Structural analysis of a set of proteins resulting from a bacterial genomics project.

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.

Folding, stability, and physical properties of the alpha subunit of bacterial luciferase.

Bacterial luciferase is a heterodimeric (alphabeta) enzyme composed of homologous subunits. When the Vibrio harveyi luxA gene is expressed in Escherichia coli, the alpha subunit accumulates to high levels. The alpha subunit has a well-defined near-UV circular dichroism spectrum and a higher intrinsic fluorescence than the heterodimer, demonstrating fluorescence quenching in the enzyme which is reduced in the free subunit [Sinclair, J. F., Waddle, J. J., Waddill, W. F., and Baldwin, T. O. (1993) Biochemistry 32, 5036-5044]. Analytical ultracentrifugation of the alpha subunit has revealed a reversible monomer to dimer equilibrium with a dissociation constant of 14.9 +/- 4.0 microM at 18 degrees C in 50 mM phosphate and 100 mM NaCl, pH 7.0. The alpha subunit unfolded and refolded reversibly in urea-containing buffers by a three-state mechanism. The first transition occurred over the range of 0-2 M urea with an associated free-energy change of 2.24 +/- 0.25 kcal/mol at 18 degrees C in 50 mM phosphate buffer, pH 7.0. The second, occurring between 2.5 and 3.5 M urea, comprised a cooperative transition with a free-energy change of 6.50 +/- 0.75 kcal/mol. The intermediate species, populated maximally at ca. 2 M urea, has defined near-UV circular dichroism spectral properties distinct from either the native or the denatured states. The intrinsic fluorescence of the intermediate suggested that, although the quantum yield had decreased, the tryptophanyl residues remained largely buried. The far-UV circular dichroism spectrum of the intermediate indicated that it had lost ca. 40% of its native secondary structure. N-Terminal sequencing of the products of limited proteolysis of the intermediate showed that the C-terminal region of the alpha subunit became protease labile over the urea concentration range at which the intermediate was maximally populated. These observations have led us to propose an unfolding model in which the first transition is the unfolding of a C-terminal subdomain and the second transition represents the unfolding of a more stable N-terminal subdomain. Comparison of the structural properties of the unfolding intermediate using spectroscopic probes and limited proteolysis of the alpha subunit with those of the alphabeta heterodimer suggested that the unfolding pathway of the alpha subunit is the same, whether it is in the form of the free subunit or in the heterodimer.

Techniques and complications of esophageal foreign body extraction in children and adults.

To assess the success rates and complications of esophageal foreign body extraction in adult and pediatric patients, the charts of 76 adults and 116 children with endoscopically or radiographically documented esophageal foreign body impaction were retrospectively reviewed. Success rates for rigid esophagoscopy (100%) and flexible endoscopy (96.2%) were not significantly different (p > 0.05). Overall, rigid esophagoscopy had a higher complication rate than flexible endoscopy (10% versus 5.1%, p > 0.05), but this trend did not reach statistical significance. The Foley catheter technique was used predominantly in children with proximally located blunt objects. Our study shows that flexible and rigid esophagoscopy are both safe and effective methods of removing esophageal foreign bodies, but rigid esophagoscopy carries a higher complication rate and therefore should be reserved for patients in whom flexible endoscopy is unsuccessful. The Foley catheter technique is suited only for proximally located blunt objects, and its routine use is not recommended.

Sterol carrier protein2-like activity in rat intestine.

A sterol carrier protein2 (SCP2)-like activity has been demonstrated in rat intestinal mucosal homogenates and in isolated intestinal cells from both crypt and villus zones. The results indicate the presence of a protein with similar molecular weight and antigenicity to that of authentic SCP2 purified from rat liver cytosol. Like liver SCP2, mucosal cytosol stimulates pregnenolone production in rat adrenal mitochondria and acyl coenzyme A:cholesterol acyltransferase activity of liver and mucosal microsomes. The distribution of SCP2-like activity as determined by radioimmunoassay indicates high levels in mitochondria and cytosol and relatively lower levels in microsomes and in brush-border membranes. The widespread distribution of SCP2-like protein in the intestine is consistent with potential transfer functions in all phases of cholesterol processing.

Primary sequence and structural analysis of sterol carrier protein 2 from rat liver: homology with immunoglobulins.

Sterol carrier protein 2 (SCP2) is involved in the later steps of cholesterol biosynthesis and in the intracellular transport of cholesterol. In the present investigation, the amino acid sequence of SCP2 from rat liver has been determined. It is a single polypeptide chain with 122 amino acid residues. Secondary structure prediction indicates an amphipathic alpha-helix region for residues 21-34 and antiparallel beta-sheet structure for residues 35-95. A major finding is the significant homology which exists over approximately 80 residues between SCP2 and the variable domains of the heavy chain of immunoglobulin G.

Sterol carrier protein2: further evidence for its role in adrenal steroidogenesis.

Homogeneous rat liver sterol carrier protein (SCP2) has been implicated in adrenal steroidogenesis by studies utilizing as a model system various sub-cellular fractions of rat adrenals. Levels of SCP2 were measured in rat adrenal subcellular fractions and various rat tissues using a highly sensitive radioimmunoassay. The levels of SCP2 in various tissues correlate well with the capacity of each tissue to either synthesize or metabolize cholesterol. The high level of SCP2 in adrenal mitochondria (46% of total tissue SCP2) is consistent with its proposed role of enhancing transfer of cholesterol from the outer to the inner mitochondrial membrane. Neither ACTH nor cycloheximide treatment of rats had a significant effect on SCP2 levels or distribution in the adrenal subcellular fractions. Western blot analysis of adrenal subcellular fractions indicates the presence of a protein of identical molecular weight and at least similar antigenicity as homogeneous rat liver SCP2. In the present studies, intact dispersed rat adrenal fasciculata cells fused with liposomal encapsulated anti-SCP2 IgG showed a 40-65% reduction in their ability to produce corticosterone when stimulated with ACTH. The steroidogenic competence of these anti-SCP2 IgG treated cells can be restored by treatment of the cells with liposomal encapsulated SCP2 prior to ACTH stimulation. These findings provide direct evidence for the involvement of SCP2 in ACTH stimulated steroidogenesis in rat adrenocortical cells, and suggests that SCP2 may not be the putative high turnover "labile protein" involved in acute steroidogenesis.

Sterol carrier and lipid transfer proteins.

The discovery of the sterol carrier and lipid transfer proteins was largely a result of the findings that cells contained cytosolic factors which were required either for the microsomal synthesis of cholesterol or which could accelerate the transfer or exchange of phospholipids between membrane preparations. There are two sterol carrier proteins present in rat liver cytosol. Sterol carrier protein 1 (SCP1) (Mr 47 000) participates in the microsomal conversion of squalene to lanosterol, and sterol carrier protein 2 (SCP2) (Mr 13 500) participates in the microsomal conversion of lanosterol to cholesterol. In addition SCP2 also markedly stimulates the esterification of cholesterol by rat liver microsomes, as well as the conversion of cholesterol to 7 alpha-hydroxycholesterol - the major regulatory step in bile acid formation. Also, SCP2 is required for the intracellular transfer of cholesterol from adrenal cytoplasmic lipid inclusion droplets to mitochondria for steroid hormone production, as well as cholesterol transfer from the outer to the inner mitochondrial membrane. SCP2 is identical to the non-specific phospholipid exchange protein. While SCP2 is capable of phospholipid exchange between artificial donors/acceptors, e.g. liposomes and microsomes, it does not enhance the release of lipids other than unesterified cholesterol from natural donors/acceptors, e.g. adrenal lipid inclusion droplets, and will not enhance exchange of labeled phosphatidylcholine between lipid droplets and mitochondria. Careful comparison of SCP2 and fatty acid binding protein (FABP) using six different assay procedures demonstrates separate and distinct physiological functions for each protein, with SCP2 participating in reactions involving sterols and FABP participating in reactions involving fatty acid binding and/or transport. Furthermore, there is no overlap in substrate specificities, i.e. FABP does not possess sterol carrier protein activity and SCP2 does not specifically bind or transport fatty acid. The results described in the present review support the concept that intracellular lipid transfer is a highly specific process, far more substrate-specific than suggested by the earlier studies conducted using liposomal techniques.

Regulation of bile-acid synthesis. Role of sterol carrier protein 2 in the biosynthesis of 7 alpha-hydroxycholesterol.

Sterol carrier protein2 (SCP2) is known to stimulate utilization of cholesterol in enzymic reactions in which cholesterol is the substrate. Substantial recent experimental evidence indicates that SCP2: activates enzymic conversion of intermediates between lanosterol and cholesterol; stimulates the microsomal conversion of cholesterol into cholesterol ester in rat liver; and enhances mitochondrial utilization of cholesterol for pregnenolone formation in the adrenals. The conversion of cholesterol into 7 alpha-hydroxycholesterol is the rate-limiting step in bile-acid synthesis. We therefore investigated the effect of SCP2 on this physiologically critical reaction by using a gas-chromatography-mass-spectrometry procedure that measures the mass of 7 alpha-hydroxycholesterol formed. The results show that SCP2 enhances 7 alpha-hydroxycholesterol formation by rat liver microsomes (microsomal fractions), utilizing either endogenous membrane cholesterol, cholesterol supplied exogenously in serum or in the form of cholesterol/phospholipid liposomes. Microsomes immunotitrated with anti-SCP2 antibody exhibited considerably less capacity to synthesize 7 alpha-hydroxycholesterol, which was restored to control levels on addition of purified SCP2. These data are consistent with the suggestion that SCP2 may be of physiological significance in the overall metabolism of cholesterol.

Sterol carrier protein 2 and fatty acid-binding protein. Separate and distinct physiological functions.

Sterol carrier protein 2 (SCP-2) participates in the microsomal conversion of lanosterol to cholesterol, in the conversion of cholesterol to cholesterol ester, and in intracellular cholesterol transfers. The stoichiometry of binding between cholesterol and SCP-2 is 1:1. However, reports have appeared attributing sterol carrier protein activity to a protein preparation identical to hepatic fatty acid-binding protein (FABP). Therefore, the present investigation was conducted to compare homogeneous preparations of FABP and SCP-2 with respect to their capacities to participate as carrier proteins in reactions involving sterols or fatty acids. The results show that SCP-2 and FABP have separate and distinct physiological functions, with SCP-2 participating in reactions involving sterols and FABP participating in reactions involving fatty acid binding and/or transport. Furthermore, there is no overlap in substrate specificities, i.e. FABP does not possess sterol carrier protein activity and SCP-2 does not specifically bind or transport fatty acid. As long as only small quantities of organic solvent (1.6 volume %) were used for substrate addition, the sterol delta 7-reductase liver microsomal assay for SCP-2 correlated well with the physiologically relevant assays employed in the reconstituted adrenal system. The sterol carrier protein activity previously attributed to rat hepatic FABP is explained by the presence of significant quantities of propylene glycol (15 volume %) or Tween 80 in the assay procedure.

Sterol carrier protein2 (SCP2)-mediated transfer of cholesterol to mitochondrial inner membranes.

Rats treated with cycloheximide accumulate cholesterol in the adrenal mitochondria. This cholesterol is largely associated with the mitochondrial outer membrane. The addition of homogeneous sterol carrier protein2 (SCP2) to these mitochondria stimulated pregnenolone production to a greater extent than with mitochondria from untreated rats. Adrenal mitochondria from cycloheximide-treated rats were incubated in the presence of aminoglutethimide and cycloheximide to prevent further utilization of cholesterol for pregnenolone synthesis. Outer and inner membrane fractions of these mitochondria incubated with and without 0.75 microM SCP2, were prepared following hypotonic disruption. Incubations of mitochondria in the presence of SCP2 caused a marked shift in cholesterol from outer to inner membrane fractions. The findings provide direct evidence for a role of SCP2 or a similar peptide in modulating transfer of cholesterol to the inner membrane site of cholesterol side chain cleavage.

Cholesterol ester hydrolase and sterol carrier proteins.

The cholesterol substrate required for sustained adrenal steroidogenesis is largely derived from the endogenous stores of cholesterol esters, which are located in large lipid inclusion droplets in the cytoplasm. In isolated adrenal cells, these esters are hydrolyzed during a variety of stimuli associated with cellular cAMP production. This largely appears to be a response to the action of a neutral cholesterol ester hydrolase, whose activity is modulated by phosphorylation of the enzyme protein, catalyzed by cAMP-dependent protein kinase. Transfer of the resulting unesterified cholesterol to mitochondria can be accomplished in a model system by sterol carrier protein2 (SCP2). This protein is distinct from fatty acid binding protein (FABP), has a Mr of 13,500 and is basic in nature. SCP2 can sequester cholesterol from lipid inclusion droplets in a stoichiometric relationship, and transfer this cholesterol to isolated adrenal mitochondria. SCP2 can also enhance the intermembrane transfer of mitochondrial cholesterol to cytochrome P 450scc, but does not directly affect cholesterol side chain cleavage. The stimulatory effect of adrenal cytosolic preparations on mitochondrial pregnenolone production can be completely abolished by pretreatment with anti SCP2 IgG.

Evidence for sterol carrier protein2-like activity in hepatic, adrenal and ovarian cytosol.

Purified sterol carrier protein2 (SCP2) from rat liver stimulated utilization of endogenous cholesterol for pregnenolone synthesis by adrenal mitochondria. Cytosolic preparations of rat liver, adrenal and luteinized ovary were also stimulatory in mitochondrial pregnenolone synthesis to different extents. Treatment of all preparations with rabbit anti-rat SCP2 IgG neutralized the stimulatory effects, and immunoprecipitated proteins gave similar patterns on SDS-gradient polyacrylamide gel electrophoresis. Treatment with rabbit pre-immune IgG had no effect on these parameters. Thus, proteins which are immunochemically compatible with hepatic SCP2 appear to be present in steroidogenic tissues and may play a role in control of mitochondrial cholesterol side chain cleavage activity.

Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization.

Addition of homogeneous rat liver sterol carrier protein2 (SCP2) or an adrenal cytosolic fraction enhanced pregnenolone production by adrenal mitochondria. Pretreatment of SCP2 or adrenal cytosol with anti-SCP2 IgG abolished the stimulatory effect of both preparations on mitochondrial pregnenolone output. Incubation of mitochondria with aminoglutethimide, which blocks interaction of cholesterol with inner membrane cytochrome P-450scc, resulted in decreased pregnenolone production and a decreased level of mitoplast cholesterol. Addition of SCP2 to the incubation media caused an almost 2-fold increase in cholesterol associated with the mitoplast, but did not enhance mitochondrial pregnenolone production. Studies with reconstituted cytochrome P-450scc in phospholipid vesicles also suggested that SCP2 did not affect interaction of cholesterol with the hemoprotein. Treatment of rats with cycloheximide alone or with adrenocorticotropic hormone resulted in a dramatic increase in mitochondrial cholesterol. However, these mitochondria did not exhibit increased levels of pregnenolone output under control incubation conditions. When SCP2 was included in the mitochondrial incubation media, pregnenolone production was significantly increased over that observed with adrenal mitochondria from untreated or adrenocorticotropic hormone-treated rats. The results imply that SCP2 enhances mitochondrial pregnenolone production by improving transfer of mitochondrial cholesterol to cytochrome P-450scc on the inner membrane, but does not directly influence the interaction of substrate with the hemoprotein.

Sterol carrier protein2. Delivery of cholesterol from adrenal lipid droplets to mitochondria for pregnenolone synthesis.

The ability of sterol carrier protein2 (SCP2) to mediate transfer of unesterified cholesterol from adrenal lipid inclusion droplets to mitochondria has been tested in an in vitro model system. Unlike mitochondrial utilization of cholesterol added in acetone or dimethyl sulfoxide, the unesterified cholesterol of lipid droplets did not provide a readily available source of substrate for mitochondrial pregnenolone production, without the addition of a transport mediator. Addition of SCP2, but not albumin, stimulated mitochondrial utilization of droplet cholesterol in a concentration-dependent manner. In the absence of mitochondria, SCP2 sequestered lipid droplet cholesterol, and in the presence of mitochondria, which were unable to convert cholesterol to pregnenolone, this cholesterol was quantitatively accumulated by mitochondria. Both processes were concentration-dependent and demonstrated a molar ratio of SCP2 and cholesterol for both binding and transport of 1. SCP2 also enhanced pregnenolone formation by mitochondria which were incubated in the absence of an extramitochondrial source of cholesterol. However, SCP2 had no effect on steroid release from a crude particulate fraction. These studies suggest that the effects of SCP2 are related to delivery of cholesterol from preformed stores to and into mitochondria for initiation of steroid hormone synthesis, and may represent an important modulator of sterol metabolism in adrenal cortical cells.

In vivo regulation of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase: immunotitration of the enzyme after short-term mevalonate or cholesterol feeding.

In recent studies using either a single dose of mevalonolactone administered by intragastric tube or a single meal containing 2% cholesterol, it was demonstrated that rat liver hydroxymethylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] (HMG-CoA reductase) the major regulatory enzyme in cholesterol biosynthesis, is subject to two phases of inhibition. The first phase of inhibition is explained by in vivo phosphorylation of the enzyme; however, the nature of the second phase of inhibition remained obscure. The present study tested two possible explanations for this second phase of inhibition--increased enzyme turnover leading to a decreased concentration of HMG-CoA reductase molecules, and further inactivation of existing enzyme molecules. The results with the technique of immunotitration of HMG-CoA reductase show that, in short-term studies conducted up to 2 hr after the administration of a single dose of mevalonolactone or up to 6 hr after a single meal of rat chow containing 2% cholesterol, the in vivo regulation of rat liver HMG-CoA reductase during the first half of the dark period does not occur by increased enzyme turnover but, instead, existing enzyme is further inactivated.

The participation of sterol carrier protein2 in the conversion of cholesterol to cholesterol ester by rat liver microsomes.

The purification of sterol carrier protein2 (SCP2), purified 1500-fold to homogeneity from the 303,000 x g supernatant (S303) of rat liver, has recently been described (Noland, B. J., Arebalo, R. E., Hansbury, E., and Scallen T. J. (1980) J. Biol. Chem, 255, 4282-4289). Since SCP2 is required for the synthesis of cholesterol by microsomal membranes, it was decided to test the hypothesis that SCP2 might also participate in enzymatic reactions which utilize cholesterol as a substrate. The reaction studied in the present investigation was the conversion of cholesterol to cholesterol ester (acyl-CoA cholesterol acyltransferase) by rat liver microsomes. The results show that when exogenously added [4-14C]cholesterol is the substrate, SCP2 produces a striking increase in cholesterol ester biosynthesis by rat liver microsomes. Although the effect of SCP2 was most clearly seen with exogenously added cholesterol, it was also demonstrated when [1-14C]oleoyl-CoA was the labeled substrate and the incorporation of labeled oleate into cholesterol ester was determined. Although it was demonstrated that microsomes could bind large amounts of cholesterol in the absence of SCP2, the bound cholesterol was ineffective as a substrate for microsomal acyl-CoA cholesterol acyltransferase. However, the microsomally bound cholesterol became an effective substrate for the enzyme upon the addition of SCP2. The results demonstrate that SCP2 participates in the utilization of cholesterol via the microsomal conversion of cholesterol to cholesterol ester. We also conclude that SCP2 may participate in the intracellular transport of cholesterol, in particular, the delivery of either exogenous (dietary) cholesterol or endogenous cholesterol to acyl-CoA cholesterol acyltransferase in the endoplasmic reticulum.

In vivo regulation of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase: enzyme phosphorylation as an early regulatory response after intragastric administration of mevalonolactone.

Although substantial evidence supports the conclusion that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] is the major regulatory enzyme in cholesterol biosynthesis, the molecular events involved in the in vivo regulation of this enzyme have remained obscure. To study this problem, rats were given a single 100-mg dose of mevalonolactone by intragastric tube. The rats were sacrificed 20 or 60 min later, and liver microsomes were prepared by ultracentrifugation. Two phases of inhibition of microsomal HMG-CoA reductase were observed. The first phase of inhibition, observed 20 min after mevalonolactone administration, was completely reversed by preincubation of the microsomes with purified phosphoprotein phosphatase. The second phase of inhibition, observed 60 min after mevalonolactone administration, was not reversed by phosphoprotein phosphatase. The reactivation of liver microsomal HMG-CoA reductase by phosphoprotein phosphatase was blocked by potassium fluoride or by phosphoprotein phosphatase inhibitor. Results obtained by immunotitration also showed that microsomal HMG-CoA reductase obtained from animals killed 20 min after mevalonolactone administration was significantly activated by phosphoprotein phosphatase treatment of the microsomes. These findings demonstrate that phosphorylation of rat liver HMG-CoA reductase is an early in vivo regulatory response after intragastric administration of mevalonolactone.