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Biomedical subjects

A H Merrill

Publications and source records attributed to A H Merrill.

At least 145 records · Page 8Linked to original sources
PubMed1983

Characterization of serine palmitoyltransferase activity in Chinese hamster ovary cells.

Serine palmitoyltransferase (palmitoyl-CoA: L-serine C-palmitoyltransferase (decarboxylating) EC 2.3.1.50) catalyzes the first unique and regulatory reaction of sphingolipid biosynthesis. Its activity was demonstrated in Chinese hamster ovary cells (CHO-K1) by measuring the incorporation of radiolabel from L-[3H]serine into 3-ketosphinganine, which was found to be the predominant chloroform-soluble product under optimal assay conditions. Most of the total activity (14.8 +/- 4.2 pmol/min per 10(6) cells, measured with sonicated cells) was recovered in particulate fractions, with the highest percentage (54%) and specific activity (102 pmol/min per mg) in the high-speed (airfuge) pellet. The greatest activity was obtained with palmitoyl-CoA; however, other fatty acyl-CoA thioesters were also utilized. Serine palmitoyltransferase required pyridoxal 5'-phosphate for activity, but was apparently fully saturated with this coenzyme when assayed with sonicated cells. Regardless of whether the CHO cells were grown in culture medium containing whole serum with or without sphinganine addition, lipoprotein-depleted serum, lipid-extracted serum, low-density lipoproteins, or no serum, the activities of this enzyme were identical. This finding was confirmed using human fibroblasts. Hence, these results establish that CHO cells, and probably others, are engaged in long-chain base synthesis de novo and that serine palmitoyltransferase activity is not regulated by the availability of such compounds in the culture media.

Acyltransferases↗
PubMed1982

Facile enzymatic synthesis of fatty acylcoenzyme A thioesters.

The fatty acid:CoA ligase (acyl-CoA synthetase, EC 6.2.1.3) of rat liver microsomes was solubilized with Triton X-100 and bound to Matrex Gel Red A. Fatty acid:CoA ligase immobilized on Matrex Gel Red A was active and proved useful for the synthesis of fatty acyl-CoA thioesters. The immobilized activity was characterized by a 3-fold higher apparent Km for ATP than the soluble activity, similar apparent Km values for CoA and palmitate, and a shift in the pH dependence. Quantitative incorporation of fatty acid or CoA was possible. Long chain-fatty acyl CoA thioesters were purified in a single step by hydrophobic chromatography on Octyl-Sepharose. In addition to producing the thioesters of typical fatty acids (e.g., myristic, palmitic, stearic, oleic, cis-vaccenic, linoleic, and arachidonic), analogs such as the fluorescent molecules beta-parinaroyl-CoA and palmitoyl-(1-N6-etheno-)CoA were easily synthesized. These procedures should be generally applicable for both the small-scale, i.e., 1 to 10 mumoles, and large-scale, i.e., 50 to 250 mumoles, scale preparation of numerous fatty acyl-CoA's and related compounds.

Acyl Coenzyme A↗
PubMed1980

Spectroscopic studies of complexes between pyridoxamine (pyridoxine)-5'-phosphate oxidase and pyridoxyl 5'-phosphate compounds differing at position 4'.

The absorbance spectrum of pyridoxamine (pyridoxine)-5'-phosphate oxidase (EC 1.4.3.5) is altered upon the binding of pyridoxal 5'-phosphate or analogs with different substituents at position 4'. The absorbance difference spectra are similar for complexes of the oxidase and pyridoxal 5'-phosphate, 4'-desoxypyridoxine 5'-phosphate, and 4-ethynyl-4-deformylpyridoxal 5'-phosphate; hence, these perturb the flavoprotein absorbance by similar interactions primarily involving the pyridoxyl 5'-phosphate moiety, and not specifically the 4-formyl group or other relatively small and uncharged functions at this position. A different type of spectral perturbation is caused by analogs with larger substituents at position 4' (i.e. 4'-methoxypyridoxine 5'-phosphate, 4-methyl-vinyl-4-deformylpyridoxal 5'-phosphate, and pyridoxal 5'-phosphate oxime and hydrazone). These analogs impose bulky groups in a region of the active site that critically influences the environment of the flavin, and, thus, may reflect positioning of this portion of the substrates close to the flavin ring, as is required for their redox interaction.

Animals↗
PubMed1980

Affinity chromatographic purification and properties of flavokinase (ATP:riboflavin 5'-phosphotransferase) from rat liver.

Flavokinase (ATP:riboflavin 5'-phosphotransferase, EC 2.7.1.26) has been purified to apparent homogeneity from rat liver by affinity chromatography using flavinyl agarose beads (agarose-OCH2CONH(CH2)2NHCO(CH2)/N10-7,8-dimethylisoalloxazine). The specific activity of the pure enzyme is 9,900 units (nmol of FMN formed/h at 37 degrees C)/mg of protein, and reflects a one-step, 7000-fold purification. Flavokinase thus obtained, unlike previous preparations from mammalian sources, is free from contaminating phosphatase and FAD synthase. The purified enzyme rapidly loses activity upon storage but is stabilized by riboflavin and thiol-protecting reagents. The apparent molecular weight, estimated by gel filtration on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 28,000 +/- 1,000. Flavokinase phosphorylates and/or is inhibited by a large number of riboflavin analogs; however, the physiologically important 8 alpha-(amino acid)riboflavins are poorly accommodated. The strongly preferred phosphate donors are ATP and dATP. Both Zn2+ and Mg2+, as well as several other divalent cations, activate flavokinase, but Zn2+ yields greatest activity (1.8 times that with Mg2+). The pH optimum for activity with either Zn2+ or Mg2+ is approximately 9.3; at pH 7.0, the activity is 40% of that at the pH optimum.

Animals↗
PubMed1979

Purification of riboflavin-binding proteins from bovine plasma and discovery of a pregnancy-specific riboflavin-binding protein.

Riboflavin-binding proteins have been purified from bovine plasma using flavinyl agarose beads. At least three major protein bands, migrating in regions assigned to the beta- and gamma-globulins of plasma, are observed by cellulose acetate electrophoresis. These proteins coelute from a calibrated Sephadex G-100 column in the volume corresponding to a molecular weight of approximately 150,000; a small amount of another riboflavin-binding protein (molecular weight approximately 37,000) is also present. Polyacrylamide gel electrophoresis of the proteins, with detection by autoradiography of those having tightly bound [2-14C]riboflavin, reveals one protein band which is present only in preparations from pregnant cows. This protein has been purified to apparent homogeneity by storing the mixture of riboflavin-binding proteins at 8 degrees C for 3 weeks, which precipitates the other, less stable proteins. Hence, bovine plasma, like that of the laying hen, contains a number of riboflavin-binding proteins, one of which correlates with pregnancy.

Animals↗
PubMed1979

Spectroscopic studies of pyridoxamine (pyridoxine) 5'-phosphate oxidase. Equilibrium dissociation constants and spectra for riboflavin 5'-phosphate and analogues.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been shown to bind 1 mol of riboflavin 5'-phosphate (FMN) per mol of apoenzyme and is active with or inhibited by numerous FMN analogues [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436--3442]. The KD values and spectra for selected apoenzyme--flavin complexes have been determined and used to elucidate some of the properties of the FMN-binding site of this flavoprotein. Alterations of the pyrimidinoid portion of the flavin ring decrease binding considerably. The absorption spectra for the protein complexes with 3-deaza-FMN and 8-hydroxy-FMN indicate the presence of a dipolar or positively charged protein group near N1 and O2. The substitution of methyl for hydrogen at N3 apparently causes distortion of the interaction between the flavin ring and an active-site aromatic amino acid residue. Although binding is also decreased somewhat by substitutions at postions 8 and 8 alpha, considerable bulk [e.g., 8-(diethylamino)-FMN and 8 alpha-S-(N-acetyl-cysteinyl)-FMN] is accommodated. Hence, this portion of the flavin ring is probably oriented toward, possibly in contact with, solvent, as has been found for the flavodoxins. The importance of optimum interactions between the flavin and the apoprotein is further emphasized by large differences in the activity of flavin analogues that have similar midpoint potentials in solution.

Apoenzymes↗
PubMed1979

Preparation and properties of immobilized flavokinase.

Flavokinase (ATP: riboflavin 5'-phosphotransferase, EC 2.7.1.26) purified from rat liver by affinity chromatography, has been immobilized by amide linkage to omega-aminoalkyl-agarose beads. The immobilized enzyme differs from the soluble enzyme in having greater stability, slightly higher Km for the substrates, riboflavin and ATP, a broader pH optimum, and a lower energy of activation. These results suggest that the immobilized enzyme is influenced by the microenvironment of the bead and is subject to some degree of internal diffusional limitation. A small (3 ml), continuous, plug-flow reactor prepared with immobilized flavokinase effects 50% conversion of riboflavin to riboflavin 5'-phosphate (FMN) with a flow rate of 0.16 ml/min, which corresponds to an output of 5 nmol FMN/min. Immobilized flavokinase is effective for phosphorylating riboflavin and numerous riboflavin analogs and provides a facile method for preparing exclusively, unlike other synthetic methods, the 5'-phosphates.

Chemical Phenomena↗