3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.     

Isolation of a soluble and membrane-associated Fe(III) reductase from the thermophile, Thermus scotoductus (SA-01)

The Fe(III) reductase activity was studied in the South African Fe(III)-reducing bacterium, Thermus scotoductus (SA-01). Fractionation studies revealed that the membrane as well as the soluble fraction contained NAD(P)H-dependent Fe(III) reductase activity. The membrane-associated enzyme was solubilized by KCl treatment and purified to electrophoretic homogeneity by hydrophobic interaction chromatography. A combination of ion-exchange and gel filtration chromatography was used to purify the soluble enzyme to apparent homogeneity. The molecular mass of the membrane-associated Fe(III) reductase was estimated to be 49 kDa, whereas the soluble Fe(III) reductase had an apparent molecular mass of 37 kDa. Optimum activity for the membrane-associated enzyme was observed at around 75 degrees C, whereas the soluble enzyme exhibited a temperature optimum at 60 degrees C.     

Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 micromol. min(-1). mg(-1). The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP(+) oxidoreductase activity and catalyzed the reduction of NADP(+) with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.     

Purification and characterization of Fe(III)-EDTA reductase from Bacillus sp. B-3

Fe(III)-EDTA reductase was purified from Bacillus sp. B-3 isolated as a Fe(III)-EDTA-degrading bacterium. The purified enzyme showed a single protein band corresponding to a molecular mass of 19 kDa on SDS-PAGE, and had FMN as cofactor. It was alkali-thermostable. Its N-terminal amino acid sequence was identical with that of NADPH azoreductase from several species of Bacillus.     

Purification and biochemical characterization of lysosomal acid phosphatases (EC 3.1.3.2) from blood stream forms, Trypanosoma brucei brucei

Three Acid phosphatases (ACP) were isolated and characterized from the lysosomes of blood stream forms of Trypanosoma brucei by a combination of isopynic and differential centrifugation through Ficoll, organic solvent precipitation, ion exchange on DEAE cellulose 52 and size exclusion chromatography on Sephadex G-75 columns. The purified ACP emerged as three distinct peaks (ACP I, ACP II and ACP III) with high specific activities and they moved homogenously on 12% SDS-PAGE each as a single band with relative molecular weight of 36 kDa, 25 kDa and 45 kDa respectively. The purified enzymes were active at an optimum pH and temperature of 5.5 and 40 °C respectively. The enzyme activities appeared to be ACP because their activities were enhanced at low pH values and inhibited by the acid phosphatase inhibitor, sodium fluoride. ACP I and ACP II were sensitive to l-tartrate while ACP III was insensitive to l tartrate. The kinetic analysis of the purified enzyme (ACP I, ACP II and ACP III) determined using para-nitrophenylphosphate as substrate gave K_M values of 0.2 mM, 0.15 mM and 0.5 mM. Monofunctional group sulfhydryl group inhibitors; HgCl₂, and AgCl₂ strongly inhibited the activity of ACP III and millimolar concentrations of dithiothreitol and iodoacetamide activated and inhibited the activity of the ACP III respectively, suggesting the involvement of thiol groups at the active site of the enzyme. Thus, differentiating it from ACP I and ACP II. The implication of these findings in relation to the pathology of trypanosomosis is discussed.     

A novel hyperthermophilic archaeal glyoxylate reductase from Thermococcus litoralis. Characterization, gene cloning, nucleotide sequence and expression in Escherichia coli.

A novel NADH-dependent glyoxylate reductase has been found in a hyperthermophilic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophilic archaea. NADH-dependent glyoxylate reductase was purified approximately 560-fold from a crude extract of the hyperthermophile by five successive column chromatographies and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodimer with a subunit molecular mass of approximately 37 kDa. The optimum pH and temperature for enzyme activity were approximately 6.5 and 90 degrees C, respectively. The enzyme was extremely thermostable; the activity was stable up to 90 degrees C. The glyoxylate reductase catalyzed the reduction of glyoxylate and hydroxypyruvate, and the relative activity for hydroxypyruvate was approximately one-quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron donor compared with NADH. The Km values for glyoxylate, hydroxypyruvate, and NADH were determined to be 0.73, 1.3 and 0.067 mM, respectively. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the glyoxylate reductase gene was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular mass of 36,807. The amino-acid sequence of the T. litoralis enzyme showed high similarity with those of probable dehydrogenases in Pyrococcus horikoshii and P. abyssi. The purification of the enzyme from recombinant E. coli was much simpler compared with that from T. litoralis; only two steps of heat treatment and dye-affinity chromatography were needed.     

Characterization of a Pseudomonas aeruginosa Fatty Acid Biosynthetic Gene Cluster: Purification of Acyl Carrier Protein (ACP) and Malonyl-Coenzyme A:ACP Transacylase (FabD)

A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding beta-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding beta-ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally coupled. The fabH gene (encoding beta-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between plsX and fabD, is absent from this gene cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal fabF insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disruption of the chromosomal fabG gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H6-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption-ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4'-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H(6)-FabD exhibited malonyl-CoA:ACP transacylase activity.     

Purification and properties of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (phenylalanine-inhibitable) of Saccharomyces cerevisiae

The phenylalanine-inhibitable 3-deoxy-D-arabino-heptulosonate-7-phosphate (dHp1P) synthase from Saccharomyces cerevisiae has been purified to apparent homogeneity by a 1250-fold enrichment of the enzyme activity present in wild-type crude extracts, employing an overproducing strain. The estimated molecular mass of 42 kDa corresponds to the calculated molecular mass of 42.13 kDa deduced from the previously determined primary sequence. Gel filtration indicates that the active enzyme is a monomer. The enzyme is an Fe protein and is inactivated by EDTA in a reaction which is reversible by several bivalent metal ions. The Michaelis constant of the enzyme is 18 microM for phosphoenolpyruvate (P-pyruvate) and 130 microM for erythrose 4-phosphate (Ery4P) and the rate constant was calculated as 10 s-1. Inhibition by phenylalanine is competitive with respect to erythrose 4-phosphate and non-competitive to phosphoenolpyruvate, with a Ki of 10 microM.     

A 27.368 kDa retinal reductase in New Zealand white rabbit liver cytosol encoded by the peroxisomal retinol dehydrogenase-reductase cDNA: purification and characterization of the enzyme.

We obtained a full-length cDNA based on a sequence deposited in GenBank (accession No. AB045133), annotated as rabbit peroxisomal NADP(H)-dependent retinol dehydrogenase-reductase (NDRD). The rabbit NDRD gene, like its mouse and human homologs, harbors 2 initiation sites, one of which theoretically encodes a 29.6 kDa protein with 279 amino acids, and the other encodes a 27.4 kDa protein with 260 amino acids. The purification of a rabbit cytosolic retinol oxidoreductase with a subunit molecular mass of 34 kDa and an N terminus that is not completely identical to that of NDRD, has been reported. An enzyme responsible for the all-trans retinal reductase activity in the liver cytosol of New Zealand white rabbit was purified to homogeneity using differential centrifugation and successive chromatographic analyses. The subunit molecular mass of the purified enzyme, revealed by SDS-PAGE, was approximately 27 kDa. The intact molecular mass, measured by MALDI-TOF mass spectrometry, was 27.368 kDa. The 60 kDa relative mobility observed in size-exclusion chromatography indicates that the native protein probably exists as a dimer. The purified enzyme was positively confirmed to be the product of NDRD by peptide mass fingerprinting, tandem mass spectrometry, and N-terminal sequencing. Taken together, the results suggested that the native protein is truncated at the N terminus.     

Triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P: purification, characterization, gene cloning, and overexpression of a functional protein in Escherichia coli

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60 degrees C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.     

Purification and characterization of methylenetetrahydrofolate reductase from human cadaver liver

Methylenetetrahydrofolate reductase from human cadaver liver was purified to homogeneity. The purified enzyme had a molecular mass of 150 kDa. On SDS-polyacrylamide gel electrophoresis it was dissociated into a single fragment with a molecular mass of 39 kDa. In contrast, fresh lymphocyte enzyme extract showed a major band with a molecular mass of 75 kDa and a minor band of 39 kDa. Fresh liver enzyme was inhibited by S-adenosylmethionine while the purified enzyme from human cadaver liver was not inhibited. These observations suggest that human methylenetetrahydrofolate reductase is composed of two identical subunits of 75 kDa each but is cleaved into a major single band due to autolysis in cadaver liver. The purified cadaver enzyme was a FAD-specific protein. The pH optimum was 6.6 for methylenetetrahydrofolate-NADPH oxidoreductase, 6.5 for methyltetrahydrofolate-menadione oxidoreductase, and 7.2 for NADP-menadione oxidoreductase. The Km values of human liver methylenetetrahydrofolate reductase were 17 microns for NADPH and 38 microns for methyltetrahydrofolate in the reduction of menadione, and 12 microns for NADPH in the reduction of methylenetetrahydrofolate.     

Purification and characterization of bisulfite reductase (desulfofuscidin) from Desulfovibrio thermophilus and its complexes with exogenous ligands

A dissimilatory bisulfite reductase has been purified from a thermophilic sulfate-reducing bacterium Desulfovibrio thermophilus (DSM 1276) and studied by EPR and optical spectroscopic techniques. The visible spectrum of the purified bisulfite reductase exhibits absorption maxima at 578.5, 392.5 and 281 nm with a weak band around 700 nm. Photoreduction of the native enzyme causes a decrease in absorption at 578.5 nm and a concomitant increase in absorption at 607 nm. When reduced, the enzyme reacts with cyanide, sulfite, sulfide and carbon monoxide to give stable complexes. The EPR spectrum of the native D. thermophilus bisulfite reductase shows the presence of a high-spin ferric signal with g values at 7.26, 4.78 and 1.92. Upon photoreduction the high-spin ferric heme signal disappeared and a typical 'g = 1.94' signal of [4Fe-4S] type cluster appeared. Chemical analyses show that the enzyme contains four sirohemes and eight [4Fe-4S] centers per mol of protein. The molecular mass determined by gel filtration was found to be 175 kDa. On SDS-gel electrophoresis the enzyme presents a main band of 44 to 48 kDa. These results suggest that the bisulfite reductase contains probably one siroheme and two [4Fe-4S] centers per monomer. The dissimilatory bisulfite reductase from D. thermophilus presents some homologous properties with desulfofuscidin, the bisulfite reductase isolated from Thermodesulfobacterium commune (Hatchikian, E.C. and Zeikus, J.G. (1983) J. Bacteriol. 153, 1211-1220).     

Triphenylmethane Reductase from Citrobacter sp. Strain KCTC 18061P: Purification, Characterization, Gene Cloning, and Overexpression of a Functional Protein in Escherichia coli

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.     

Acetoacyl-acyl carrier protein synthase from avocado: its purification,characterisation and clear resolution from acetyl CoA:ACP transacylase

-ketoacyl-ACP synthetase III (KAS III) has been purified from avocado using a six-step purification procedure. The enzyme, which is cerulenin-insensitive and thiolactomycin-sensitive, was assayed using a partial component reaction: acetyl CoA:ACP transacylase (ACAT) activity. KAS III activity is distinguished from ACAT activity on the basis that the former is highly stimulated by the addition of malonyl CoA in the presence of malonyl-CoA:ACP transacylase, and the latter is not. KAS III and ACAT activity have been separated from each other thus providing the first evidence that these two discrete activities exist in higher plants. Both of these enzymes have been implicated in the initial reactions of fatty acid synthesis.KAS III was purified 134-fold using a combination of PEG precipitation, Fast Q, ammonium sulphate precipitation, Phenyl Sepharose and ACP-affinity chromatography. The enzyme requires Triton X-100 for solubility and is highly salt sensitive. The subunit molecular mass of 37 kDa has been identified by SDS-PAGE. The results of gel filtration analysis are consistent with the native enzyme being homodimeric. The native molecular mass of KAS III is 69 kDa and that of ACAT 18.5 kDa. The enzyme has a pH optimum of 7.0–7.5, which is similar to the pH optimum of the ACAT reaction. The Km for acetyl CoA is 12.5 M and the Km for malonyl-ACP is 14M. Both KAS III and ACAT are sensitive to thiolactomycin inhibition. The results are discussed with respect to the potential role of acetyl CoA:ACP transacylase in plants.     

Characterisation of the specific p-nitrophenylphosphatase gene and protein of Schizosaccharomyces pombe.

Cloning and sequencing of the pho2 gene which codes for a specific p-nitrophenylphosphatase from Schizosaccharomyces pombe is described. The gene has an open contiguous reading frame of 269 amino acids corresponding to a protein with a molecular mass of 29.5 kDa and a calculated pI of 6.6. The sequence reveals four regions that share significant sequence similarity with the corresponding gene PHO13 of Saccharomyces cerevisiae. Purification of the enzyme to apparent homogeneity is reported. The amino acid composition of the purified protein matches well the values predicted from the nucleotide sequence. On SDS/polyacrylamide gels, the enzyme runs as a protein with a molecular mass of 33 kDa, and by Sephadex chromatography under nondenaturing conditions as 70 kDa. This indicates that the enzyme is a homodimer in its native form. The enzyme is not glycosylated. Its activity is stimulated by Mg2+ and inhibited by Zn2+. The available data on p-nitrophenylphosphatase do not give any clues to its biological role and its physiological substrates.     

The NADH-dependent reductase of a putative multicomponent tetrahydrofuran mono-oxygenase contains a covalently bound FAD.

NADH-cytochrome c oxidoreductase activity specifically expressed during growth on tetrahydrofuran was detected in cell extracts of Pseudonocardia sp. strain K1. The enzyme catalyzing this reaction was purified to apparent homogeneity by a three-step purification procedure. It was characterized as a monomer of apparent molecular mass 40 kDa. Spectroscopic studies indicated that it contains an iron-sulfur cluster and a flavin cofactor. An amount of 1 mol of flavin and 1 mol of iron was determined per mol of homogeneous protein. The N-terminal amino-acid sequence exhibited great similarity to the reductase component of various oxygenases. Cloning and sequencing of the corresponding gene designated as thmD revealed an ORF encoding a protein of 360 amino acids. An overall similarity of up to 38% was obtained to the NAD(P)H-acceptor reductase of several binuclear iron-containing mono-oxygenases. Conserved sequence motifs were identified that were similar to the chloroplast-type ferredoxin 2Fe-2S centre and to nucleotide-binding domains. Studies on the flavin cofactor showed that it could not be removed from the protein by denaturation, indicating a covalent attachment. Spectroscopic studies revealed that the flavin is at the FAD level and covalently bound to the protein via the flavin 8alpha-methyl group. Thus, the isolated reductase component is the first enzyme of this type for which a covalent attachment of the flavin has been observed.     

Purification of hydroxyquinol 1,2-dioxygenase and maleylacetate reductase: the lower pathway of 2,4,5-trichlorophenoxyacetic acid metabolism by Burkholderia cepacia AC1100.

The enzyme hydroxyquinol 1,2-dioxygenase, which catalyzes ortho cleavage of hydroxyquinol (1,2,4-trihydroxybenzene) to produce maleylacetate, was purified from Escherichia coli cells containing the tftH gene from Burkholderia cepacia AC1100. Reduction of the double bond in maleylacetate is catalyzed by the enzyme maleylacetate reductase, which was also purified from E. coli cells, these cells containing the tftE gene from B. cepacia AC1100. The two enzymes together catalyzed the conversion of hydroxyquinol to 3-oxoadipate. The purified hydroxyquinol 1,2-dioxygenase was specific for hydroxyquinol and was not able to use catechol, tetrahydroxybenzene, 6-chlorohydroxyquinol, or 5-chlorohydroxyquinol as its substrate. The native molecular mass of hydroxyquinol 1,2-dioxygenase was 68 kDa, and the subunit size of the protein was 36 kDa, suggesting a dimeric protein of identical subunits.     

Molecular cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was 85 degrees C and the enzyme exhibited a high level of heat stability.     

Isolation and purification of a rat liver 3-hydroxy-3-methylglutaryl-coenzyme reductase activating protein (RAP)

A protein with an estimated subunit mass of 19 kDa was isolated and purified from perfused rat liver cytosol. This protein activates hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase (NADPH) (EC 1.1.1.34), the rate-limiting enzyme in the cholesterol biosynthetic pathway. The activation process by this HMG-CoA reductase activating protein (RAP) is time-dependent and requires NADPH. Maximal activity of HMG-CoA reductase induced by RAP is comparable to that obtained in the presence of thiols, such as GSH, and can exceed 100-fold the activity obtained when thiols are omitted. Purified RAP lacks ability to reduce 5,5'-dithiobis-(2-nitrobenzoic acid). RAP was purified to homogeneity utilizing DEAE- and phenyl-Sepharose CL-4B column chromatography. The purified RAP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shows multiple interconvertible aggregational forms on native polyacrylamide gel electrophoresis. A monospecific antibody against RAP was prepared by immunization of hens and extracted from either their egg yolks or serum. The catalytic activity of RAP might be responsible for the physiological activation of HMG-CoA reductase and regulation of its activity.