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Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).

ABSTRACT: gamma-Butyrobetaine hydroxylase [4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] from human kidney was resolved into three forms by chromatofocusing. After further chromatography on an anion-exchanger, each form appeared as a single band on electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate. The isoelectric points of isoenzymes 1, 2 and 3 were 5.6, 5.7 and 5.8 respectively, as estimated by isoelectric focusing. Their specific activities were 17-29 mu kat/g of protein. The concentrations of the three isoenzymes were about equal, possibly slightly lower for isoenzyme 1. The requirement for Fe2+ and the Km values for gamma-butyrobetaine and 2-oxoglutarate were about the same for the different enzyme forms. L- and D-Carnitine caused decarboxylation of 2-oxoglutarate to the same extent (8 and 29%) with the three forms. The enzyme forms had the same mass, 64 kDa, as determined by gel filtration in nondenaturing media. The same subunit mass, 42 kDa, was obtained for the multiple forms by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Isoenzyme 2 was resolved into two protein bands by isoelectric focusing in polyacrylamide gels containing urea. Isoenzyme 1 contained only one of these bands and isoenzyme 3 the other. The three enzyme forms of gamma-butyrobetaine hydroxylase thus appear to be dimeric combinations of two subunits differing in charge but not in size. gamma-Butyrobetaine hydroxylase from crude extracts of human, rat and calf liver was also separated into multiple forms by a chromatofocusing technique. The isoenzyme pattern was the same in human liver and kidney. The technique used to resolve the mammalian enzymes gave no evidence for the presence of multiple forms of the bacterial enzyme from Pseudomonas sp. AK 1.

SUBMITTER: Lindstedt S

PROVIDER: S-EPMC1144272 | biostudies-other | 1984 Oct

REPOSITORIES: biostudies-other

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Project description:Background: The plant-based form of vitamin K (phylloquinone, vitamin K-1) has been well quantified in the US diet. Menaquinones (vitamin K-2) are another class of vitamin K compounds that differ from phylloquinone in the length and saturation of their side chain, but they have not been well characterized in foods. Objectives: The objectives of this study were to 1) quantify phylloquinone and the different forms of menaquinones [menaquinone (MK) 4-MK13] in milk, yogurt, Greek yogurt, creams, and cheeses and 2) compare the menaquinone contents of full-fat, reduced-fat, and nonfat dairy products. Methods: All dairy samples were either obtained from the USDA National Food and Nutrient Analysis Program or purchased from retail outlets. Phylloquinone and menaquinone concentrations in these dairy products were quantified by mass spectrometry technology. Results: Full-fat dairy products contained appreciable amounts of menaquinones, primarily in the forms of MK9, MK10, and MK11. We also measured modest amounts of phylloquinone, MK4, MK8, and MK12 in these products. In contrast, there was little MK5-7 or MK13 detected in the majority of dairy products. The total vitamin K contents of soft cheese, blue cheese, semi-soft cheese, and hard cheese were (means ± SEMs): 506 ± 63, 440 ± 41, 289 ± 38, and 282 ± 5.0 µg/100 g, respectively. Nonfermented cheeses, such as processed cheese, contained lower amounts of vitamin K (98 ± 11 µg/100 g). Reduced-fat or fat-free dairy products contained ∼5-22% of the vitamin K found in full-fat equivalents. For example, total vitamin K contents of full-fat milk (4% fat), 2%-fat milk, 1%-fat milk, and nonfat milk were 38.1 ± 8.6, 19.4 ± 7.7, 12.9 ± 2.0, and 7.7 ± 2.9 µg/100 g, respectively. Conclusions: To the best of our knowledge, this is the first report of menaquinone contents of US dairy products. Findings indicate that the amount of vitamin K contents in dairy products is high and proportional to the fat content of the product.

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Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).

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