Purification and partial characterization of thioredoxin reductase from Streptomyces aureofaciens

Soluble carbonic anhydrase (CA, EC 4.2.1.1) inducible by low levels of CO2 was purified from the unicellular green alga Chlorella sorokiniana grown at alkaline pH. The purified CA had a specific activity of 2,300 units (mg protein)-1. The molecular mass of the CA was found to be 100 kDa by non-dissociating (native)-polyacrylamide gel electrophoresis and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 50-kDa subunit was recognized by concanavalin A. These results suggest that the protein has a dimeric form with two 50-kDa subunits that are glycosylated in an asparagine-linked manner. The native CA was revealed by isoelectric focusing to be a very acidic protein with an isoelectric point of 4.2. About 60% of the CA activity was inhibited by 0.5 M NaCl. The enzyme was inactivated over 95% by preincubation with 50 mM dithiothreitol but not with 1 mM dithiothreitol. After partial amino acid sequence analysis, a cDNA clone of the CA was isolated and characterized. The cloned cDNA fragment encoded a 348-amino-acid polypeptide (36,709 Da) including an NH2-terminal hydrophobic signal peptide composed of 35 amino acids (3,725 Da). Conserved regions of sequences found in animal CAs, in the periplasmic (pCA) and the intracellular CAs of Chlamydomonas, and in the plasma-membrane-bound CA of Dunaliella (Dca) were also found in this Chlorella CA. The signal sequence was significantly homologous to the pCA and the Dca. The internal signal sequence between the large and the small subunits reported for pCA was not found in this Chlorella CA. The soluble CA of this alga was an alpha-type CA with salt-sensitive, periplasm-locating and acidic properties and very different from pCA and Dca with their salt-sensitive/neutral and salt-resistant/acidic properties, respectively.     

Assembly of amino-terminal fibronectin dimers into the extracellular matrix

Fibronectin is a dimeric adhesion molecule that consists of three types of repeating modules. Adherent cells bind soluble fibronectin and incorporate it into insoluble fibrils in the extracellular matrix. The amino-terminal 70-kDa portion of fibronectin mediates binding to the cell surface, but amino-terminal fragments do not accumulate in the extracellular matrix. The ninth type I and first type III modules, the cell adhesion region, and the cysteines that form the interchain disulfide bonds have also been implicated in matrix assembly. To further define which regions of fibronectin are essential for matrix assembly, we generated a dimeric protein (d70 kDa) in which the 70-kDa amino terminus is directly linked to the last 51 amino acids of fibronectin, which contain the cysteines involved in interchain disulfide bonding. d70 kDa bound to cells and accumulated in the extracellular matrix. Incorporation of d70 kDa into the extracellular matrix was dependent upon protein synthesis; in cycloheximide-treated cultures that lacked a pre-existing matrix, d70 kDa accumulated in the extracellular matrix only in the presence of intact fibronectin. Monomeric 70-kDa protein was not incorporated into the matrix in the presence or absence of cycloheximide. These data indicate that fibronectin molecules containing only the amino-terminal 70-kDa region and the carboxyl-terminal 51 amino acids can become assembled into the extracellular matrix.     

Human lymphocyte cDNA ordered library analyzed by 2D gel electrophoresis. 1. Pooling strategy and matching of gel patterns

beta-Citryl-L-glutamate-hydrolysing enzyme (beta-CGHE) was purified from rat testis particulate fraction 13,000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, beta-CG-Sepharose affinity chromatography and Sephacryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiff's-stained bands on native polyacrylamide gel-electrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both bands hydrolyzed beta-citryl-L-glutamate (beta-CG) to citrate and glutamate. The 420 kDa band was changed by digestion with N-glycosidase F, into a 350 kDa band on native PAGE. The purified enzyme was composed of 90, 100, 115 and 130 kDa subunits on SDS-PAGE under non-reduced conditions. The purified enzyme was pharmacologically similar to the beta-CGHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucleotides such as ATP or GTP and phosphate ions. beta-CGHE was also potently inhibited by an excitatory amino acid agonist, L-quisqualate, but not by another agonists, N-methyl-D-aspartate and kinate. It had high substrate specificity for beta-CG. The antibodies against the purified enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precipitated the enzyme activity from the crude and purified enzyme solution.     

Alternate amino terminal processing of surfactant protein A results in cysteinyl isoforms required for multimer formation

The biological functions of rat surfactant protein A (SP-A), an oligomer composed of 18 polypeptide subunits derived from a single gene, are dependent on intact disulfide bonds. Reducible and collagenase-reversible covalent linkages of as many as six or more subunits in the molecule indicate the presence of at least two NH2-terminal interchain disulfide bonds. However, the reported primary structure of rat SP-A predicts that only Cys6 in this region is available for interchain disulfide formation. Direct evidence for a second disulfide bridge was obtained by analyses of a set of three mutant SP-As with telescoping deletions from the reported NH2-terminus. Two of the truncated recombinant proteins formed reducible dimers despite deletion of the domain containing Cys6. Edman degradation revealed that each mutant protein was a mixture of two isoforms with and without an isoleucine-lysine-cysteine (IKC) extension at the NH2-terminus, which was derived from the COOH-terminal end of the reported signal peptide. Large variations in the abundance of the IKC isoforms between truncated SP-As suggested that the amino acid sequences located downstream from the signal peptide modulated alternate-site cleavage by signal peptidase. Elution of the newly identified cysteine in the position of DiPTH-Cys indicated participation in disulfide linkage, which was interchain based on the direct correlation between prevalence of the IKC variant and the extent of dimerization for each truncated protein. Sequencing of both native rat SP-A and human SP-A also revealed isoforms with disulfide-forming NH2-terminal extensions. The extended rat SP-A isoforms were enriched in the more fully glycosylated and multimeric SP-A species separated on SDS-PAGE gels. Thus, a novel post translational modification results in naturally occurring cysteinyl isoforms of rat SP-A, which are essential for multimer formation.     

A comparison of fluvoxamine and fluoxetine in the treatment of major depression

Two monoclonal antibodies (MAbs) to human placenta laminin (pl-LAM), 1D8 (IgG1) and 6G5 (IgG2b) were generated and shown by ELISA and immunoblot analysis to recognize only native pl-LAM, but not denatured, reduced pl-LAM or mouse EHS laminin. Intact pl-LAM was easily isolated and purified in large scale from human placenta by 1D8-conjugated affinity chromatography. Electrophoretic analysis of the purified pl-LAM revealed the presence of a major 750-kDa component composed of 320-, 220-, and 200-kDa polypeptides and a minor 800-kDa component composed of 320-, 240-, and 220-kDa polypeptides. Neither molecule had a 400-kDa component corresponding to the A chain. It has already been shown that the 320-kDa polypeptide is identical to the M chain of human merosin (Hori et al. J. Biochem. 1994;116:1212-1219). Electron microscopy revealed that isolated merosin was composed of three short arms and one long arm. By immunohistochemistry, MAbs showed positive staining in human adult kidney and liver. These results indicate that these MAbs recognize only native merosin and can be used to study merosin structure and function by rapid purification of native merosin and by immunohistochemical analysis.     

Intact proinsulin, des 31,32 proinsulin, and specific insulin concentrations among nondiabetic and diabetic subjects in populations at varying risk of type 2 diabetes

Starting with crude yeast mitochondria, the intron homing endonuclease, I-SecIV, was purified to near homogeneity. This highly purified enzyme differs from some other well-characterized yeast mitochondrial intron-encoded endonucleases in terms of its structure and DNA cleavage specificity. The enzyme is a heterodimer with a native molecular mass of 92 kDa. A small catalytic subunit (32 kDa) is probably encoded largely or entirely by intron 5 alpha of the cytochrome oxidase subunit I gene. A larger polypeptide subunit (60 kDa) may be a nuclear factor necessary for intron mobility. I-SceIV exhibits a low DNA sequence specificity as it cleaves a variety of DNA substrates. Analysis of kinetic parameters shows that the purified enzyme has a very high affinity for DNA and exhibits low turnover which may have implications for subsequent steps in the intron homing process.     

A 17-amino acid insert changes UDP-N-acetylhexosamine pyrophosphorylase specificity from UDP-GalNAc to UDP-GlcNAc

We previously reported the purification of a UDP-N-acetylhexosamine (UDP-HexNAc) pyrophosphorylase from pig liver that catalyzed the synthesis of both UDP-GlcNAc and UDP-GalNAc from UTP and the appropriate HexNAc-1-P (Szumilo, T., Zeng, Y., Pastuszak, I., Drake, R., Szumilo, H., and Elbein, A. D. (1996) J. Biol. Chem. 271, 13147-13154). Both sugar nucleotides were synthesized at nearly the same rate, although the Km for GalNAc-1-P was about 3 times higher than for GlcNAc-1-P. Based on native gels and SDS-polyacrylamide gel electrophoresis, the enzyme appeared to be a dimer of 120 kDa composed of two subunits of about 57 and 64 kDa. Three peptides sequenced from the 64-kDa protein and two from the 57-kDa protein showed 100% identity to AGX1, a 57-kDa protein of unknown function from human sperm. An isoform called AGX2 is identical in sequence to AGX1 except that it has a 17-amino acid insert near the carboxyl terminus. We expressed the AGX1 and AGX2 genes in Escherichia coli. The protein isolated from the AGX1 clone comigrated on SDS gels with the liver 57-kDa pyrophosphorylase subunit and was 2-3 times more active with GalNAc-1-P than with GlcNAc-1-P. On the other hand, the protein from the AGX2 clone migrated with the liver 64-kDa pyrophosphorylase subunit and had 8-fold better activity with GlcNAc-1-P than with GalNAc-1-P. These results indicate that insertion of the 17-amino acid peptide modifies the specificity of the pyrophosphorylase from synthesis of UDP-GalNAc to synthesis of UDP-GlcNAc.     

Biosynthesis of chondroitin sulfate. Purification of glucuronosyl transferase II and use of photoaffinity labeling for characterization of the enzyme as an 80-kDa protein

A photoaffinity analogue, [beta-32P]5-azido-UDP-GlcA, was used to photolabel the enzymes that utilize UDP-GlcA in cartilage microsomes and rat liver microsomes. SDS-polyacrylamide gel electrophoresis analysis of photolabeled cartilage microsomes, which are specialized in chondroitin sulfate synthesis, showed a major radiolabeled band at 80 kDa and other minor radiolabeled bands near 40 and 60 kDa. Rat liver microsomes, which are enriched for enzymes of detoxification by glucuronidation, had a different pattern with multiple major labeled bands near 50-60 and 35 kDa. To determine that the photolabeled 80-kDa protein is the GlcA transferase II, we have purified the enzyme from cartilage microsomes. This membrane-bound enzyme, involved in the transfer of GlcA residues to non-reducing terminal GalNAc residues of the chondroitin polymer, has now been solubilized, stabilized, and then purified greater than 1350-fold by sequential chromatography on Q-Sepharose, heparin-Sepharose, and WGA-agarose. The purified enzyme exhibited a conspicuous silver-stained protein band on SDS-polyacrylamide gel electrophoresis that coincided with the major radiolabeled band of 80 kDa. SDS-polyacrylamide gel analysis of photoaffinity-labeled active fractions from the Q-Sepharose, heparin-Sepharose, and WGA-agarose also indicated only the single radiolabeled band at 80 kDa. Intensity of photolabeling in each of the fractions examined coincided with enzyme activity. The photolabeling of this 80-kDa protein was saturable with the photoprobe and could be inhibited by the addition of UDP-GlcA prior to the addition of the photoprobe. Thus, the photolabeling with [beta-32P]5-azido-UDP-GlcA has identified the GlcA transferase II as an 80-kDa protein. The purified enzyme was capable of transferring good amounts of GlcA residues to chondroitin-derived pentasaccharide with negligible transfer to pentasaccharides derived from hyaluronan or heparan.     

O-Acetylserine and O-acetylhomoserine sulfhydrylase of yeast. Subunit structure

The molecular weight of O-acetylserine (OAS)-O-acetylhomoserine (OAH) sulfhydrylase purified from yeast was estimated to be about 200,000 by Sephadex G-200 gel chromatography in various buffers. The S20, w value of this protein was determined to be about 9.0 by sucrose density gradient centrifugation. The calculated molecular weight based on this value was similar to that estimated by gel chromatography. Treatment with 1% sodium dodesylsulfate (SDS) or 6 M urea dissociated the enzyme into 4 subunits; these had a molecular weight estimated to be 51,000 by SDS-poly-acrylamide gel electrophoresis and to be 57,000 by Sephadex G-100 gel chromatography in the presence of 6 M urea and 0.5% beta-mercaptoethanol. The 4 subunits appeared to be identical, based on the symmetric subunit elution pattern from a Sephadex column, a single peptide band on SDS-polyacrylamide gel, and the detection of histidine as the sole N-terminal amino acid in the native enzyme. Since dissociation into the subunits occurred without the use of reducing agents, the association of the subunits seems to require no disulfide linkage. One mole of the subunit contained one mole of sulfhydryl group which appeared to be buried inside the molecule. Partial restoration of the catalytic activity was observed when the urea-denatured enzyme was dialyzed to remove urea, especially in the presence of reducing agents such as dithiothreitol. The urea-denatured enzyme showed a tendency in the absence of reducing agents to form a subunit dimer linked by a disulfide bond between the cystine residues exposed by denaturation. The amino acid composition of the enzyme was determined; it contained one half-cystine residue per subunit, and the content of acidic residues was much higher than that of basic residues. Based on these findings, the subunit structure of the enzyme is discussed.     

Hominid thumb strength predicted by high resolution magnetic resonance imaging and force measurements in living subjects

The domestic cat (Felis domesticus) is an important source of indoor allergens, the major allergen being Fel d1 (formerly cat allergen 1). Fel d1 is responsible for cat allergy and has also been established to cause cat-induced asthma. The allergen is a 38 kDa dimer composed of two 19 kDa subunits. Each 19 kDa subunit comprises two disulfide linked polypeptide chains, a light alpha-chain and a heavy beta-chain containing an N-linked oligosaccharide. In this study a variety of endoproteinase digestions of the native allergen in combination with HPLC and matrix-assisted laser desorption mass spectrometry was used to determine the position of the disulfide bridges and to demonstrate that the peptide chains are linked in an anti parallel way. Enzymatic digestion of the reduced and alkylated peptides located the N-glycan to Asn33. Moreover, Fel d1 is found to be partially truncated and to exist in several isoforms. Sequential degradation of the glycosylated peptide with specific glycosidases monitored by mass spectrometry, shows that the glycan is a heterogeneous triantennary complex type structure. The heterogeneity is caused by terminal sialic acid and a fucose residue attached to a beta-galactose residue.     

Cysteinyl-tRNA synthetase from Saccharomyces cerevisiae. Purification, characterization and assignment to the genomic sequence YNL247w

Cysteinyl-tRNA synthetase (CRS) from Saccharomyces cerevisiae was purified 2300-fold with a yield of 33%, to a high specific activity (kcat4.3 s-1 at 25 degrees C for the aminoacylation of yeast tRNACys). SDS-PAGE revealed a single polypeptide corresponding to a molecular mass of 86 kDa. Polyclonal antibodies to the purified protein inactivated CRS activity and detected only one polypeptide of 86 kDa in a yeast extract subjected to SDS-PAGE followed by immunoblotting. In contrast to bacterial CRS which is a monomer of about 50 kDa, the native yeast enzyme behaved as a dimer, as assessed by gel filtration and cross-linking. Its subunit molecular mass is in good agreement with the value of 87.5 kDa calculated for the protein encoded by the yeast genomic sequence YNL247w. The latter was previously tentatively assigned to CRS, based on limited sequence similarities to the corresponding enzyme from other sources. Determination of the amino acid sequence of internal polypeptides derived from the purified yeast enzyme confirmed this assignment. Alignment of the primary sequences of prokaryotic and yeast CRS reveals that the larger size of the latter is accounted for mostly by several insertions within the sequence.     

Regulation of ribosomal RNA gene transcription during retinoic acid-induced differentiation of mouse teratocarcinoma cells

The human cytomegalovirus glycoprotein B gene (gB; gpUL55) was truncated at amino acid 692 and recombined into Autographa californica nuclear polyhedrosis virus (baculovirus). Infection of insect cells with the recombinant baculovirus resulted in high-level expression and secretion of the truncated gB protein (gB-S) into the culture medium. Purification of gB-S by monoclonal antibody affinity chromatography yielded a protein of ca. 200 kDa. Characterization of the 200-kDa purification product indicated that the recombinant gB protein retained many structural and functional features of the viral gB. Comparison of electrophoretic migration patterns in reduced versus nonreduced protein samples and immune blotting analysis with antibodies specific for the amino or carboxy-terminus of gB demonstrated that the recombinant protein was composed of disulfide linked 69 kDa amino terminal and 35-kDa carboxy-terminal fragments. In addition, recognition of the 200-kDa gB-S by a conformational-dependent, oligomer-specific monoclonal antibody suggested that gB-S was properly folded and dimeric. Like the viral gB, gB-S had heparin binding ability. One heparin binding site was found to reside within the 35-kDa carboxy-terminal fragment (aa 492-692). Heparin binding was abolished when gB-S was denatured. These data suggest that gB contains a novel heparin binding motif that is at least partially conformational dependent.     

Biochemical and genetic characterization of trans-2'-carboxybenzalpyruvate hydratase-aldolase from a phenanthrene-degrading Nocardioides strain

trans-2'-Carboxybenzalpyruvate hydratase-aldolase was purified from a phenanthrene-degrading bacterium, Nocardioides sp. strain KP7, and characterized. The purified enzyme was found to have molecular masses of 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography. Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme. The Km and kcat values of this enzyme for trans-2'-carboxybenzalpyruvate were 50 microM and 13 s(-1), respectively. trans-2'-Carboxybenzalpyruvate was transformed to 2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The structural gene for this enzyme was cloned and sequenced; the length of this gene was 996 bp. The deduced amino acid sequence of this enzyme exhibited homology to those of trans-2'-hydroxybenzalpyruvate hydratase-aldolases from Pseudomonas putida PpG7 and Pseudomonas sp. strain C18.     

Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus

Cell-free extracts of cellobiose-grown cells of the hyperthermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a beta-glucosidase. The cytoplasmic enzyme was purified 22-fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein. The native beta-glucosidase has a molecular mass of 230 +/- 20 kDa, composed of 58 +/- 2-kDa subunits. The enzyme has a pI of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH 5.0 and 102-105 degrees C. From Lineweaver-Burk plots, Vmax values of 470 U/mg and 700 U/mg were found for cellobiose (Km = 20 mM) and p-nitrophenyl-beta-D-glucopyranoside (Km = 0.15 mM), respectively. The purified enzyme also exhibits high beta-galactosidase activity and beta-xylosidase activity, but shows no activity towards alpha-linked disaccharides or beta-linked polymers, like cellulose. The purified beta-glucosidase shows a remarkable thermostability with a half life of 85 h at 100 degrees C and 13 h at 110 degrees C.     

Evidence that the thyrotropin receptor ectodomain contains not one, but two, cleavage sites

TSH receptor (TSHR) cleavage into two subunits (A and B) was explored using two new mammalian cell lines expressing the recombinant receptor; 1) TSHR-10,000 CHO cells overexpressing the TSHR; 2) TSHRmyc cells with a c-myc epitope inserted at residues 338-349. Immunoprecipitation or immunoblotting of TSHR-10,000 cells with mAb to either the A subunit or the B subunit revealed multiple forms of the TSHR: 1) uncleaved receptors of approximately 115 kDa and approximately 100 kDa with complex carbohydrate and high mannose carbohydrate, respectively; 2) two subunit TSHR with an approximately 62 kDa A subunit containing complex carbohydrate. The A subunit was approximately 35 kDa after enzymatic deglycosylation (predicted C-terminus near residue 330). The nonglycosylated B subunit was evident primarily as an approximately 42 kDa band (predicted N terminus near residue 380). The sum of the A and B subunit polypeptide backbones was smaller than the predicted size of the TSHR, a polypeptide backbone (84.5 kDa), raising the possibility that an approximately 5-kDa polypeptide fragment was excised during intramolecular cleavage. This hypothesis was supported by data obtained with the TSHRmyc cells. Thus, mAb to the c-myc epitope and to amino acid residues 22-35 (mAb A10) were equally effective in detecting the single chain forms of the TSHR in these cells. However, the 35 kDa, deglycosylated A subunit was clearly visible on immunoprecipitation with mAb A10 to the TSHR amino terminus, but not with the anti-myc mAb, indicating loss of the c-myc epitope at residues 338-349. Further, even though the A subunit was not detected in TSHRmyc cells with anti-myc mAb, 125I-TSH cross-linking to the cell surface showed similar A subunit expression in TSHRmyc and wild-type TSHR expressing cells. In summary, our study provides a surprising and novel finding for G protein-coupled receptors. Contrary to the prevailing concept of one cleavage site in the TSHR, we present evidence that there are, in fact, two such sites. The TSHR, like insulin, may release a C peptide during intramolecular cleavage into two subunits.     

Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme

A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min-1 (mg protein)-1. The dismutase obeyed Michaelis-Menten kinetics. The Vmax and Km calculated for chlorite were 2,200 U (mg protein)-1 and 170 microM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole. Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase displayed maxima for activity at pH 6.0 and 30 degrees C.     

Continuous administration of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate produces striatal lesion

Mammalian phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in the regulation of various cellular, receptor tyrosine kinase-mediated processes, such as mitogenesis and transformation. PI 3-kinase is composed of a 110-kDa catalytic subunit and a regulatory subunit of 85 kDa or 55 kDa. We have cloned a gene for a regulatory subunit from Drosophila melanogaster, named droPIK57, from head-specific cDNA libraries. The droPIK57 gene encodes a protein containing two SH2 domains with significant sequence homology to those in p85 and p55. Like the p55 subunits, DroPIK57 is missing the SH3 domain and the bcr homology region of the p85 subunit. The short N-terminus as well as the C-terminus of the DroPIK57 protein show no identity to the known PI 3-kinase subunits, suggesting that it is a new member in the family of regulatory subunits. In-situ hybridization and Northern blot analysis indicate a widespread function of this gene during embryogenesis and in the CNS.     

Purification and characterization of an NAD-malic enzyme from Bradyrhizobium japonicum A1017

An NAD-malic enzyme was purified to homogeneity from Bradyrhizobium japonicum A1017, and its molecular characteristics were surveyed. The enzyme exhibited native and subunit molecular masses of 388 and 85 kDa, respectively, suggesting that it exists as a homotetramer, and was activated by metabolic intermediates in glycolysis. The role of the enzyme in bacteroids' carbon metabolism is discussed.     

Trypsin cleavage of human cystathionine beta-synthase into an evolutionarily conserved active core: structural and functional consequences

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine and serine to cystathionine-an irreversible step in the eukaryotic transsulfuration pathway. The native enzyme is a homotetramer or multimer of 63-kDa (551 amino acids) subunits and is activated by S-adenosyl-l-methionine (AdoMet) or by partial cleavage with trypsin. Amino-terminal analysis of the early products of trypsinolysis demonstrated that the first cleavages occur at Lys 30, 36, and 39. The enzyme still retains the subunit organization as a tetramer or multimer composed of 58-kDa subunits. Analysis by electrospray ionization mass spectrometry showed that further trypsin treatment cleaves CBS in its COOH-terminal region at Arg 413 to yield 45-kDa subunits. This 45-kDa active core is the portion of CBS most conserved with the evolutionarily related enzymes isolated from plants, yeast, and bacteria. The active core of CBS forms a dimer of approximately 85 kDa. The dimer is about twice as active as the tetramer. It binds both pyridoxal 5'-phosphate and heme cofactors but is no longer activated by AdoMet. Further analysis suggests that the dissociation of CBS to dimers causes a decrease in enzyme thermostability and a threefold increase in affinity toward the sulfhydryl-containing substrate-homocysteine. We found that the COOH-terminal region, residues 414-551, is essential for maintaining the tetrameric structure and AdoMet activation of the enzyme. The inability of the active core to form multimeric aggregates has facilitated its crystallization and X-ray diffraction studies.     

Human thioredoxin reductase from HeLa cells: selective alkylation of selenocysteine in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin

Human thioredoxin reductase (TR) contains selenocysteine (Secys) in a redox center [cysteine (Cys)-497,Secys-498] near the C-terminus. The essential role of Secys in TR isolated from HeLa cells was demonstrated by the alkylation studies. Reaction of native NADPH reduced enzyme with bromoacetate at pH 6.5 inhibited enzyme activity 99%. Of the incorporated carboxymethyl (CM) group, 1.1 per subunit, >90% was in CM-Secys-498. Alkylation at pH 8 increased the stoichiometry to 1.6 per subunit with additional modification of the Cys-59, Cys-64 disulfide center. A minor tryptic peptide containing both CM-Cys-497 and CM-Secys-498 was isolated from enzyme alkylated at pH 6.5 or at pH 8. Preparations of TR isolated from HeLa cells grown in a fermentor under high aeration contained selenium-deficient enzyme species that had 50% lower activity. Decreasing oxygen to an optimal level increased cell yield, and fully active TR containing one Se per subunit was present. Reduction of fully active enzyme with tris-(2-carboxyethyl) phosphine converted it from a low to a high heparin affinity form. The tris-(2-carboxyethyl) phosphine-reduced enzyme was oxygen-sensitive and lost selenium and catalytic activity unless maintained under strictly anaerobic conditions. This enzyme could be converted to an oxygen-insensitive species by addition of NADPH, indicating that bound pyridine nucleotide is important for enzyme stability. An induced enzyme conformation in which the essential Secys is shielded from oxidative damage could explain these effects.