Enzymes responsible for chlorate reduction by Pseudomonas sp. are different from those used for perchlorate reduction by Azospira sp

Pseudomonas sp. PDA is an unusual bacterium due to its ability to respire using chlorate under aerobic conditions. The chlorate reductase produced by PDA was shown to be intrinsically different from the enzyme responsible for chlorate and perchlorate [(per)chlorate] reduction produced by Azospira sp. KJ based on subunit composition and other enzyme properties. The perchlorate reductase from strain KJ appeared to have two subunits (100 and 40 kDa) while the chlorate reductase from PDA had three subunits (60, 48, and 27 kDa). N-terminal amino acid sequencing of the 100 kDa protein from strain KJ showed a 77% similarity with the perchlorate reductase alpha subunit from another perchlorate-respiring bacterium, Dechloromonas agitata, while the N-terminus amino acid sequence of the 60 kDa protein from strain PDA did not show a similarity to previously isolated chlorate or perchlorate reductases.     

Sequence Analysis of Cloned cDNA and Proteolytic Fragments for Nitrate Reductase from Spinacia oleracea L.

Proteolytic fragments were obtained by limited proteolysis of120 kDa nitrate reductase from Spinacia oleracea L. using trypsinand Staphylococcus aureus V8 protease. Determination of NH₂-terminalsequences in 9 to 14 Edman degradation steps allowed the exactlocalization of the fragments within the amino-acid sequenceof spinach nitrate reductase was deduced from the nucleotidesequence of cDNA clone pSPNR117 which was initially identifiedby hybridization to squash nitrate reductase cDNA clone [Crawford,¹N. M., Campbell, W. H. and Davis, R. W. (1986) Proc. Natl. Acad.Sci. USA 83: 8073] and anti spinach nitrate reductase polyclonalantibodies. This clone has a 2324 base insert, and the aminoacid sequence deduced from its open reading frame, which contains640 residues. The predicted sizes 42.5 and 30 kDa were in reasonableagreement with previous determination of the apparent molecularsizes of the FAD-cyt-chrome b557-binding, and FAD-binding fragments,respectively. Arginine residue was the cleavage site for trypsin and glutamicacid was for S. aureus V8 protease. The amino acid residueswithin the linker regions which connect the functional domains,could be cleaved with trypsin or S. aureus V8 protease may bewell conserved in the amino acid sequences deduced from thenitrate reductase cDNA sequences. A sequence identity of 61.2-80.1 % was found in the amino acidsequences deduced from the cDNA sequences as obtained by spinachand other higher plant nitrate reductases. However, the aminoacid sequences surrounding the proteolytic cleavage sites ofnitrate reductase had poor homology. (Received March 30, 1991; Accepted July 24, 1991)     

Mitochondrial thioredoxin reductase in bovine adrenal cortex its purification, properties, nucleotide/amino acid sequences, and identification of selenocysteine.

Mitochondrial thioredoxin reductase was purified from bovine adrenal cortex. The enzyme is a first protein component in the mitochondrial thioredoxin-dependent peroxide reductase system. The purified reductase exhibited an apparent molecular mass of 56 kDa on SDS/PAGE, whereas the native protein was about 100 kDa, suggesting a homodimeric structure. It catalysed NADPH-dependent reduction of 5, 5'dithiobis(2-nitrobenzoic acid) and thioredoxins from various origins but not glutathione, oxidized dithiothreitol, DL-alpha-lipoic acid, or insulin. Amino acid and nucleotide sequence analyses revealed that it had a presequence composed of 21 amino acids which had features characteristic of a mitochondrial targeting signal. The amino acid sequence of the mature protein was similar to that of bovine cytosolic thioredoxin reductase (57%) and of human glutathione reductase (34%) and less similar to that of Escherichia coli (19%) or yeast (17%) enzymes. Human and bovine cytosolic thioredoxin reductase were recently identified to contain selenocysteine (Sec) as one of their amino acid constituents. We also identified Sec in the C-terminal region of mitochondrial (mt)-thioredoxin reductase by means of MS and amino acid sequence analyses of the C-terminal fragment. The four-amino acid motif, Gly-Cys-Sec-Gly, which is conserved among all Sec-containing thioredoxin reductases, probably functions as the third redox centre of the enzyme, as the mitochondrial reductase was inhibited by 1-chloro-2,4-dinitrobenzene, which was reported to modify Sec and Cys covalently. It is known that mammalian thioredoxin reductase is different from bacterial or yeast enzyme in, for example, their subunit molecular masses and domain structures. These two different types of enzymes with similar activity are suggested to have evolved convergently. Our data clearly show that mitochondria, which might have originated from symbiotic prokaryotes, contain thioredoxin reductase similar to the cytosolic enzyme and different from the bacterial one.     

Complete amino acid sequence of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes

The complete amino acid sequence of porcine hepatic microsomal NADPH-cytochrome P-450 reductase has been determined by microsequence analysis on several sets of proteolytic fragments. Sequence studies were performed initially on a 20-kilodalton (kDa) fragment and then on 80-kDa fragment. The amino-terminal end of the mature protein was blocked with an acetyl group, followed by 676 amino acid residues. It has been revealed that the COOH-terminal 20-kDa fragment has been derived from original enzyme by cleavage at the Asn-Gly (residues 502-503) linkage by an unknown mechanism. An NADPH-protected cysteine residue is located at residue 565, near a region exhibiting high sequence homology with ferredoxin-NADP+ reductase. The FMN and FAD binding regions are possibly located in the amino-terminal region and the middle part of the protein molecule, respectively, as suggested by Porter and Kasper [Porter, T. D., & Kasper, C. B. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 973-977]. When this sequence is compared with that of rat enzyme, 60 amino acid residues are substituted, probably due to species differences. However, total sequence homology between these enzymes is 90%. Hydropathy plot analysis reveals that two regions from residues 27-43 and from residues 523-544 exhibit a high degree of hydrophobicity, suggesting membrane binding or interaction with cytochrome P-450.     

IgE-reactive 60 kDa glycoprotein occurring in wheat flour.

A new IgE-reactive glycoprotein with a molecular size of 60 kDa was isolated from wheat flour. The N-terminal amino acid sequence of the protein was LDPDESEXVTRYFRIR. The 8th amino acid residue would have been Asn to which the peroxidase-type glycochain was attached. The IgE-binding activity of the glycoprotein was rendered negligible by the enzymatic treatment applied for hypoallergenic flour production.     

IgE-reactive 60 kDa Glycoprotein Occurring in Wheat Flour

A new IgE-reactive glycoprotein with a molecular size of 60 kDa was isolated from wheat flour. The N-terminal amino acid sequence of the protein was LDPDESEXVTRYFRIR. The 8th amino acid residue would have been Asn to which the peroxidase-type giycochain was attached. The IgE-binding activity of the glycoprotein was rendered negligible by the enzymatic treatment applied for hypoallergenic flour production.     

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) in mitochondrial ubiquinol-cytochrome c reductase: isolation, ubiquinone-binding domain, and immunoinhibition

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) was purified to homogeneity from 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinol+ ++- labeled bovine heart mitochondrial ubiquinol-cytochrome c reductase. The N-terminal amino acid sequence of the isolated protein is Gly-Arg-Gln-Phe-Gly-His-Leu-Thr-Arg-Val-Arg-His-, which is identical with that of a Mr = 9500 protein in the reductase [Borchart et al. (1986) FEBS Lett. 200, 81-86]. A ubiquinone-binding peptide was prepared from [3H]azidoubiquinol-labeled QPc-9.5 kDa protein by trypsin digestion followed by HPLC separation. The partial N-terminal amino acid sequence of this peptide, Val-Ala-Pro-Pro-Phe-Val-Ala-Phe-Tyr-Leu-, corresponds to amino acid residues 48-57 in the reported Mr = 9500 protein. According to the proposed structural model for the Mr = 9500 protein, the azido-Q-labeled peptide is located in the membrane on the matrix side. These results confirm our previous assessment that the Mr = 13,400 subunit is not the small molecular weight Q-binding protein. Purified antibodies against QPc-9.5 kDa have a high titer with isolated QPc-9.5 kDa protein and complexes that contain it. Although antibodies against QPc-9.5 kDa do not inhibit intact succinate- and ubiquinol-cytochrome c reductases, a decrease of 85% and 20% in restoration of succinate- and ubiquinol-cytochrome c reductases, respectively, is observed when delipidated succinate- or ubiquinol-cytochrome reductases are incubated with antibodies prior to reconstitution with ubiquinone and phospholipid, indicating that epitopes at the catalytic site of QPc-9.5 kDa are buried in the phospholipid environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of Saccharomyces cerevisiae NADPH-cytochrome P450 reductase deduced from nucleotide sequence of its cloned gene

We isolated cDNA (pgCYR, about 2.1 kb) and genomic DNA (pgGYR, about 4 kb) clones coding for NADPH-cytochrome P450 reductase by immunoscreening of yeast Saccharomyces cerevisiae cDNA and genomic DNA libraries in phage lambda gt11. The clones were sequenced and found to encode a protein of 691 amino acid residues with a calculated molecular weight of 76,737 daltons. The amino-terminal sequence (excluding the initial methionine residue) deduced therefrom was in agreement with the protein sequence of the yeast reductase. In addition, the deduced sequence included the partial amino acid sequence determined with the papain-solubilized reductase. The total amino acid sequence of the yeast reductase showed 33-34% similarity with those of the rat, rabbit, pig, and trout reductases. In spite of low similarity in the total amino acid sequences, the possible functional domains related to binding of FAD, FMN, and NADPH were well conserved among all five species compared.     

NAD(P)H:menadione oxidoreductase. Novel purification of enzyme cDNA and complete amino acid sequence, and gene regulation

NAD(P)H:menadione oxidoreductase induction by polycyclic hydrocarbons is known to be governed by the aromatic hydrocarbon-responsive (Ah) locus. This cytosolic enzyme was isolated from 3-methylcholanthrene-treated rat liver by a rapid two-step procedure with the use of affinity gel purification and fast-protein liquid chromatography. Polyclonal antiserum to menadione reductase was raised in rabbits. On Western (immuno) blot analysis, large increases in this hepatic menadione reductase protein (NMOR1) of 3-methylcholanthrene-treated C57BL/6N but not DBA/2N mice confirmed that induction of this enzyme by 3-methyl-cholanthrene is regulated by the Ah receptor. A cDNA expression library was constructed in lambda gt11 and screened with antiserum. Positive cDNA clones were plaque purified and further characterized by showing enhanced hybridization to 3-methylcholanthrene-induced poly(A+) RNA from rats; the longest cDNA clone (1,501 base pairs) has an open reading frame (bases 75-899) and a nucleotide sequence consistent with a new gene family. On Northern blot analysis, a single 3-methylcholanthrene-inducible rat liver mRNA (approximately 1.6 kilobases) hybridizes to the cDNA probe. On Southern blot analysis a total of 14-16 kilobases of rat genomic DNA fragments hybridize to the cDNA probe, indicating one or a small number of menadione reductase genes in this family. The amino acid sequence (274 residues) and Mr of 30,946 compare well with the size of the rat enzyme (32 kDa) estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of two internal fragments of the trypsin-digested purified NMOR1 protein is in complete agreement with that deduced from the cDNA nucleotide sequence. This study represents the first cloning and sequencing of a cDNA encoding a Phase II drug-metabolizing enzyme under control of the Ah locus.     

Identification of the acyl transfer site of fatty acyl-protein synthetase from bioluminescent bacteria

Fatty acid activation, transfer, and reduction by the fatty acid reductase multienzyme complex from Photobacterium phosphoreum to generate fatty aldehydes for the luminescence reaction is regulated by the interaction of the synthetase and reductase subunits of this complex. Identification of the specific site involved in covalent transfer of the fatty acyl group between the sites of activation and reduction on the synthetase and reductase subunits, respectively, is a critical step in understanding how subunit interactions modulate the flow of fatty acyl groups through the fatty acid reductase complex. To accomplish this goal, the nucleotide sequence of the luxE gene coding for the acyl-protein synthetase subunit (373 amino acid residues) was determined and the conserved cysteinyl residues implicated in fatty acyl transfer identified. Using site-specific mutagenesis, each of the five conserved cysteine residues was converted to a serine residue, the mutated synthetases expressed in Escherichia coli, and the properties of the mutant proteins examined. On complementation of four of the mutants with the reductase subunit, the synthetase subunit was acylated and the acyl group could be reversibly transferred between the reductase and synthetase subunits, and fatty acid reductase activity was fully regenerated. As well, sensitivity of the acylated synthetases to hydroxylamine cleavage (under denaturation conditions to remove any conformational effects on reactivity) was retained, showing that a cysteine and not a serine residue was still acylated. However, substitution of a cysteine residue only ten amino acid residues from the carboxyl terminal (C364S) prevented acylation of the synthetase and regeneration of fatty acid reductase activity. Moreover, this mutant protein preserved its ability to activate fatty acid to fatty acyl-AMP but could not accept the acyl group from the reductase subunit, demonstrating that the C364S synthetase had retained its conformation and specifically lost the fatty acylation site. These results provide evidence that the flow of fatty acyl groups in the fatty acid reductase complex is modulated by interaction of the reductase subunit with a cysteine residue very close to the carboxyl terminal of the synthetase, which in turn acts as a flexible arm to transfer acyl groups between the sites of activation and reduction.     

Cloning and expression of human aldose reductase

The complete amino acid sequence of human retina and muscle aldose reductase was determined by nucleotide analysis of cDNA clones isolated using synthetic oligonucleotide probes based on partial amino acid sequences of purified human psoas muscle aldose reductase. The cDNA sequence differs substantially in the noncoding and coding regions of recently published sequences of this enzyme. The mRNA for aldose reductase was abundantly expressed in HeLa cells, but only scarcely in a neuroblastoma cell line. Recombinant baculovirus containing one of the muscle cDNA clones was constructed and used to infect Spodoptera frugiperda (SF9) cells. A prominent protein with an apparent molecular size of 36 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the culture medium as well as in the homogenate of SF9 cells after 2 days of infection. Culture medium or the supernatant fraction of cell homogenates containing this protein had high aldose reductase activity which showed characteristics of the reported human enzyme. These findings indicate that the amino acid sequence reported in this paper represents human retina and muscle aldose reductase and that functional human aldose reductase can be expressed in large amounts in a baculovirus expression system. The result should facilitate refined structural analysis and the development of new specific aldose reductase inhibitors for the treatment of diabetic complications.     

Sarcosine reductase of Tissierella creatinophila: purification and characterization of its components

Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate. The acetyl-phosphate-forming component protein C was purified to homogeneity. SDS-PAGE of the purified protein revealed two protein bands with apparent mol. masses of 62 and 50 kDa. The N-terminal amino acid sequence of the two subunits was determined. Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T. creatinophila, Clostridium litorale and Clostridium sporogenes. The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit. Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate. The protein A component of the sarcosine reductase of T. creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR. Protein A had an apparent mol. mass of 21 kDa. Its N-terminal amino acid sequence showed high similarities to that of other proteins A. Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein B_sarcosine component of T. creatinophila indicated the involvement of a 50-kDa protein. Received: 18 May 1998 / Accepted: 5 August 1998     

Cleavage of the PO glycoprotein of the rat peripheral nerve myelin: Tentative identification of cleavage site and evidence for the precursor-product relationship

The incubation of sciatic nerve slices in Krebs Ringer bicarbonate (KRB) buffer (pH 7.4) at 37°C, or the incubation of freshly isolated myelin in ammonium bicarbonate buffer (pH 8), resulted in the generation of a 24kDa protein with a concomitant decrease of PO protein. The conversion of PO into 24kDa protein was blocked by heating isolated myelin at 100°C for 5 min suggesting that the reaction is enzyme mediated. Inclusion of the protease inhibitors and chelating agent to isolated myelin did not prevent the formation of 24kDa protein. Similarly, addition of CaCl₂ to isolated myelin did not accentuate the formation of 24kDa protein suggesting that the conversion of PO into 24kDa protein may not be due to Ca²⁺ activated protease. It is postulated that the formation of 24kDa protein may be due to neutral protease and/or metalloproteinase associated with the PNS myelin. 24kDa protein was purified and characterized. The N-terminal sequence of 1–17 amino acid residues of 24kDa protein was identical to PO. 24kDa protein was immunostained and immunoprecipitated with anti-PO antiserum indicating the immunological similarities between PO and 24kDa protein. Labeling of 24kDa protein with [³⁵S]methionine provided evidence that PO may be in all probability cleaved between Met-168 and Met-193. Further studies were carried out to demonstrate that 24kDa protein was phosphorylated, glycosylated and acylated like PO. Phosphorylation of 24kDa protein in the nerve slices was increased five-fold by phorbol esters and phosphoserine was the only phosphoamino acid identified after partial acid hydrolysis of 24kDa protein. These results suggested that serine residue phosphorylated by protein kinase C may be located in amino acid residues 1-168. 24kDa protein was stained with periodic Schiff reagent. In addition, 24kDa protein was fucosylated and the fucosylation of 24kDa protein was inhibited (70%) by tunicamycin, providing evidence that it is N-glycosylated. Recently, it was demonstrated that both PO and 24kDa protein were fatty acylated with [³H]palmitic acid in the nerve slices and fatty acids are covalently linked to these proteins (Agrawal, H.C. and Agrawal, D. 1989, Biochem. J. 263:173–177). The time course of inhibition of acylation by cycloheximide of 24kDa protein was identical to PO. Cycloheximide inhibited acylation of PO and 24kDa protein by 61% and 58% respectively, whereas, monensin had little affect on the fatty acylation of these proteins. Less [³H]palmitic acid and¹⁴C-amino acids were incorporated into 24kDa protein when compared to PO between 5–30 min after incubation of the nerve slices. However, more radioactivity was incorporated into 24kDa protein after 60 min when compared to PO under identical conditions. These results provided evidence of a precursor-product relationship between PO and 24kDa protein. Therefore, PO may be cleaved into 24kDa protein in the myelin membrane following its acylation and glycosylation in the Schwann cells.     

Structural homology of human complement component C8 gamma and plasma protein HC: identity of the cysteine bond pattern

Anti-C8 alpha-gamma specific antibodies were used to isolate cDNA clones from a human liver expression library. Antibodies affinity-purified on the expressed hybrid protein of one clone bound exclusively to the gamma-chain of reduced C8 alpha-gamma. This clone, as well as a second full length cDNA clone obtained by hybridization screening, were sequenced and the complete primary structure for C8 gamma was established. Cyanogen bromide cleavage of C8 alpha-gamma released a 12 kDa carboxy-terminal C8 gamma fragment under both reducing and nonreducing conditions which was identified by fragment-specific, affinity-purified antibodies. Our data clearly show that C8 gamma has one internal disulfide bridge between cys-76 and cys-168 within the carboxy-terminal 12 kDa fragment, whereas the remaining cysteine residue 40 forms the disulfide bridge with C8 alpha. The overall sequence homology to plasma protein HC (23% amino acid identities) and the conservation of one internal cysteine bond and one free, surface-located cysteine residue suggests a highly conserved three-dimensional structure of C8 gamma and protein HC and also a possible functional relationship between these proteins.     

cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A 64 kD protein was enriched from rat liver mito-chondria during the purification of choline dehydro-genase (CHDH)[1]. Homologous comparison and func-tional experiments demonstrated that the protein was electron-transfer flavoprotein-ubiquinone oxidoreduc-tase protein (ETF-QO). The N-terminal sequence determination of rat liver ETF-QO protein purified by various methods did not provide unequivocal result. However, when the protein was digested with V8 protease, peptide fragments could b…     

The complete amino acid sequence of a bacteriochlorophyll a binding polypeptide isolated from the cytoplasmic membrane of the green photosynthetic bacterium Chloroflexus aurantiacus

A polypeptide soluble in organic solvents was isolated from whole membrane fractions of the green thermophilic bacterium Chloroflexus aurantiacus by chromatography on Sephadex LH-60, Whatman DE-32 and Bio Gel P-10. The complete amino acid sequence of this 4.9 kDa polypeptide (44 amino acid residues) was determined. The polypeptide shows a 3-domain structure, similar to the domain structure of the antenna BChI polypeptides of purple photosynthetic bacteria, and sequence homologies (27–39%) to the light-harvesting α-polypeptides of the B870 (890) antenna complexes from purple bacteria. Therefore, the 4.9 kDa polypeptide is designated B(808-866)-α. The typical His residue (conserved His residue identified in all antenna polypeptides of purple bacteria as possible BChI binding site) is found within the hydrophobic domain, which extends from Asn 10 to Leu 30.     

Identification of the serine residue phosphorylated by protein kinase C in vertebrate nonmuscle myosin heavy chains

Two-dimensional mapping of the tryptic phosphopeptides generated following in vitro protein kinase C phosphorylation of the myosin heavy chain isolated from human platelets and chicken intestinal epithelial cells shows a single radioactive peptide. These peptides were found to comigrate, suggesting that they were identical, and amino acid sequence analysis of the human platelet tryptic peptide yielded the sequence -Glu-Val-Ser-Ser(PO4)-Leu-Lys-. Inspection of the amino acid sequence for the chicken intestinal epithelial cell myosin heavy chain (196 kDa) derived from cDNA cloning showed that this peptide was identical with a tryptic peptide present near the carboxyl terminal of the predicted alpha-helix of the myosin rod. Although other vertebrate nonmuscle myosin heavy chains retain neighboring amino acid sequences as well as the serine residue phosphorylated by protein kinase C, this residue is notably absent in all vertebrate smooth muscle myosin heavy chains (both 204 and 200 kDa) sequenced to date.     

NADH-ferric reductase activity associated with dihydropteridine reductase

In mammals dietary ferric iron is reduced to ferrous iron for more efficient absorption by the intestine. Analysis of a pig duodenal membrane fraction revealed two NADH-dependent ferric reductase activities, one associated with a b-type cytochrome and the other not. Purification and characterization of the non-cytochrome ferric reductase identified a 31 kDa protein. MALDI-MS analysis and amino acid sequencing identified the ferric reductase as being related to the 26 kDa liver NADH-dependent quinoid dihydropteridine reductase (DHPR). The NADH-dependent DHPR ferric reductase activity was found to be pteridine-independent since exhaustive dialysis did not reduce activity and heat-inactivation destroyed activity. In intestinal Caco-2 cells, DHPR mRNA levels were found to be regulated by iron. Thus, DHPR appears to be a dual function enzyme, a NADH-dependent dihydopteridine reductase and an iron-regulated, NADH-dependent, pteridine-independent ferric reductase.     

A protein kinase activated by darkness phosphorylates nitrate reductase in Komatsuna (Brassica campestris) leaves

Although it has been shown that leaf nitrate reductase (NR: EC 1.6.6.1) is phosphorylated by subjecting plants to darkness, there is no evidence for the existence of dark-activated or dark-induced NR kinase. This study was undertaken to investigate the occurrence of a protein kinase phosphorylating NR in response to dark treatments. Immediately after transferring Komatsuna (Brassica campestris L.) plants to darkness, we observed rapid increases in the phosphorylating activity of the synthetic peptide, which is designed for the amino acid sequence surrounding the regulatory serine residue of the hinge 1 region of Komatsuna NR, in crude extracts from leaves. The activity reached a maximum after 10 min of darkness. Inactivation states of NR estimated from relative activities with or without Mg2+ were correlated to activities of the putative dark-activated protein kinase. Using the synthetic peptide as a substrate, we purified a protein kinase from dark-treated leaves by means of successive chromatographies on Q-Sepharose, Blue Sepharose, FPLC Q-Sepharose, and ATP-gamma-Sepharose columns. The purified kinase had an apparent molecular mass of 150 kDa with a catalytic subunit of 55 kDa, and it was Ca2+-independent. The purified kinase phosphorylated a recombinant cytochrome c reductase protein, a partial protein of NR, and holo NR, and inactivated NR in the presence of both 14-3-3 protein and Mg2+. The kinase also phosphorylated synthetic peptide substrates designed for sucrose phosphate synthase and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase. Among inhibitors tested, only K252a, a potent and specific serine/threonine kinase inhibitor, completely inhibited the activity of the dark-activated kinase. The activity of the purified kinase was also specifically inhibited by K252a. Taken together with these findings, results obtained suggest that the putative dark-activated protein kinase may be the purified kinase itself, and may be responsible for in vivo phosphorylation of NR and its inactivation during darkness.