Inhibition of human squalene monooxygenase by tellurium compounds: evidence of interaction with vicinal sulfhydryls

Squalene monooxygenase is a flavin adenine dinucleotide-containing, microsomal enzyme that catalyzes the second step in the committed pathway for cholesterol biosynthesis. Feeding weanling rats a diet containing 1% elemental tellurium causes a transient, peripheral demyelination due to the disruption of cholesterol synthesis in Schwann cells secondary to inhibition of squalene monooxygenase. The tellurium species responsible for the inhibition is unknown, as is the mechanism of inhibition. To study the potential mechanisms of tellurium toxicity in humans, three likely in vivo metabolites of tellurium (tellurite, dimethyltellurium dichloride, and dimethyltelluride) were tested as inhibitors of purified human squalene monooxygenase. All three inhibitors reacted with the enzyme slowly and the resulting interaction was not freely reversible. The 50% inhibitory concentration for the methyltellurium compounds (approximately 100 nM) after a 30-min preincubation was 100-fold lower than that of tellurite, indicating a role for hydrophobicity in the enzyme-inhibitor interaction. The ability of glutathione and 2,3-dimercaptopropanol to prevent and reverse the inhibition indicated that the tellurium compounds were reacting with sulfhydryls on squalene monooxygenase, and the ability of phenylarsine oxide, which reacts specifically with vicinal sulfhydryls, to inhibit the enzyme indicated that these sulfhydryls are located proximal to one another on the enzyme. These results suggest that the unusual sensitivity of squalene monooxygenase to tellurium compounds is due to the binding of these compounds to vicinal cysteines, and that methylation of tellurium in vivo may enhance the toxicity of tellurium for this enzyme.     

Inhibition of human squalene monooxygenase by selenium compounds

Selenosis in animals is characterized by a variety of neurological abnormalities, but the chemical species of selenium and the molecular targets that mediate this neurotoxicity are unknown. We have previously shown that selenite is a potent inhibitor of squalene monooxygenase, the second enzyme in the committed pathway for cholesterol biosynthesis; inhibition of this enzyme by dimethyltellurium leads to a peripheral demyelinating neuropathy similar to that seen in selenosis. To evaluate the role methylation plays in selenium toxicity, we examined the ability of three methylselenium compounds, methylselenol, dimethylselenide, and trimethylselenonium iodide, to inhibit purified recombinant human squalene monooxygenase. IC(50) values for methylselenol (95 microM) and dimethylselenide (680 microM) were greater than that previously obtained for selenite (37 microM), and inhibition by trimethylselenonium iodide was evident only at concentrations above 3 mM. Inhibition by methylselenol as well as by selenite was slow and irreversible, suggestive of covalent binding to the enzyme, and thiol-containing compounds could prevent and reverse this inhibition, indicating that these compounds were reacting with sulfhydryl groups on the protein. Monothiols such as glutathione and beta-mercaptoethanol provided better protection than did dithiols, suggesting that these selenium compounds bind to only one of the two proposed vicinal cysteines on squalene monooxygenase. Unexpectedly, the inhibition by selenite was significantly enhanced by dithiols, indicating that a more toxic species, possibly selenide, was formed in the presence of these dithiol reductants.     

Structure and properties of the recombinant NADH-cytochrome b5 reductase of Physarum polycephalum

A cDNA for NADH-cytochrome b(5) reductase of Physarum polycephalum was cloned from a cDNA library, and the nucleotide sequence of the cDNA was determined (accession no. AB259870). The DNA of 943 base pairs contains 5'- and 3'-noncoding sequences, including a polyadenylation sequence, and a coding sequence of 843 base pairs. The amino acid sequence (281 residues) deduced from the nucleotide sequence was 25 residues shorter than those of vertebrate enzymes. Nevertheless, the recombinant Physarum enzyme showed enzyme activity comparable to that of the human enzyme. The recombinant Physarum enzyme showed a pH optimum of around 6.0, and apparent K(m) values of 2 microM and 14 microM for NADH and cytochrome b(5) respectively. The purified recombinant enzyme showed a typical FAD-derived absorption peak of cytochrome b(5) reductase at around 460 nm, with a shoulder at 480 nm. These results suggest that the Physarum enzyme plays an important role in the organism.     

Human pancreas protein 2 (PAN2) has a retinal reductase activity and is ubiquitously expressed in human tissues

Human gene for pancreas protein 2 (PAN2) is a novel member of the short-chain dehydrogenase/reductase gene superfamily. The properties of PAN2 protein have not yet been characterized. We present the first evidence that human PAN2 is a ubiquitously expressed microsomal enzyme that recognizes retinoids but not steroids as substrates with the apparent K(m) values between 0.08 microM and 0.4 microM. PAN2 is approximately 4-fold more efficient in the reductive than in the oxidative direction. The apparent K(m) values for NADP(+) and NADPH are 0.65 microM and 0.32 microM versus 1200 microM and 1060 microM for NAD(+) and NADH, respectively. Kinetic constants and expression pattern of PAN2 suggest that it is likely to function as a reductase in vivo and might contribute to the reduction of retinaldehyde to retinol in most human tissues.     

Tellurite Specifically Affects Squalene Epoxidase: Investigations Examining the Mechanism of Tellurium-Induced Neuropathy

Abstract: A peripheral neuropathy characterized by a transient demyelinating/remyelinating sequence results when young rats are fed a tellurium-containing diet. The neuropathy occurs secondary to a systemic block in cholesterol synthesis. Squalene accumulation suggested the lesion was at the level of squalene epoxidase, a microsomal monooxygenase that uses NADPH cytochrome P450 reductase to receive its necessary reducing equivalents from NADPH. We have now demonstrated directly specificity for squalene epoxidase; our in vitro studies show that squalene epoxidase is inhibited 50% in the presence of 5 µ M tellurite, the presumptive in vivo active metabolite. Under these conditions, the activities of other monooxygenases, aniline hydroxylase and benzo( a )pyrene hydroxylase, were inhibited less than 5%. We also present data suggesting that tellurite inhibits squalene epoxidation by interacting with highly susceptible -SH groups present on this monooxygenase. In vivo studies of specificity were based on the compensatory response to feeding of tellurium. Following tellurium intoxication, there was up-regulation of squalene epoxidase activity both in liver (11-fold) and sciatic nerve (fivefold). This induction was a specific response, as demonstrated in liver by the lack of up-regulation following exposure to the nonspecific microsomal enzyme inducer, phenobarbital. As a control, we also measured the microsomal monooxygenase activities of aniline hydroxylase and benzo( a )pyrene hydroxylase. Although they were induced following phenobarbital exposure, activities of these monooxygenases were not affected following tellurium intoxication, providing further evidence of specificity of tellurium intoxication for squalene epoxidase.     

Conversion of 4-hydroxyacetophenone into 4-phenyl acetate by a flavin adenine dinucleotide-containing Baeyer-Villiger-type monooxygenase

An arylketone monooxygenase was purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baeyer-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetophenone, into 4-hydroxyphenyl acetate with the consumption of one molecule of oxygen and oxidation of one molecule of NADPH per molecule of substrate. The enzyme was a monomer with an M(r) of about 70,000 and contained one molecule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADPH as the electron donor, and spectral studies showed rapid reduction of the FAD by NADPH but not by NADH. Other arylketones were substrates, including acetophenone and 4-hydroxypropiophenone, which were converted into phenyl acetate and 4-hydroxyphenyl propionate, respectively. The enzyme displayed Michaelis-Menten kinetics with apparent K(m) values of 47 microM for 4-hydroxyacetophenone, 384 microM for acetophenone, and 23 microM for 4-hydroxypropiophenone. The apparent K(m) value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 microM. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the proposed NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone monooxygenase from an Acinetobacter sp.     

Supernatant protein factor stimulates HMG-CoA reductase in cell culture and in vitro

Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase in vitro and, unexpectedly, cholesterol synthesis in cell culture. Because squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, we investigated the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway. Substitution of [(14)C]mevalonate for [(14)C]acetate in McARH7777 hepatoma cells expressing SPF reduced the 1.8-fold increase in cholesterol synthesis by half, suggesting that SPF acted on or prior to mevalonate synthesis. This conclusion was supported by the finding that substitution with [(14)C]mevalonate completely blocked an SPF-induced increase in squalene synthesis. Evaluation of 2,3-oxidosqualene synthesis from [(14)C]mevalonate demonstrated that SPF also stimulated squalene monooxygenase (1.3-fold) in hepatoma cells. Immunoblot analysis showed that SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels, indicating a direct effect on enzyme activity. Addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold, and the SPF-concentration/activation curve paralleled that for the SPF-mediated stimulation of squalene monooxygenase. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.     

Purification and partial characterization of squalene epoxidase from rat liver microsomes

Squalene epoxidase (EC 1.14.99.7, squalene 2,3-monooxygenase (epoxidizing) was purified to an apparent homogeneity from rat liver microsomes. The purification was carried out by solubilization of microsomes by Triton X-100, fractionation with ion exchangers, hydroxyapatite, Cibacron Blue Sepharose 4B, and chromatofocusing column chromatography. A total purification of 143-fold over the first DEAE-cellulose fraction was achieved. The purified enzyme gave a single major band on SDS-polyacrylamide gel electrophoresis and the Mr was estimated to be 51 000 as a single polypeptide chain. The enzyme showed no distinct absorption spectrum in the visible regions. The squalene epoxidase activity was reconstituted with the purified enzyme, NADPH-cytochrome P-450 reductase (EC 1.6.2.4), FAD, NADPH and molecular oxygen in the presence of Triton X-100. The apparent Michaelis constants for squalene and FAD were 13 microM and 5 microM, respectively. The Vmax was about 186 nmol per mg protein per 30 min for 2,3-oxidosqualene. The enzyme activity was not inhibited by potent inhibitors of cytochrome P-450. It is suggested that squalene epoxidase is distinct from cytochrome P-450 isozymes.     

Purification and characterization of rat sterol 14-demethylase P450 (CYP51) expressed in Escherichia coli.

Sterol 14-demethylase P450 (CYP51) is an essential enzyme for sterol biosynthesis by eukaryotes. We have cloned rat and human CYP51 cDNAs [Aoyama, Y., Noshiro, M., Gotoh, O., Imaoka, S., Funae, Y., Kurosawa, N., Horiuchi, T., and Yoshida, Y. (1996) J. Biochem. 119, 926-933]. The cloned rat CYP51 cDNA was expressed in Escherichia coli with modification of the N-terminal amino acid sequence, and the expressed protein (CYP51m) was purified to gel-electrophoretic homogenity. The spectrophotometrically determined specific content of CYP51m was 16 nmol/mg protein and the apparent molecular weight was estimated to be 53,000 on SDS-PAGE. Soret peaks of the oxidized and reduced CO-complex of CYP51m were observed at 417 and 447 nm, respectively. The purified CYP51m catalyzed the 14-demethylation of lanosterol and 24,25-dihydrolanosterol upon reconstitution with NADPH-P450 reductase purified from rat liver microsomes. The apparent K(m) and V(max) values for lanosterol were 10.5 microM and 13.9 nmol/min/nmol P450, respectively, and those for 24, 25-dihydrolanosterol were 20.0 microM and 20.0 nmol/min/nmol P450, respectively. The lanosterol demethylase activity of the reconstituted system of CYP51m was inhibited by ketoconazole, itraconazole and fluconazole with apparent IC(50) values of 0.2, 0.7, and 160 microM, respectively.     

cDNA cloning, overproduction and characterization of rat adrenodoxin reductase.

We isolated a full-length cDNA clone for rat adrenodoxin reductase (AdR). The precursor of rat AdR was predicted to consist of 34 amino-terminal residues of extrapeptide for transport into mitochondria and the following 460 residues of the mature peptide region. The deduced amino acid sequence was 70.8 and 61.8% homologous to those of bovine and human AdRs in the extrapeptide region, respectively, and 88.5% homologous to both the sequences of bovine and human AdRs in the mature peptide region. The predicted mature form of rat AdR was directly expressed in Escherichia coli, using cDNA, and was purified with a yield of 32 mg/l of culture. The purified recombinant rat AdR showed an absorption spectrum characteristic of a flavoprotein with peaks at 270, 378 and 450 nm and shoulders at 280, 425 and 474 nm. The extinction coefficient was estimated to be 10.9 mM(-1) cm(-1) at 450 nm. The absorbance ratio at 270 nm/450 nm was 7.1. From the θ(208) value in the circular dichroism spectrum, the alpha-helix content in the rat AdR was calculated to be 30%. In NADPH-cytochrome c reductase activity reconstituted with adrenodoxin (Ad), the apparent K(m) value of rat AdR for NADPH was 0.32 microM, a value significantly lower than that of bovine AdR (1.4 microM). The rat AdR showed a higher affinity to the heterologous redox partner (bovine Ad, K(m)=9.3 nM) than to the native partner (rat Ad, K(m)=16.7 nM), whereas the affinity of bovine AdR was slightly higher to the native partner (bovine Ad, K(m)=37.1 nM) than to the heterologous partner (rat Ad, K(m)=46.8 nM). The K(m) values showed a reverse correlation to the difference of pI values between the redox partners. These results indicate that AdR binds to Ad mainly by ionic interaction.     

Kinetic and mechanistic properties of biotin sulfoxide reductase

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia coli, have demonstrated that the purified protein utilizes NADPH as a facile electron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. Biol. Chem. 272, 3355-3362] that, at pH 8 and in the presence of saturating concentrations of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app) = 500 s(-1), K(m,app) = 269 microM, and k(cat,app)/K(m,app) = 1.86 x 10(6) M(-1) s(-1)) compared to NADH (k(cat,app) = 47 s(-1), K(m,app) = 394 microM, and k(cat,app)/K(m,app) = 1.19 x 10(5) M(-1) s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzyme also utilized ferricyanide as an artificial electron acceptor, which effectively suppressed biotin sulfoxide reduction and biotin formation. Analysis of the enzyme isolated from tungsten-grown cells yielded decreased reduced methyl viologen:BSO reductase, NADPH:BSO reductase, and NADPH:FR activities, confirming that Mo is required for all activities. Kinetic analyses of substrate inhibition profiles revealed that the enzyme followed a Ping Pong Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive substrate inhibition. Replots of the 1/v-axes intercepts of the parallel asymptotes obtained at several low concentrations of fixed substrate yielded a K(m) for BSO of 714 and 65 microM for NADPH. In contrast, utilizing NADH as an electron donor, the replots yielded a K(m) for BSO of 132 microM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yielded a K(i) for BSO of 6.10 mM and 900 microM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibited NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpyrocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histidine residues are involved in cofactor-binding.     

Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver

Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme, respectively.     

Biochemical characteristics of purified beef liver NADPH-cytochrome P450 reductase

NADPH-cytochrome P450 reductase, an obligatory component of the cytochrome P450 dependent monooxygenase system, was purified to electrophoretic homogeneity from beef liver microsomes. The purification procedure involved the ion exchange chromatography of the detergent-solubilized microsomes on first and second DEAE-cellulose columns, followed by 2',5'-ADP Sepharose affinity chromatography. Further concentration of the enzyme and removal of Emulgen 913 and 2'-AMP were accomplished on the final hydroxylapatite column. The enzyme was purified 239-fold and the yield was 13.5%. Monomer molecular weight of the enzyme was estimated to be 76000 +/- 3000 (N = 5) by SDS-PAGE. The absolute absorption spectrum of beef reductase showed two peaks at 455 and 378 nm, with a shoulder at 478 nm, characteristics of flavoproteins. The effects of cytochrome c concentration, pH, and ionic strength on enzyme activity were studied. Reduction of cytochrome c with the enzyme followed Michaelis-Menten kinetics, and the apparent K(m) of the purified enzyme was found to be 47.7 microM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer (pH 7.7). Stability of cytochrome c reductase activity was examined at 25 and 37 degrees C in the presence and absence of 20% glycerol. The presence of glycerol enhanced the stability of cytochrome c reductase activity at both temperatures. Sheep lung microsomal cytochrome P4502B and NADPH-cytochrome P450 reductase were also purified by the already existing methods developed in our laboratory. Both beef liver and sheep lung reductases were found to be effective in supporting benzphetamine and cocaine N-demethylation reactions in the reconstituted systems containing purified sheep lung cytochrome P4502B and synthetic lipid, phosphatidylcholine dilauroyl.     

Purification, characterization, and overexpression of flavin reductase involved in dibenzothiophene desulfurization by Rhodococcus erythropolis D-1

The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the K(m) values for NADH and FMN were 208 and 10.8 microM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35 degrees C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80 degrees C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705-1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.     

Expression, purification, and characterization of His-tagged human mitochondrial 2,4-dienoyl-CoA reductase

Mitochondrial 2,4-dienoyl-CoA reductase is a key enzyme for the beta-oxidation of unsaturated fatty acids. The cDNA of the full-length human mitochondrial 2,4-dienoyl-CoA reductase was previously cloned as pUC18::DECR. PCR methodologies were used to subclone the genes encoding various truncated human mitochondrial 2,4-dienoyl-CoA reductases from pUC18::DECR with primers that were designed to add six continuous histidine codons to the 3' or 5' primer. The PCR products were inserted into pLM1 expression vectors and overexpressed in Escherichia coli. A highly active truncated soluble protein was expressed and purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity based on Coomassie blue-stained SDS-PAGE. The molecular weight of the protein subunit was 34 kDa. The purified protein is highly stable at room temperature, which makes it potentially valuable for protein crystallization. KM of 26.5 +/- 3.8 microM for 2,4-hexadienoyl-CoA, KM of 6.22 +/- 2.0 microM for 2,4-decadienoyl-CoA, and KM of 60.5 +/- 19.7 microM for NADPH, as well as Vmax of 7.78 +/- 1.08 micromol/min/mg for 2,4-hexadienoyl-CoA and Vmax of 0.74 +/- 0.07 micromol/min/mg for 2,4-decadienoyl-CoA were determined on kinetic study of the purified protein. The one-step purification of the highly active human mitochondrial 2,4-dienoyl-CoA reductase will greatly facilitate further investigation of this enzyme through site-directed mutagenesis and enzyme catalyzed reactions with substrate analogs as well as protein crystallization for solving its three-dimensional structure.     

Hepatic cytochrome P450 reductase-null mice reveal a second microsomal reductase for squalene monooxygenase

Squalene monooxygenase is a microsomal enzyme that catalyzes the conversion of squalene to 2,3(s)-oxidosqualene, the immediate precursor to lanosterol in the cholesterol biosynthesis pathway. Unlike other flavoprotein monooxygenases that obtain electrons directly from NAD(P)H, squalene monooxygenase requires a redox partner, and for many years it has been assumed that NADPH-cytochrome P450 reductase is this requisite redox partner. However, our studies with hepatic cytochrome P450-reductase-null mice have revealed a second microsomal reductase for squalene monooxygenase. Inhibition studies with antibody to P450 reductase indicate that this second reductase supports up to 40% of the monooxygenase activity that is obtained with microsomes from normal mice. Studies carried out with hepatocytes from CPR-null mice demonstrate that this second reductase is active in whole cells and leads to the accumulation of 24-dihydrolanosterol; this lanosterol metabolite also accumulates in the livers of CPR-null mice, indicating that cholesterol synthesis is blocked at lanosterol demethylase, a cytochrome P450.     

Purification and characterization of recombinant human alpha-N-acetylglucosaminidase secreted by Chinese hamster ovary cells

alpha-N-Acetylglucosaminidase (EC 3.2.1.50) is a lysosomal enzyme that is deficient in the genetic disorder Sanfilippo syndrome type B. To study the human enzyme, we expressed its cDNA in Lec1 mutant Chinese hamster ovary (CHO) cells, which do not synthesize complex oligosaccharides. The enzyme was purified to apparent homogeneity from culture medium by chromatography on concanavalin A-Sepharose, Poros 20-heparin, and aminooctyl-agarose. The purified enzyme migrated as a single band of 83 kDa on SDS-PAGE and as two peaks corresponding to monomeric and dimeric forms on Sephacryl-300. It had an apparent K(m) of 0.22 mM toward 4-methylumbelliferyl-alpha-N-acetylglucosaminide and was competitively inhibited by two potential transition analogs, 2-acetamido-1,2-dideoxynojirimycin (K(i) = 0.45 microM) and 6-acetamido-6-deoxycastanospermine (K(i) = 0.087 microM). Activity was also inhibited by mercurials but not by N-ethylmaleimide or iodoacetamide, suggesting the presence of essential sulfhydryl residues that are buried. The purified enzyme preparation corrected the abnormal [(35)S]glycosaminoglycan catabolism of Sanfilippo B fibroblasts in a mannose 6-phosphate-inhibitable manner, but its effectiveness was surprisingly low. Metabolic labeling experiments showed that the recombinant alpha-N-acetylglucosaminidase secreted by CHO cells had only a trace of mannose 6-phosphate, probably derived from contaminating endogenous CHO enzyme. This contrasts with the presence of mannose 6-phosphate on naturally occurring alpha-N-acetylglucosaminidase secreted by diploid human fibroblasts and on recombinant human alpha-l-iduronidase secreted by the same CHO cells. Thus contrary to current belief, overexpressing CHO cells do not necessarily secrete recombinant lysosomal enzyme with the mannose 6-phosphate-targeting signal; this finding has implications for the preparation of such enzymes for therapeutic purposes.     

Assay optimization and kinetic profile of the human and the rabbit isoforms of 11beta-HSD1

Assay conditions for the 11beta-hydroxysteroid dehydrogenase have been optimized by adding phospholipids in the media buffer to increase and stabilize the enzymatic activity. The presence of phospholipids greatly facilitates the study of the binding of cortisone and NADPH at the enzyme catalytic site. Kinetic analyses conducted with the human and rabbit enzyme isoforms suggest that both enzymes behave according to an ordered sequential bi-bi mechanism where the NADPH is the first to bind at the active site followed by cortisone. The equilibrium dissociation constant, K(i)a as well as the apparent Michaelis-Menten constants K(m)a, K(m)b, k(cat)a, and k(cat)b for NADPH and cortisone, have been determined to be 147.5 microM, 14.4 microM, 43.8 nM, 0.21 min(-1), and 0.27 min(-1), respectively, for the human enzyme and 41.1 microM, 3.1 microM, 161.7 nM, 0.49 min(-1), and 0.52min(-1), respectively, for the rabbit enzyme.     

NADPH-dependent GMP reductase isoenzyme of human (GMPR2). Expression,purification, and kinetic properties

GMP reductase (EC 1.6.6.8) is the only known metabolic step by which guanine nucleotides can be converted to the pivotal precursor of both adenine and guanine nucleotides. Human GMP reductase has been previously partially purified from erythrocytes and a chromosome 6-linked cDNA has been identified to correspond for encoding human GMP reductase. Here, we reported a distinct cDNA for human GMP reductase isoenzyme isolated from a human fetal brain library, and the GenBank accession number is AF419346. The deduced protein shows 90% identity with human GMP reductase reported (named GMPR1 compared with GMPR2 of this paper) and 69% with E. coli GMP reductase. Comparison of GMPR2 cDNA sequence with human genome indicates the corresponding gene spans about 6.6kb on chromosome 14, which encodes 348 amino acid residues. Northern hybridization analysis indicates a differential and disproportionate expression of mRNAs for GMPR1 and GMPR2, suggesting the existence of distinct molecular species of GMP reductase in human. The apparent Km of GMPR2 for NADPH and GMP are 26.6 and 17.4 microM, respectively. This is the first report suggesting the existence of two distinct types of human GMP reductase molecular species, which can be used to explain the bimodal saturation curve noted with the purified human erythrocyte GMP reductase.     

Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase.

Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa.