Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 18 de 18
Filter
Add more filters










Publication year range
1.
J Mol Biol ; 400(3): 518-30, 2010 Jul 16.
Article in English | MEDLINE | ID: mdl-20546754

ABSTRACT

Yeast flavocytochrome b(2) (Fcb2) is an L-lactate:cytochrome c oxidoreductase in the mitochondrial intermembrane space participating in cellular respiration. Each enzyme subunit consists of a cytochrome b(5)-like heme domain and a flavodehydrogenase (FDH) domain. In the Fcb2 crystal structure, the heme domain is mobile relative to the tetrameric FDH core in one out of two subunits. The monoclonal antibody B2B4, elicited against the holoenzyme, recognizes only the native heme domain in the holoenzyme. When bound, it suppresses the intramolecular electron transfer from flavin to heme b(2), hence cytochrome c reduction. We report here the crystal structure of the heme domain in complex with the Fab at 2.7 A resolution. The Fab epitope on the heme domain includes the two exposed propionate groups of the heme, which are hidden in the interface between the domains in the complete subunit. The structure discloses an unexpected plasticity of Fcb2 in the neighborhood of the heme cavity, in which the heme has rotated. The epitope overlaps with the docking area of the FDH domain onto the heme domain, indicating that the antibody displaces the heme domain in a movement of large amplitude. We suggest that the binding sites on the heme domain of cytochrome c and of the FDH domain also overlap and therefore that cytochrome c binding also requires the heme domain to move away from the FDH domain, so as to allow electron transfer between the two hemes. Based on this hypothesis, we propose a possible model of the Fcb2.cytochrome c complex. Interestingly, this model shares similarity with that of the cytochrome b(5) x cytochrome c complex, in which cytochrome c binds to the surface around the exposed heme edge of cytochrome b(5). The present results therefore support the idea that the heme domain mobility is an inherent component of the Fcb2 functioning.


Subject(s)
L-Lactate Dehydrogenase (Cytochrome)/chemistry , L-Lactate Dehydrogenase (Cytochrome)/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Antibodies, Fungal/immunology , Antibodies, Fungal/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Binding Sites , Crystallography, X-Ray , Electron-Transferring Flavoproteins/chemistry , Electron-Transferring Flavoproteins/immunology , Electron-Transferring Flavoproteins/metabolism , Heme/chemistry , Heme/metabolism , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/metabolism , L-Lactate Dehydrogenase (Cytochrome)/immunology , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/immunology , Mitochondrial Proteins/metabolism , Models, Biological , Models, Chemical , Models, Molecular , Motion , Protein Binding , Protein Conformation , Protein Structure, Quaternary , Saccharomyces cerevisiae Proteins/immunology
2.
Biochemistry ; 48(45): 10803-9, 2009 Nov 17.
Article in English | MEDLINE | ID: mdl-19821613

ABSTRACT

Each flavocytochrome b(2) (l-lactate cytochrome c oxidoreductase) subunit consists of an N-terminal cytochrome domain and a C-terminal flavodehydrogenase (FDH) domain. In the enzyme crystal structure, only two heme domains are visible per enzyme tetramer, because of the mobility of the other two heme domains relative to the FDH domains. Evidence was subsequently provided that this mobility also exists in solution. Numerous kinetic studies showed that, during the catalytic cycle, electrons are transferred one by one from the reduced flavin to heme b(2) in the same subunit. In previous work, we provided evidence that a monoclonal antibody that abolishes flavin to heme electron transfer uses part of the interdomain interface for binding to its antigen, the native heme domain. In this work, we use a number of heme domain side chain substitutions in and around the interface to probe their effect on flavin to heme electron transfer. Using steady-state and pre-steady-state kinetics, as well as redox potential determinations and EPR measurements, we define several hydrophobic interactions and van der Waals contacts that are important for a catalytically competent docking of the heme domain onto the FDH domain. In addition, with several extremely slow mutant enzymes, we propose an isosbestic wavelength between oxidized and reduced heme for specifically following the kinetics of flavosemiquinone formation from two-electron reduced flavin.


Subject(s)
L-Lactate Dehydrogenase (Cytochrome)/metabolism , Binding Sites , Electron Spin Resonance Spectroscopy , Flavins/metabolism , Kinetics , L-Lactate Dehydrogenase (Cytochrome)/genetics , Mutagenesis, Site-Directed , Oxidation-Reduction
3.
Biochemistry ; 46(15): 4661-70, 2007 Apr 17.
Article in English | MEDLINE | ID: mdl-17373777

ABSTRACT

Saccharomyces cerevisiae flavocytochrome b2 (L-lactate:cytochrome c oxido reductase, EC 1.1.2.3) is a homotetramer, with FMN and protoheme IX binding on separate domains. The flavin-binding domains form the enzyme tetrameric core, while the cytochrome b2 domains appear to be mobile around a hinge region (Xia, Z. X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 867-863). The enzyme catalyzes electron transfer from L-lactate to cytochrome c, or to nonphysiological acceptors such as ferricyanide, via FMN and heme b2. The kinetics of this multistep reaction are complex. In order to clarify some of its aspects, the tetrameric FMN-binding domain (FDH domain) has been independently expressed in Escherichia coli (Balme, A., Brunt, C. E., Pallister, R., Chapman, S. K., and Reid, G. A. (1995) Biochem. J. 309, 601-605). We present here an additional characterization of this domain. In our hands, it has essentially the same ferricyanide reductase activity as the holo-enzyme. The comparison of the steady-state kinetics with ferricyanide as acceptor and of the pre-steady-state kinetics of flavin reduction, as well as the kinetic isotope effects of the reactions using L-2-[2H]lactate, indicates that flavin reduction is the limiting step in lactate oxidation. During the oxidation of the reduced FDH domain by ferricyanide, the oxidation of the semiquinone is much faster than the oxidation of two-electron-reduced flavin. This order of reactivity is reversed during flavin to heme b2 transfer in the holo-enzyme. Potentiometric studies of the protein yielded a standard redox potential for FMN at pH 7.0, E(o)7, of -81 mV, a value practically identical to the published value of -90 mV for FMN in holo-flavocytochrome b2. However, as expected from the kinetics of the oxidative half-reaction, the FDH domain was characterized by a significantly destabilized flavin semiquinone state compared with holo-enzyme, with a semiquinone formation constant K of 1.25-0.64 vs 33.5, respectively (Tegoni, M., Silvestrini, M. C., Guigliarelli, B., Asso, M., and Bertrand, P. (1998) Biochemistry, 37, 12761-12771). As in the holo-enzyme, the semiquinone state in the FDH domain is significantly stabilized by the reaction product, pyruvate. We also studied the inhibition exerted in the steady and pre steady states by the reaction product pyruvate and by anions (bromide, chloride, phosphate, acetate), with respect to both flavin reduction and reoxidation. The results indicate that these compounds bind to the oxidized and the two-electron-reduced forms of the FDH domain, and that excess L-lactate also binds to the two-electron-reduced form. These findings point to the existence of a common or strongly overlapping binding site. A comparison of the effect of the anions on WT and R289K holo-flavocytochromes b2 indicates that invariant R289 belongs to this site. According to literature data, it must also be present in other members of the family of L-2-hydroxy acid-oxidizing enzymes.


Subject(s)
Anions/chemistry , Flavins/chemistry , L-Lactate Dehydrogenase (Cytochrome)/chemistry , Saccharomyces cerevisiae/enzymology , Algorithms , Anions/metabolism , Binding Sites , Ferricyanides/metabolism , Flavin Mononucleotide/chemistry , Flavin Mononucleotide/metabolism , Flavin-Adenine Dinucleotide/analogs & derivatives , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Flavins/metabolism , Kinetics , L-Lactate Dehydrogenase (Cytochrome)/metabolism , Models, Chemical , Models, Molecular , NADH, NADPH Oxidoreductases/metabolism , Oxidation-Reduction , Protein Binding , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism
4.
Biochimie ; 88(12): 1961-71, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17010495

ABSTRACT

Lafora disease is a progressive myoclonus epilepsy with an early fatal issue. Two genes were identified thus far, the mutations of which cause the disease. The first one, EPM2A, encodes the consensus sequence of a protein tyrosine phosphatase. Its product, laforin, is the object of the present work. We analysed in detail the amino acid sequence of this protein. This suggested, as also observed by others, that it could present two domains, a carbohydrate-binding domain (CBM20, known as a starch-binding domain) and the catalytic domain of a dual-specificity protein phosphatase. We produced the enzyme as two different GST-fused proteins and as an N-terminally His-tagged protein. Differences in solubility were observed between the constructs. Moreover, the N-terminal carbohydrate-binding domain contains a thrombin cleavage site, which is hidden in the simplest GST-fusion protein we produced, but was accessible after introducing a five-residue linker between the engineered cleavage site and the enzyme N-terminus. The two types of constructs hydrolyse pNPP and OMFP with kinetic parameters consistent with those of a dual-specificity phosphatase. We show in addition that the protein not only binds glycogen, but also starch, amylose and cyclodextrin. Neither binding of glycogen nor of beta-cyclodextrin appreciably affects the phosphatase activity. These results suggest that the role of the N-terminal domain is rather that of targeting the protein in the cell, probably to glycogen and the protein complexes attached to it, rather than that of directly modulating the catalytic activity.


Subject(s)
Lafora Disease/enzymology , Protein Tyrosine Phosphatases/metabolism , Amino Acid Sequence , Blotting, Western , Glycogen/metabolism , Humans , Kinetics , Lafora Disease/genetics , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Tertiary , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases, Non-Receptor , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity
5.
Biochemistry ; 44(5): 1521-31, 2005 Feb 08.
Article in English | MEDLINE | ID: mdl-15683236

ABSTRACT

Long chain hydroxy acid oxidase (LCHAO) is a member of an FMN-dependent enzyme family that oxidizes L-2-hydroxy acids to ketoacids. LCHAO is a peroxisomal enzyme, and the identity of its physiological substrate is unclear. Mandelate is the most efficient substrate known and is commonly used in the test tube. LCHAO differs from most family members in that one of the otherwise invariant active site residues is a phenylalanine (Phe23) instead of a tyrosine. We now report the crystal structure of LCHAO. It shows the same beta8alpha8 TIM barrel structure as other structurally characterized family members, e.g., spinach glycolate oxidase (GOX) and the electron transferases yeast flavocytochrome b2 (FCB2) and Pseudomonas putida mandelate dehydrogenase (MDH). Loop 4, which is mobile in other family members, is visible in part. An acetate ion is present in the active site. The flavin interacts with the protein in the same way as in the electron transferases, and not as in GOX, an unexpected observation. An interpretation is proposed to explain this difference between GOX on one hand and FCB2 and LCHAO on the other hand, which had been proposed to arise from the differences between family members in their reactivity with oxygen. A comparison of models of the substrate bound to various published structures suggests that the very different reactivity with mandelate of LCHAO, GOX, FCB2, and MDH cannot be rationalized by a hydride transfer mechanism.


Subject(s)
Alcohol Oxidoreductases/chemistry , Kidney/enzymology , Recombinant Proteins/chemistry , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Isoenzymes/chemistry , Molecular Sequence Data , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity
6.
Biochem J ; 373(Pt 1): 115-23, 2003 Jul 01.
Article in English | MEDLINE | ID: mdl-12646042

ABSTRACT

Flavocytochrome b(2) (yeast L-lactate dehydrogenase) carries one FMN and one protohaem IX on each of its four subunits. The prosthetic groups are bound to separate domains, the haem domain (residues 1-99) and the flavin domain (residues 100-485), which interact for electron transfer between lactate-reduced FMN and haem b(2); in vivo, the latter reduces cytochrome c. In the crystal structure, one haem domain out of two is mobile. Previously we have described a monoclonal antibody, raised against the tetramer, that only recognizes the native haem domain and prevents electron transfer between flavin and haem, while having no effect on flavin reduction by the substrate [Miles, Lederer and Lê (1998) Biochemistry 37, 3440-3448]. In order to understand the structural basis of the uncoupling between the domains, we proceeded to site-directed mutagenesis, so as to map the epitope on the surface of the haem domain. We analysed the effects of 14 mutations at 12 different positions, located mostly in the domain interface or at its edge; we also analysed the effect of replacing protohaem IX with its dimethyl ester. We used as criteria the antibody-mediated inhibition of cytochrome c reduction by flavocytochrome b(2), competitive ELISA tests and surface plasmon resonance. We have thus defined a minimal epitope surface on the haem domain; it encompasses positions 63, 64, 65, 67, 69 and 70 and one or both haem propionates. When the haem and flavin domains are docked for electron transfer, the 65, 67 and 70 side chains, as well as the haem propionates, are excluded from solvent. The present results thus indicate that, when bound, the antibody acts as a wedge between the domains and constitutes a physical barrier to electron transfer.


Subject(s)
Antibodies, Monoclonal/pharmacology , L-Lactate Dehydrogenase/chemistry , L-Lactate Dehydrogenase/metabolism , Amino Acid Substitution , Base Sequence , Crystallography, X-Ray , DNA Primers , Electron Transport/drug effects , Enzyme-Linked Immunosorbent Assay , Epitopes/analysis , Epitopes/chemistry , Kinetics , L-Lactate Dehydrogenase/antagonists & inhibitors , L-Lactate Dehydrogenase (Cytochrome) , Models, Molecular , Molecular Weight , Mutagenesis, Site-Directed , Oxidation-Reduction , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Restriction Mapping
7.
J Vet Med Sci ; 61(12): 1313-5, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10651052

ABSTRACT

A serosurvey of feline herpesvirus type 1 (FHV-1), feline calicivirus (FCV), and feline parvovirus (FPV) in cats from Ho Chi Minh City area in southern Vietnam was conducted in December 1998, and we compared the results with our previous results in northern Vietnam (Hanoi area). The positive rate of FHV and FCV in domestic cats were 44% and 74%, respectively. They were rather higher than those in Hanoi area, while the seropositivity of FPV (44%) was similar to that in Hanoi area. In leopard cats, the positive rate of FPV was high (3/4) and it indicated that FPV was prevailing in leopard cats in Vietnam.


Subject(s)
Caliciviridae Infections/veterinary , Calicivirus, Feline , Carnivora , Cat Diseases/epidemiology , Feline Panleukopenia/epidemiology , Herpesviridae Infections/veterinary , Animals , Animals, Domestic , Animals, Wild , Antibodies, Viral/blood , Caliciviridae Infections/epidemiology , Calicivirus, Feline/immunology , Cat Diseases/virology , Cats , Feline Panleukopenia Virus/immunology , Female , Fluorescent Antibody Technique, Indirect/veterinary , Herpesviridae/immunology , Herpesviridae Infections/epidemiology , Male , Prevalence , Seroepidemiologic Studies , Vietnam/epidemiology
9.
Biochemistry ; 37(10): 3440-8, 1998 Mar 10.
Article in English | MEDLINE | ID: mdl-9521665

ABSTRACT

Flavocytochrome b2 or L-lactate dehydrogenase from yeast is a tetrameric enzyme which oxidizes lactate at the expense of cytochrome c or artificial electron acceptors. The prosthetic group FMN is reduced by the substrate and then transfers sequentially the reducing equivalents to heme b2 in the same subunit. The latter is reoxidized by cytochrome c. The crystal structure of the enzyme indicates that each subunit is composed of a flavodehydrogenase domain (FDH) and a cytochrome b2 domain; the latter, which encompasses the first 99 residues of the peptide chain, is mobile relative to the tetrameric FDH assembly. We describe here the properties of a monoclonal antibody elicited against the holoenzyme. It only recognizes the heme-binding domain, with a Kd lower than 10(-7) M, and its epitope is conformational. In the enzyme-IgG complex, flavin is reduced normally and can be reoxidized by ferricyanide, but no longer by heme b2. Stopped-flow experiments in the absence of electron acceptors give no indication of flavin to heme electron transfer in the enzyme-antibody complex. In other words, the two domains are functionally uncoupled. The binding stoichiometry is 1/1 for the Fab fragment with respect to the isolated, monomeric, heme-binding domain, but 2/4 with respect to the enzyme tetramer; furthermore, binding of two Fab fragments per tetramer is sufficient to cause inhibition of intra-subunit flavin to heme electron transfer in all four subunits. Altogether these results can only be rationalized by considering that mobility of the cytochrome domain with respect to the FDH is an essential component of the catalytic cycle. The first experiment designed to locate the epitope shows it does not encompass the interdomain peptide linker (so-called hinge region, centered on residues 99-100).


Subject(s)
Antibodies, Monoclonal , L-Lactate Dehydrogenase/chemistry , L-Lactate Dehydrogenase/metabolism , Animals , Binding Sites , Cytochrome c Group/metabolism , Electron Transport , Epitopes/chemistry , Escherichia coli/genetics , Ferricyanides/metabolism , Heme/chemistry , Immunochemistry , Kinetics , L-Lactate Dehydrogenase/immunology , L-Lactate Dehydrogenase (Cytochrome) , Mice , Models, Molecular , Pichia/enzymology , Pichia/genetics , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics
11.
Protein Sci ; 4(5): 925-35, 1995 May.
Article in English | MEDLINE | ID: mdl-7663348

ABSTRACT

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from Saccharomyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable level as recombinant proteins in Escherichia coli (20-25 mg per liter culture) with a full complement of prosthetic groups. At the same expression level, active-site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN. Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis. Flavin-free and holo-forms for both mutants could be separated on a Blue-Trisacryl M column. The far-UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild-type enzyme, suggesting the existence of only local conformational differences between the active holo-enzymes and the nonreconstitutable flavin-free forms. Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo-enzymes as in the wild-type one, in the protease-sensitive loop. In contrast, for the flavin-free forms of both mutants, cleavage occurred at more than a single bond. Identification of the cleaved bonds suggested that the structural differences between the mutant flavin-free and holo-forms are located mostly at the C-terminal end of the barrel, which carries the prosthetic group and the active site. Altogether, these findings suggest that the two mutations induce an alteration of the protein-folding process during biosynthesis in E. coli; as a result, the synchrony between folding and flavin insertion is lost. Finally, a preliminary kinetic characterization of the mutant holo-forms showed the Km value for lactate to be little affected; kcat values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild-type enzyme.


Subject(s)
Flavin Mononucleotide/metabolism , L-Lactate Dehydrogenase/biosynthesis , Protein Folding , Amino Acid Sequence , Apoproteins/chemistry , Apoproteins/genetics , Apoproteins/metabolism , Base Sequence , Binding Sites , Chymotrypsin/metabolism , Circular Dichroism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial/genetics , Hemeproteins/chemistry , Hemeproteins/genetics , Kinetics , L-Lactate Dehydrogenase/chemistry , L-Lactate Dehydrogenase/genetics , L-Lactate Dehydrogenase/metabolism , L-Lactate Dehydrogenase (Cytochrome) , Models, Chemical , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation/genetics , Protein Denaturation , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Saccharomyces cerevisiae/chemistry
12.
Eur J Biochem ; 214(1): 17-25, 1993 May 15.
Article in English | MEDLINE | ID: mdl-8508789

ABSTRACT

Long-chain L-alpha-hydroxy acid oxidase from rat kidney is a member of the family of FMN-dependent alpha-hydroxy-acid-oxidizing enzymes. With the knowledge of the recently determined amino acid sequence, the cDNA encoding the enzyme has now been cloned using the polymerase chain reaction. The 1648-bp cDNA contains an open reading frame coding for the 352 residues of the previously determined sequence, preceded by a methionine codon. In addition, several clones were found to present a nine-base insertion, predicting the existence of an isoform with a tripeptide VRK inserted between residues 188 and 189 of the mature protein. The presence of about 10% of this isoform in the oxidase purified from rat kidney was indeed identified by amino acid sequencing. A recombinant active enzyme was obtained as a protein fused to glutathione S-transferase using the bacterial expression plasmid pGEX-3X. Physico-chemical characterization indicated, for the fused enzyme, properties similar to those of the rat kidney protein. When the chimaera was submitted to factor Xa, proteolysis at the engineered cleavage point was poor. Separation of hydroxy acid oxidase from glutathione S-transferase could not be achieved with trypsin either. With both proteases, the initial cleavage point appeared to be in a peptide loop internal to the hydroxy acid oxidase sequence, close to or in the tripeptide insertion locus and not at the engineered factor-Xa-cleavage point. Comparative tryptic proteolysis of the rat kidney enzyme yielded a form cleaved in the same loop.


Subject(s)
Alcohol Oxidoreductases/biosynthesis , Cloning, Molecular , DNA/genetics , Kidney/enzymology , Recombinant Fusion Proteins/biosynthesis , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/isolation & purification , Alcohol Oxidoreductases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , DNA/chemistry , DNA/isolation & purification , Escherichia coli/enzymology , Escherichia coli/genetics , Glutathione Transferase , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Restriction Mapping , Tissue Distribution
13.
J Biol Chem ; 266(31): 20877-81, 1991 Nov 05.
Article in English | MEDLINE | ID: mdl-1939137

ABSTRACT

The complete amino acid sequence of rat kidney long chain alpha-hydroxy acid oxidase has been determined by microsequencing, using a number of standard enzymatic and chemical cleavages. Peptides were purified by high pressure liquid chromatography or by gel electrophoresis followed by electrotransfer. The sequence comprises 352 residues and ends with a peroxisomal targeting sequence SRL. The present work definitely establishes that hydroxy acid oxidase is a member of the family of FMN-dependent alpha-hydroxy acid-oxidizing enzymes. The family includes lactate oxidase, short chain alpha-hydroxy acid oxidase (glycolate oxidase), flavocytochrome b2, and mandelate dehydrogenase. There are altogether 45 totally conserved positions among the six sequences known. The sequence similarities are analyzed in light of the known three-dimensional structure of flavocytochrome b2 and glycolate oxidase. It is concluded that long chain hydroxy acid oxidase should be folded as a beta 8 alpha 8 barrel and should dehydrogenate alpha-hydroxy acids according to the same chemical mechanism as other enzymes of the family, in spite of a Tyr----Phe substitution at the active site.


Subject(s)
Alcohol Oxidoreductases/chemistry , Kidney/enzymology , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Binding Sites , Glucose Oxidase/chemistry , L-Lactate Dehydrogenase/chemistry , L-Lactate Dehydrogenase (Cytochrome) , Mixed Function Oxygenases/chemistry , Molecular Sequence Data , Peptides/chemistry , Rats , Sequence Alignment
14.
Biochem J ; 277 ( Pt 1): 39-45, 1991 Jul 01.
Article in English | MEDLINE | ID: mdl-1854346

ABSTRACT

Protamine P2, the major basic chromosomal protein of mouse spermatozoa, is synthesized as a precursor almost twice as long as the mature protein, its extra length arising from an N-terminal extension of 44 amino acid residues. This precursor is integrated into chromatin of spermatids, and the extension is processed during chromatin condensation in the haploid cells. We have studied processing in the mouse and have identified two intermediates generated by proteolytic cleavage of the precursor. H.p.l.c. separated protamine P2 from four other spermatid proteins, including the precursor and three proteins known to possess physiological characteristics expected of processing intermediates. Peptide mapping indicated that all of these proteins were structurally similar. Two major proteins were further purified by PAGE, transferred to poly(vinylidene difluoride) membranes and submitted to automated N-terminal sequence analysis. Both sequences were found within the deduced sequence of the precursor extension. The N-terminus of the larger intermediate, PP2C, was Gly-12, whereas the N-terminus of the smaller, PP2D, was His-21. Both processing sites involved a peptide bond in which the carbonyl function was contributed by an acidic amino acid.


Subject(s)
Epididymis/metabolism , Protamines/genetics , Protein Precursors/genetics , Protein Processing, Post-Translational , Testis/metabolism , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Molecular Weight , Peptide Fragments/isolation & purification , Peptide Mapping
15.
Biochem J ; 263(3): 929-35, 1989 Nov 01.
Article in English | MEDLINE | ID: mdl-2532007

ABSTRACT

A 32 kDa protein isolated from human mononuclear cells is a member of the lipocortin family, a new group of Ca2+-dependent lipid-binding proteins thought to be involved in the regulation of phospholipase A2, in exocytosis and in membrane-cytoskeleton interactions. Purification of this protein was based on its ability to associate with membrane phospholipids in a Ca2+-dependent manner and its capacity to inhibit purified phospholipase A2 from pig pancreas. Using immunological detection, we show that it is present in various cells involved in the inflammatory and coagulation processes. We present extensive amino acid data that strongly suggest that this protein is identical with a recently described inhibitor of blood coagulation, with endonexin II and with lipocortin V. Sequence alignment with other known proteins show a significant degree of homology with lipocortins I and II, the substrates of the epidermal-growth-factor receptor tyrosine kinase and the oncogene pp60src tyrosine kinase respectively, and with protein II. The possible physiological role of this 32 kDa lipocortin is discussed.


Subject(s)
Anticoagulants , Calcium-Binding Proteins/metabolism , Leukocytes, Mononuclear/analysis , Amino Acid Sequence , Annexin A5 , Blotting, Western , Calcium-Binding Proteins/blood , Cell Membrane/metabolism , Escherichia coli/metabolism , Molecular Sequence Data , Molecular Weight , Phospholipids/metabolism
16.
Science ; 246(4931): 803-6, 1989 Nov 10.
Article in English | MEDLINE | ID: mdl-2683083

ABSTRACT

Thymotaxin, an 11-kilodalton protein chemotactic for rat bone marrow hematopoietic precursors, was purified from media conditioned by a rat thymic epithelial cell line. The NH2-terminal sequence of thymotaxin was identical to that of rat beta 2-microglobulin (beta 2m). Antibodies to beta 2m removed thymotaxin activity from the fraction containing the 11-kilodalton protein. Chemotactic activity was observed with rat plasma beta 2m, human beta 2m, and mouse recombinant beta 2m, further supporting the identity of thymotaxin with beta 2m. The directional migration, as opposed to random movement, of the cells was also confirmed. The only rat bone marrow cells that migrated toward beta 2m were Thy1+ immature lymphoid cells devoid of T cell, B cell, and myeloid cell differentiation markers.


Subject(s)
Chemotactic Factors/pharmacology , Chemotaxis , beta 2-Microglobulin/pharmacology , Animals , Bone Marrow Cells , Cell Line , Cell Movement/drug effects , Chromatography, Gel , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Granulocytes/drug effects , Hematopoietic Stem Cells/drug effects , Rats
17.
EMBO J ; 2(11): 1909-14, 1983.
Article in English | MEDLINE | ID: mdl-16453480

ABSTRACT

Assimilatory nitrate reductase has been purified with 55% recovery from a Neurospora crassa nmr-1 nit-6 mutant, using a modification of a published procedure. It possesses one heme per 240 000 g, and subunits of mol. wt. 68 000. Upon digestion with chymotrypsin, a heme-binding domain was isolated by gel filtration; its visible spectrum was highly similar to that of cytochrome b(5). On SDS gels, the fraction showed two heme-containing bands of 10 000 and 12 5000 daltons; their amino acid composition was not very different, suggesting that they originated from the same region of the polypeptide chain. After S-carboxymethylation, the mixture of bands was submitted to cyanogen bromide cleavage, and the fragments were separated by h.p.l.c. The two largest fragments yielded an identical sequence upon automated degradation. This sequence (39 residues with some gaps) could be easily aligned with that of cytochrome b(5) starting close to the N terminus. These results are discussed in terms of the possible quaternary structure of N. crassa nitrate reductase, whose heme-binding domain proves to be another member of the family of b(5)-like cytochromes.

18.
Biochim Biophys Acta ; 524(2): 288-96, 1978 Jun 09.
Article in English | MEDLINE | ID: mdl-149561

ABSTRACT

epsilon-N-Trimethyllysine L-amino oxidase from Neurospora crassa has been purified to electrophoretic homogeneity. A 1500-fold purification was obtained by centrifugation and successive column chromatography on ion-exchange and gel filtration supports. The enzyme has an estimated molecular weight of 160 000. It transforms epsilon-N-trimethyllysine into alpha-keto, epsilon-N-trimethylhexanoic acid by oxidative deamination. Kinetic studies of this new enzyme are reported and its probable physiological role is discussed.


Subject(s)
Amino Acid Oxidoreductases/metabolism , Neurospora crassa/enzymology , Neurospora/enzymology , Amino Acid Oxidoreductases/isolation & purification , Amino Acids/analysis , Kinetics , Lysine/analogs & derivatives , Molecular Weight , Substrate Specificity
SELECTION OF CITATIONS
SEARCH DETAIL
...