Kerry T. Yasunobu

Amine oxidase: XIV. Isolation and characterization of the multiple beef liver amine oxidase components

Abstract By modifying a previously published purification procedure, three fractions of liver mitochondrial amine oxidase were isolated in sufficient yields to characterize them. All components were bright yellow and showed a pH optima near 9.2. None of the components was inhibited by aldehyde reagents and all components showed similar substrate specificities. The sedimentation coefficients were 14.4 and 20.6, and the molecular weights about 405,000 and 1,280,000, respectively, for components 1 and 2. Component 1 contained 4 and component 2 contained 12 FAD or FAD-like substance per mole of enzyme. In addition, components 1 and 2 contained 24 and 106 moles of phospholipid per mole of enzyme, respectively. The studies indicate that a part of the multiplicity of the enzyme reported by other workers is due to the isolation of the enzyme in different molecular weight forms. In fact component 2 may be a trimer of component 1. Since the higher molecular weight form has a higher specific activity as well as a higher phospholipid content, it may represent a larger fragment from the outer membrane of the mitochondria.

Structure of oxidized flavodoxin from Anacystis nidulans

The structure of oxidized flavodoxin from the cyanobacterium Anacystis nidulans has been determined at 2.5 A resolution with phases calculated from ethylmercury phosphate and dimercuriacetate derivatives. The determination of partial sequences, including a total of 85 residues, has assisted in the interpretation of the electron density. Preliminary refinement of a partial model (1072 atoms) has reduced R to 0.349 for the 10.997 reflections between 2.0 and 5.0 A with 1 greater than 2 sigma. The polypeptide backbone, which comprises 167 residues in the current model, adopts the familiar beta-alpha-beta conformation found in other flavodoxins and in the nucleotide-binding domains of the pyridine-nucleotide dehydrogenases, with five parallel strands in the central sheet. Comparison with flavodoxin from Clostridium MP (138 residues) shows that extra residues of A. nidulans flavodoxin are accommodated in a major insertion about 20 residues in length, which forms a lobe adjacent to the fifth strand of parallel sheet, and in additions to several external segments. Residues added between the fourth sheet strand and the start of the third helix alter the environment of the pyrimidine end of the flavin mononucleotide ring. The flavin mononucleotide phosphate binds to the start of helix 1, interacting with hydroxyamino acids and with main-chain amide groups. Two hydrophobic residues, both tentatively identified as Trp, enclose the isoalloxazine ring; the solvent-exposed Trp is nearly parallel to the flavin ring. The hydrophobic environment provided by these residues must be partly responsible for the pronounced vibrational resolution of the flavin spectrum near 450 nm. The flavin ring is tilted relative to its orientation in Clostridium MP flavodoxin. In addition, atoms N-3 and O-2 alpha of the isoalloxazine appear to form hydrogen bonds to the backbone at CO97 and NH99 in a conformation entirely different from that found in Clostridium MP flavodoxin but structurally analogous to Desulfovibrio vulgaris flavodoxin.

Bovine liver monoamine oxidase. A modified purification procedure and preliminary evidence for two subunits and one FAD.

Abstract Bovine liver mitochondrial monoamine oxidase was isolated in a more active state and in higher yields by an improved purification method which utilized β-mercaptoethanol and which contained several other important modifications. The subunit structure of the purified enzyme components was investigated by chemical and enzymatic methods. The subunit molecular weight of the three enzyme components isolated was estimated to be 52,000 by sodium dodecyl sulfate disc electrophoresis and by exclusion-diffusion chromatography on Biogel A-5m with 6 m guanidine HCl as the solvent. The number of peptides observed in the peptide map of the tryptic digest of the S -β-carboxymethylcysteine derivative of the enzyme also showed that the subunit molecular weight was about 52,000. Since it was previously reported that the monomer molecular weight of the enzyme was about 110,000, the active enzyme is made up of two subunits. The NH 2 -terminus of the enzyme of both subunits is blocked since Edman degradation and aminopeptidase failed to release an NH 2 -terminal amino acid. The COOH-terminal amino acid of both subunits was shown to be leucine by carboxypeptidase digestion of the enzyme since it was liberated quantitatively. From the FAD content of the enzyme and the subunit data, it is proposed that the enzyme probably consists of two subunits which differ possibly in that only one subunit contains 8-α-cysteinyl FAD.

THE MOLECULAR, MECHANISTIC AND IMMUNOLOGICAL PROPERTIES OF AMINE OXIDASES*

SummaryAmine oxidases can be classified as Cu and FAD-amine oxidases. Representatives from each of these types have now been isolated in a pure form. Current results which are concerned with the molecular, mechanistic and preliminary immunological properties of the amine oxidases are presented and are discussed in this review article.

Mechanistic studies of beef liver mitochondrial amine oxidase: XVIII. Amine oxidase☆

Abstract A possible mechanism for the action of beef liver MAO on benzylamine at pH 7.4 and 25 ° is presented. The pros and cons of the proposed formal mechanism are discussed. The mechanism was derived from the chemical, kinetic, and product inhibition data. If all the data reported by the various investigators to date are correct, and the different substrates used do not account for the observed differences in their results, it appears that the MAO isolated from different species proceed by different ping pong mechanisms.

Mammalian lipoic acid activating enzyme

Abstract The presence of a lipoic acid activating enzyme has been demonstrated in animal liver and a 50-fold increase in the specific activity of the enzyme was achieved. Some of the general properties of the labile enzyme were determined on the partially purified preparations. The substrate specificity of the enzyme is rather broad and a number of lipoic acid analogues can be converted to the hydroxamate derivative. The enzyme catalyzes a P 32 P-ATP exchange reaction and the cofactor requirements are the same for both the forward and exchange reactions confirming the occurrence of the enzyme in animal liver.

The Purification and Properties of Beef Liver Mitochondrial Monoamine Oxidase

Publisher Summary Despite many difficulties, some progress in beef liver mitochondrial monoamine oxidase purification has occurred in recent years. This chapter discusses some experiments in this regard. In the experiments, the enzyme was assayed by the spectrophotometric method using benzylamine as substrate. All enzyme purification steps were conducted at 4 ° C. Enzyme preparations when treated with ammonium sulfate were centrifuged at 12,000 X g for 20 minutes in the Servall centrifuge. Triton X-100, although very effective in disrupting mitochondria, can denature the enzyme when used at high concentrations and if left in contact with the enzyme in solution for more than a day. The homogeneity of purified enzyme is based on the following criteria: (1) the purified enzyme is homogeneous when analyzed by starch block electrophoresis, (2) the purified enzyme runs as a single chromatographic component on DEAE-cellulose or hydroxylapatite, (3) rechromatography on Sephadex G-200 shows one component, (4) analysis for the NH,-terminal amino acid has disclosed one main component, and (5) analysis of the purified enzyme by means of free boundary electrophoresis at pH 7.4 has showed the presence of one symmetrical component.

The primary structure of bovine adrenodoxin

Abstract The amino acid sequence of bovine adrenodoxin has been detemined except for the assignment of a few acid or amide groups. The protein contains 118 amino acids in the form of a single polypeptide chain. The five cysteine residues, most of which are involved in iron binding, are located at position 46, 52, 55, 95 and 98. The molecular weight of the protein from the amino acid content and the labile sulfide and the iron content is about 13,094.

Amine oxidase: XV. The sulfhydryl groups of beef liver mitochondrial amine oxidase☆

Abstract Beef liver mitochondrial amine oxidase components 1 and 2 contain about 28 and 86 sulfhydryl groups per mole of enzyme or 7 sulfhydryl groups per 100,000 g of protein. The rate of reaction of p -chloromercuribenzoate with the enzyme was complete in about 30 min and 8 m urea had no effect on the rate of reaction. After reaction of all the sulfhydryl groups with p -chloromercuribenzoate, the enzyme was not completely inhibited and removal of the excess sulfhydryl reagent led to reactivation of the enzyme. At higher p -chloromercuribenzoate concentrations, the enzyme was inhibited noncompetitively. Iodoacetic acid and iodoacetamide did not inhibit the enzyme appreciably even at a concentration of 1 × 10 −3 m . Certain heavy metal salts inhibited the enzyme noncompetitively. The sulfhydryl groups probably are required for conformational stability rather than being required for catalysis.

The amino acid sequence of Clostridium butyricum ferredoxin

Abstract The amino acid sequence of Clostridium butyricum ferredoxin, determined by the application of conventional methods of sequence determination primarily to the S -β-aminoethyl protein, was found to be: Ala-Phe-Val-Ile-Asn-Asp-Ser-Cys-Val-Ser- Cys-Gly-Ala-Cys-Ala-Gly-Glu-Cys-Pro-Val-Ser-Ala-Ile-Thr-Gln-Gly-Asp-Thr-Gln- Phe-Val-Ile-Asp-Ala-Asp-Thr-Cys-Ile-Asp-Cys-Gly-Asn-Cys-Ala-Asn-Val-Cys-Pro- Val-Gly-Ala-Pro-Asn-Gln-Glu. This sequence differs in nine positions from that of Clostridium pasteurianum ferredoxin. The positions of the cysteine and proline residues are invariant in these two species, as is the sequence including residues 30–52.