lipolytic enzymes.     

A comparison of molecular properties of hepatic triglyceride lipase and lipoprotein lipase from human post-heparin plasma

Hepatic triglyceride lipase was isolated from human post-heparin plasma by the method of Ehnholm et al. using modifications which increased the specific activity 12-fold to approximately 3,000 mumol of free fatty acid/h/mg of protein. Lipoprotein lipase with similar specific activity was prepared from the same plasma samples using heparin and concanavalin A affinity chromatography. The molecular weight of hepatic triglyceride lipase (69,000) was slightly greater than that of lipoprotein lipase (67,000) as determined by polyacrylamide electrophoresis in sodium dodecyl sulfate-containing buffers. These proteins had identical amino acid compositions, terminal amino acid residues, and tryptic peptide maps. However, the differences previously described regarding optima of pH and ionic strength and the requirement for apolipoprotein CII (only for lipoprotein lipase) were maintained in the highly purified state. It was found that both proteins contain approximately 8% carbohydrate. Antisera prepared in goats selectively precipitated each activity. Other antisera prepared in chickens reacted with both enzymes, suggesting a common antigenic determinant.     

Some chemical and physical properties of extracellular polysaccharides produced by Butyrivibrio fibrisolvens strains

Most strains of Butyrivibrio fibrisolvens are known to produce extracellular polysaccharides (EPs). However, the rheological and functional properties of these EPs have not been determined. Initially, 26 strains of Butyrivibrio were screened for EP yield and apparent viscosities of cell-free supernatants. Yields ranged from less than 1.0 to 16.3 mg per 100 mg of glucose added to the culture. Viscosities ranged from 0.71 to 5.44 mPa.s. Five strains (CF2d, CF3, CF3a, CE51, and H10b) were chosen for further screening. The apparent viscosity of the EP from each of these strains decreased by only 50 to 60% when the shear rate was increased from 20 to 1,000 s-1. Strain CE51 produced the EP having the highest solution viscosity. A detailed comparison of shear dependency of the EP from strain CF3 with xanthan gum showed that this EP was less shear sensitive than xanthan gum and, at a shear rate of 1,000 s-1, more viscous. EPs from strains CF3 and H10b were soluble over a wide range of pH (1 to 13) in 80% (vol/vol) ethanol-water or in 1% (wt/vol) salt solutions. The pH of 1% EP solutions was between 4.5 and 5.5. Addition of acid increased solution viscosities, whereas addition of base decreased viscosity. EPs from strains CF3, CE51, and H10b displayed qualitatively similar infrared spectra. Calcium and sodium were the most abundant minerals in the three EPs. The amounts of magnesium, calcium, and iron varied considerably among the EPs, but the potassium contents remained relatively constant.     

Hydrolysis of chylomicron arachidonate and linoleate ester bonds by lipoprotein lipase and hepatic lipase

Chylomicrons labeled with [3H]arachidonic and [14C]linoleic acid were incubated with bovine milk lipoprotein lipase or rat postheparin plasma, containing both lipoprotein lipase and hepatic lipase. During incubation with bovine lipoprotein lipase, [3H]arachidonic acid was released from chylomicron triacylglycerols at a slower rate than [14C]linoleic acid. Only small amounts of [14C]linoleic acid were found as 1,2(2,3)-diacylglycerols, whereas a transient accumulation as [14C]monoacylglycerols was observed. In contrast, significantly more [3H]arachidonic acid was found as 1,2(2,3)-diacylglycerols than as monoacylglycerols at all time intervals investigated. The initial pattern of triacylglycerol hydrolysis by postheparin plasma was similar to that of bovine lipoprotein lipase. However, in contrast to the results obtained with bovine lipoprotein lipase, little [3H]1,2(2,3)-diacylglycerol accumulated. The addition of antiserum to hepatic lipase increased the amount of 3H found in 1,2(2,3)-diacylglycerols and inhibited the formation of free [3H]arachidonic acid. The antiserum also caused a significant inhibition of the hydrolysis of [3H]-but not of [14C]triacylglycerol. With regard to chylomicron phospholipids, the rate of hydrolysis of [14C]linoleoyl phosphatidylcholine with milk lipoprotein lipase was twofold higher than that of the [3H]arachidonyl phosphatidylcholine. However, the hepatic lipase of postheparin plasma had similar activity towards the two phosphatidylcholine species. Postheparin plasma rapidly hydrolyzed chylomicron 3H-labeled and 14C-labeled phosphatidylethanolamine to the same degree, and lipoprotein lipase similarly hydrolyzed 3H-labeled and 14C-labeled phosphatidylethanolamine at approximately equal rates. Antiserum to hepatic lipase inhibited the postheparin plasma hydrolysis of phosphatidylethanolamine and 3H-labeled phosphatidylcholine by about 60%, but the 14C-labeled phosphatidylcholine by only 27%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical, genetic, and zoosporicidal properties of cyclic lipopeptide surfactants produced by Pseudomonas fluorescens

Zoospores play an important role in the infection of plant and animal hosts by oomycetes and other zoosporic fungi. In this study, six fluorescent Pseudomonas isolates with zoosporicidal activities were obtained from the wheat rhizosphere. Zoospores of multiple oomycetes, including Pythium species, Albugo candida, and Phytophthora infestans, were rendered immotile within 30 s of exposure to cell suspensions or cell culture supernatants of the six isolates, and subsequent lysis occurred within 60 s. The representative strain SS101, identified as Pseudomonas fluorescens biovar II, reduced the surface tension of water from 73 to 30 mN m-1. The application of cell suspensions of strain SS101 to soil or hyacinth bulbs provided significant protection against root rot caused by Pythium intermedium. Five Tn5 mutants of strain SS101lacked the abilities to reduce the surface tension of water and to cause lysis of zoospores. Genetic characterization of two surfactant-deficient mutants showed that the transposons had integrated into condensation domains of peptide synthetases. A partially purified extract from strain SS101 reduced the surface tension of water to 30 mN m-1 and reached the critical micelle concentration at 25 micrograms ml-1. Reverse-phase high-performance liquid chromatography yielded eight different fractions, five of which had surface activity and caused lysis of zoospores. Mass spectrometry and nuclear magnetic resonance analyses allowed the identification of the main constituent as a cyclic lipopeptide (1,139 Da) containing nine amino acids and a 10-carbon hydroxy fatty acid. The other four zoosporicidal fractions were closely related to the main constituent, with molecular massesranging from 1,111 to 1,169 Da.     

Studies on the hyperglycemia and hepatic glycogenolysis produced by alpha and beta adrenergic agonists in the cat

Previous studies in this laboratory showed that both alpha and beta receptors can mediate adrenergically-induced hyperglycemia in the cat. In the present study, the results of experiments on the isolated perfused cat liver provide affirmation that hepatic glycogenolysis in this species can be subserved by both types of receptors. Thus, the acute hepatic release of glucose induced by isoproterenol was found to be antagonized by propranolol but not by phentolamine or phenoxybenzamine. The opposite was found for the glycogenolytic action of phenylephrine. Experiments $\text{in}$$\text{vivo}$ showed that the hyperglycemic response to the beta agonist was associated with activation of hepatic phosphorylase and increased intracellular cAMP content while the hyperglycemia induced by the alpha agonist was associated with an activation of phosphorylase which was independent of cAMP.     

Some chemical and physical properties of extracellular polysaccharides produced by Butyrivibrio fibrisolvens strains.

Most strains of Butyrivibrio fibrisolvens are known to produce extracellular polysaccharides (EPs). However, the rheological and functional properties of these EPs have not been determined. Initially, 26 strains of Butyrivibrio were screened for EP yield and apparent viscosities of cell-free supernatants. Yields ranged from less than 1.0 to 16.3 mg per 100 mg of glucose added to the culture. Viscosities ranged from 0.71 to 5.44 mPa.s. Five strains (CF2d, CF3, CF3a, CE51, and H10b) were chosen for further screening. The apparent viscosity of the EP from each of these strains decreased by only 50 to 60% when the shear rate was increased from 20 to 1,000 s-1. Strain CE51 produced the EP having the highest solution viscosity. A detailed comparison of shear dependency of the EP from strain CF3 with xanthan gum showed that this EP was less shear sensitive than xanthan gum and, at a shear rate of 1,000 s-1, more viscous. EPs from strains CF3 and H10b were soluble over a wide range of pH (1 to 13) in 80% (vol/vol) ethanol-water or in 1% (wt/vol) salt solutions. The pH of 1% EP solutions was between 4.5 and 5.5. Addition of acid increased solution viscosities, whereas addition of base decreased viscosity. EPs from strains CF3, CE51, and H10b displayed qualitatively similar infrared spectra. Calcium and sodium were the most abundant minerals in the three EPs. The amounts of magnesium, calcium, and iron varied considerably among the EPs, but the potassium contents remained relatively constant.     

Selective carboxymethylation of cysteine-174 of the beta 2 beta 2 and beta 1 beta 1 human liver alcohol dehydrogenase isoenzymes by iodoacetate

The beta 1 beta 1 and beta 2 beta 2 human liver alcohol dehydrogenase isoenzymes differ by only one residue at the coenzyme-binding site; Arg-47 in beta 1 is replaced by His in the beta 2 subunit. Since Arg-47 is thought to facilitate the carboxymethylation of Cys-46 in horse liver alcohol dehydrogenase by binding halo acids in a Michaelis-Menten complex prior to inactivation, the specificity and kinetics of modification of the two human liver beta beta isoenzymes with iodoacetate were compared. Both of the beta beta isoenzymes were inactivated by treatment with iodo[14C]acetate, and one Cys per subunit was carboxymethylated. Cys-174, which is a ligand to the active-site zinc atom in horse liver alcohol dehydrogenase, was selectively carboxymethylated in each of the human beta beta isoenzymes; less than 15% of the iodo[14C]acetate incorporated into the enzyme appeared in Cys-46. Therefore, the three-dimensional structure of the basic amino acids in the anion-binding site of the human beta beta isoenzymes appears to be different from that of horse liver alcohol dehydrogenase. The kinetics of alkylation are consistent with the formation of a Michaelis-Menten complex before inactivation of the isoenzymes. The average Ki values for iodoacetate were 10 and 16 mM for beta 1 beta 1 and beta 2 beta 2, respectively, and maximal rate constants for inactivation were 0.22 and 0.17 min-1, respectively. From these data, it can be concluded that there is a relatively minor effect of the substitution of His for Arg at position 47 on the kinetics of inactivation.     

Hydrolysis of lipid mixtures by rat hepatic lipase

The hydrolysis of phospholipid mixtures by purified rat hepatic lipase, also known as hepatic triglyceride lipase, was studied in a Triton X-100/lipid mixed micellar system. Column chromatography of the mixed micelles showed elution of Triton X-100 and binary lipid mixtures of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine as a single peak. This indicated that the mixed micelles were homogenous and contained all components in the designated molar ratios. The molar ratio of Triton X-100 to lipid was kept constant at 4 to 1. Labeling one lipid with 3H and the other lipid with 14C enabled us to determine the hydrolysis of both components of these binary lipid mixed micelles. We found that the hydrolysis of phosphatidylcholine was activated by the inclusion of small amounts of phosphatidic acid (2.5-fold), phosphatidylethanolamine (1.5-fold) or phosphatidylserine (1.4-fold). The maximal activation of phosphatidylcholine hydrolysis was observed when 5 mol% of phosphatidylethanolamine, 7.5 mol% phosphatidic acid or 5 mol% phosphatidylserine was added to Triton X-100 mixed micelles. The hydrolysis of phosphatidic acid was activated 30%, and that of phosphatidylserine was inhibited 30% when the molar proportion of phosphatidylcholine was less than 50 mol%. The hydrolysis of phosphatidylethanolamine was slightly activated when the mol% of phosphatidylcholine was below 5. The hydrolysis of phosphatidylserine was inhibited by phosphatidylethanolamine when the mol% of the latter was 50 or less whereas phosphatidylethanolamine hydrolysis was not affected by phosphatidylserine. Under the conditions used sphingomyelin and cholesterol did not have a significant effect on the hydrolysis of the phospholipids studied. In agreement with our previous study (Kucera et al. (1988) J. Biol. Chem. 263, 1920-1928) these studies show that the phospholipid polar head group is an important factor which influences the action of hepatic lipase and that the interfacial properties of the substrate play a role in the expression of the activity of this enzyme. The molar ratios of phosphatidic acid, phosphatidylethanolamine and phosphatidylserine which activated phosphatidylcholine hydrolysis correspond closely to the molar ratios of these lipids found in the surface lipid film of lipoproteins e.g., high density lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydrophobic properties of the beta 1 and beta 2 subunits of the rat brain sodium channel

Voltage-sensitive sodium channels purified from rat brain in functional form consist of a stoichiometric complex of three glycoprotein subunits, alpha of 260 kDa, beta 1 of 36 kDa, and beta 2 of 33 kDa. The alpha and beta 2 subunits are linked by disulfide bonds. The hydrophobic properties of these three subunits were examined by covalent labeling with the photoreactive hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) which labels transmembrane segments in integral membrane proteins. All three subunits of the sodium channel were labeled by [125I]TID when the purified protein was solubilized in mixed micelles of Triton X-100 and phosphatidylcholine (4:1). The half-time for photolabeling was approximately 7 min consistent with the half-time of 9 min for photolysis of TID under our conditions. Comparable amounts of TID per mg of protein were incorporated into each subunit. Purified sodium channels reconstituted in phosphatidylcholine vesicles were also labeled by TID with comparable incorporation per mg of protein into all three subunits. The efficiency of photolabeling of the three subunits was reduced from 39 to 44% by a 2-fold expansion of the hydrophobic phase of the reaction mixture but was unaffected by a 2-fold expansion of the aqueous phase, confirming that the photolabeling reaction took place in the lipid phase of the vesicle bilayer. The hydrophobic properties of the sodium channel subunits were examined further using phase separation in the nonionic detergent Triton X-114. Under conditions in which beta 1 is dissociated from alpha, the beta 1 subunit was preferentially extracted into the Triton X-114 phase, and the disulfide-linked alpha beta 2 complex was retained in the aqueous phase. When the disulfide bonds between the alpha and beta 2 subunits were reduced with dithioerythritol, the beta 2 subunit was also preferentially extracted into the Triton X-100 phase leaving the free alpha subunit in the aqueous phase. A preparative method for isolation of the beta 1 and beta 2 subunits was developed based on this technique. Considered together, the results of our hydrophobic labeling and phase separation experiments indicate that the alpha, beta 1, and beta 2 subunits all have substantial hydrophobic domains that may interact with the hydrocarbon phase of phospholipid bilayer membranes. Since the alpha subunit is known to be a transmembrane protein with many potential membrane-spanning segments, we conclude that the beta 1 and beta 2 subunits are likely to also be integral membrane proteins with one or more membrane-spanning segments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical characterization of CD1d expression in the absence of beta2-microglobulin

CD1d is a major histocompatibility complex class I-like molecule that exhibits a distinct antigen processing pathway that functions in the presentation of hydrophobic antigens to T cells. CD1d has been previously shown to be expressed on the cell surface of human intestinal epithelial cell lines in vivo and a transfected cell line in vitro independently of beta2-microglobulin (beta2m). To define the relationship between CD1d and beta2m and characterize the biochemical structure of CD1d in the absence of beta2m, we have used a newly generated series of CD1d transfectants and CD1d-specific antibodies. These studies show that in the absence of beta2m, CD1d is expressed on the cell surface as a 45-kDa glycoprotein that is sensitive to endoglycosidase-H and is reduced to 37-kDa after N-glycanase digestion. In contrast, in the presence of beta2m, CD1d is expressed on the cell surface as a 48-kDa endoglycosidase-H-resistant glycoprotein. Pulse-chase metabolic labeling studies demonstrate that acquisition of endoglycosidase-H resistance of CD1d is observed in the presence of beta2m but not in the absence of beta2m even after a 24-h chase period. Thus, CD1d is able to be transported to the cell surface independently of beta2m; however, in the absence of beta2m, the glycosylation pattern of CD1d is altered and consistent with an immature glycoprotein.