Subcellular localization of 3-methylcrotonyl-coenzyme A carboxylase in bovine kidney

The intracellular localization of 3-methylcrotonyl-CoA carboxylase (MCase), an enzyme of the leucine oxidative pathway, was studied in bovine kidney. Differential centrifugation of kidney homogenates demonstrated that the majority of the enzyme was associated with the mitochondrial and cytosolic fractions. Isopycnic centrifugation of the mitochondrial fraction demonstrated cofractionation of MCase with mitochondrial markers, but not with lysosomal markers, consistent with a mitochondrial location for the enzyme. Using different homogenization techniques and comparing the fractional extraction of MCase and mitochondrial and cytosolic marker enzymes, the appearance of MCase in the “cytosolic” fraction was shown to be due to mitochondrial damage. The intramitochondrial distribution of MCase was determined using a digitonin procedure, and indicated that the enzyme is associated with the inner mitochondrial membrane. Although a fraction of MCase (30–40%) was “solubilized” by homogenization of whole mitochondria, the remaining MCase (60–70%) was tightly associated with the mitochondrial membrane fraction. Release and “solubilization” of this latter fraction was achieved by polyethylene glycol treatment. The “solubilized” MCase was stabilized in a glycerol-containing buffer.     

Characterization of biotin and 3-methylcrotonyl-coenzyme a carboxylase in higher plant mitochondria

Mitochondria from green pea (Pisum sativum) leaves were purified free of peroxisomes and chlorophyll contamination and examined for their biotin content. The bulk of the bound biotin detected in plant mitochondria was shown to be associated with the matrix space to a concentration of about 13 micromolar, and no free biotin was detected. Western blot analysis of mitochondrial polypeptides using horseradish peroxidase-labeled streptavidin revealed a unique biotin-containing polypeptide with a molecular weight of 76,000. This polypeptide was implicated as being the biotinylated subunit of 3-methylcrotonyl-coenzyme A (CoA) carboxylase. Fractionation of pea leaf protoplasts demonstrated that this enzyme activity was located largely in mitochondria. The 3-methylcrotonyl-CoA carboxylase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of mitochondria. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8.3 in the presence of Mg²⁺. Kinetic constants (apparent K_m values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 0.05 millimolar; ATP, 0.16 millimolar; HCO₃⁻, 2.2 millimolar. The involvement of 3-methylcrotonyl-CoA carboxylase in the leucine degradation pathway in plant mitochondria is proposed.     

Isolation and partial characterization of a vitamin K-dependent carboxylase from bovine aortae.

Vitamin K-dependent carboxylase activity has been demonstrated in the crude microsomal fraction of the intima of bovine aortae. The procedure for the isolation of vessel wall carboxylase is a slight modification of the general preparation procedure for tissue microsomes. The highest activity of the non-hepatic enzyme was observed at 25 degrees C and hardly any NADH-dependent vitamin K reductase could be demonstrated. The optimal reaction conditions for both vessel wall as well as liver carboxylase were similar: 0.1 M-NaCl/0.05 M-Tris/HCl, pH 7.4, containing 8 mM-dithiothreitol, 0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (CHAPS), 0.4 mM-vitamin K hydroquinone and 2 M-(NH4)2SO4. Warfarin inhibits the hepatic and non-hepatic carboxylase/reductase enzyme complex more or less to a similar degree. We have measured the apparent Km values for the following substrates: Phe-Leu-Glu-Glu-Leu ('FLEEL'), decarboxylated osteocalcin, decarboxylated fragment 13-29 from descarboxyprothrombin and decarboxylated sperm 4-carboxyglutamic acid-containing (Gla-)protein. The results obtained demonstrated that liver and vessel wall carboxylase may be regarded as isoenzymes with different substrate specificities. The newly discovered enzyme is the first vitamin K-dependent carboxylase which shows an absolute substrate specificity: FLEEL and decarboxylated osteocalcin were good substrates for vessel wall carboxylase, but decarboxylated fragment 13-29 and decarboxylated sperm Gla-protein were not carboxylated at all.     

Isolation and properties of bovine kidney aminopeptidase A]

Aminopeptidase A, which specifically hydrolyses N-terminal dicarbonic amino acid residues containing free alpha-amino groups, is isolated from bovine kidney. The enzyme is 500-fold purified and is homogenous under electrophoresis and ultracentrifugation. Aminopeptidase A has pH optimum of 7.5, it is activated with Ca2+ and inactivated with EDTA. Its molecular weight is 53000. The enzyme hydrolyses alpha-L-aspartyl-beta-naphtylamide and splits peptides having N-terminal glycine, lysine, arginine and alanine are hydrolyzed by the enzyme much slower. Aminopeptidase A does not attack alpha-L-alanyl-beta-naphtylamide, leucineamide, insulin, peptides with blocked N-terminal amino acid and peptides which have proline to be the second N-terminal amino acid.     

Adenosine deaminase-complexing protein from bovine kidney. Isolation of two distinct subunits

We have isolated a complex of two proteins from bovine kidney that bind to adenosine deaminase immobilized on Sepharose 4B. One protein, with Mr = 110,000, comigrates on both PAGE and SDS-PAGE gels with complexing protein isolated from rabbit kidney by the method of Schrader et al. (Schrader, W.P., Harder, C. M., and Schrader, D. K. (1983) Comp. Biochem. Physiol. B Comp. Biochem. 75, 119-126). The second protein has a Mr = 70,000. Both proteins bind to the adenosine deaminase-Sepharose but not to a control resin of bovine serum albumin bound to Sepharose. Based on a comparison of partial and complete denaturation on SDS-PAGE the two proteins appear to be bound to each other. At adenosine concentrations of 0.5-1 mM the isolated complexing protein increases small subunit adenosine deaminase catalytic activity by 20-30%. There may be some inhibition of catalytic activity at low adenosine concentrations. We have designated the 110,000 Mr protein CP-I, the 70,000 Mr protein CP-II and the complex of these two CP.     

Steady-state kinetics of 3-mercaptopyruvate sulfurtransferase from bovine kidney

Mammalian 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2), purified to apparent homogeneity by a new procedure, was studied by steady-state kinetic methods. The enzyme-catalyzed transfer of a sulfur atom from 3-mercaptopyruvate either to 2-mercaptoethanol or to a second molecule of 3-mercaptopyruvate was found to proceed by a sequential formal mechanism. An overall mechanism incorporating both of these transfers was shown to be capable of generating all of the initial velocity and product inhibition behavior observed.     

Molecular structure of aminopeptidase A from bovine kidney

Using gel-filtration through Sephadex G-100 and polyacrylamide gel electrophoresis in the presence of 0,5% sodium dodecyl sulfate, it was found that aminopeptidase A has a molecular weight of 65 000 +/- 2000 and is made up of two subunits with mol. weights of 33 000 +/- 2000. Each subunit consists of two polypeptide chains with mol. weights of 22 000 +/- 2000 and 12 000. During enzyme dissociation into subunits the aspartylnaphtylamidase activity is lost, while the glutamylnaphtylamidase activity is retained.     

Isolation and characterization of a new atrial peptide-degrading enzyme from bovine kidney.

An endopeptidase isolated from bovine kidney displays high affinity and selectivity for the Ser-Phe bond located in the C-terminal region of atrial peptides. Enzymatic activity converts APIII and APII to the less active peptide API. This peptidase is inhibited by both metal chelators and sulfhydryl-reactive agents, suggesting both a tightly bound metal and a cysteine residue are important for enzymatic activity. This enzyme may be important for the processing and/or degradation of atrial peptides.     

Isolation and characterization of an acyl-coenzyme A carboxylase from an erythromycin-producing Streptomyces erythreus

Streptomyces erythreus produces erythromycin presumably from methylmalonyl-coenzyme A, (CoA) which might be generated by carboxylation of propionyl-CoA. A biotin-containing enzyme which carboxylates acetyl-CoA, propionyl-CoA, and butyryl-CoA was purified to near homogeneity from S. erythreus using DEAE-cellulose, affinity chromatography on monomeric avidin-Sepharose, and blue Sepharose. The enzyme carboxylates propionyl-CoA (100%) with a K_m of 0.09 mm and V of 0.86μmol/mg/min, acetyl-CoA (16%) with a K_m of 0.17 mm and V of 0.08μmol/mg/min, and butyryl-CoA (7.7%) with a K_m of 0.67 mm and V of 0.044 μmol/mg/min. The native enzyme has a molecular weight of 537,000 and consists of two types of subunits with molecular weights of 67,000 and 61,000, respectively, indicating an octameric α₄β₄ type of structure. Biotin is associated with the large subunit (α). The enzyme has a pH optimum between 7.5 and 7.8. It is stimulated (three- to fourfold) by K⁺, Rb⁺ and Cs⁺ but not by Na⁺ or Li⁺ and is inhibited by high concentrations of NH₄⁺ and C1^?. Neither citrate nor free CoA stimulated the enzyme. The enzyme was shown to be stereospecific and generated onlyS-methylmalonyl-CoA from the carboxylation of propionyl-CoA. The present case appears to be the first enzyme possibly involved in erythromycin production to be isolated in homogeneous form.     

Isolation and characterization of pyruvate carboxylase from Azotobacter vinelandii OP

Pyruvate carboxylase has been detected in, and partially purified from, cell-free extracts of Azotobacter vinelandii OP. The best preparations obtained have specific activities in the range of 4 units/mg and appear approximately 15% pure when analyzed by polyacrylamide gel electrophoresis. The partially purified enzyme is activated by both univalent and divalent cations, contains one or more functional biotinyl residues, and exhibits apparent Michaelis constants for the substrates (pyruvate, Mg-ATP^2?, and HCO₃^?) which are in the same range as those observed for other pyruvate carboxylases. However, A. vinelandii pyruvate carboxylase is fully active in the absence of added acetyl-coenzyme A and is insensitive to inhibition by dicarboxylic acids such as l-aspartate, l-glutamate, and α-ketoglutarate. The molecular weight of the catalytically active species is obtained as 296,000.The level of pyruvate carboxylase is highest in extracts of A. vinelandii grown on pyruvate or l-lactate as sole carbon source and this level is further enhanced on addition of succinate to the medium. The enzyme is absent from cells grown on succinate and is present at intermediate levels in cells grown on sucrose, glucose, glycerol, or acetate. In contrast, the level of phosphoenolypyruvate carboxylase in these extracts is essentially independent of the carbon source. These data suggest that pyruvate carboxylase in A. vinelandii is induced by pyruvate or some closely related metabolite.     

Isolation of an acyl-CoA carboxylase from the tapeworm spirometra mansonoides

An acyl-CoA carboxylase, which catalyzes the carboxylation of acetylpropionyl-, and butyryl-CoA, has been isolated from the tapeworm $\text{Spirometra}\text{mansonoides}$. The enzyme has an absolute requirement for ATP, Mg²⁺, and HCO₃${}^{\mathrm{?}}$ and, in addition, requires K⁺ for full catalytic activity. The enzyme has been purified 50-fold by a combination of calcium phosphate gel adsorption, ion-exchange column chromatography, and gel filtration. In its substrate specificity, K⁺ requirement, molecular size, and antigenic behavior, the tapeworm enzyme is similar to the acyl-CoA carboxylase of another helminth— the free-living nematode $\text{Turbatrix}\text{aceti}$.     

Isolation of queuine from bovine amniotic fluid

A facile method for isolation of large quantities of queuine from bovine amniotic fluid is described. Queuine was sequentially purified by cation-exchange chromatography, adsorption chromatography on Sephadex G-25, and size-exclusion chromatography on Sephadex G-10. The queuine isolate was identified by its participation in the queuine-guanine tRNA transglycosylase reaction and comparisons with authentic queuine.     

Isolation of lipofuscin from the bovine myocardium

The paper describes the procedure of lipofuscin isolation from bovine myocardium with the use of 15-45% (weight/volume) linear gradient of sucrose density and differential centrifugation. Two coloured populations of lipofuscin granules with different density have been distinguished. Electron microscopic studies of the isolated lipofuscin have revealed the homogeneity of fractions and good maintenance of the granules. The electron microscopic, absorptive and luminescent spectral properties of lipofuscin preparations correspond to its properties in situ. The isolation of lipofuscin preparations suggests the possibility of studying the chemical composition and intramolecular structure of the granules.