Purification and Mechanism of Action of Hog Liver Mevalonic Kinase

Beytía, Enrique; Dorsey, Jill; Marr, J. Joseph; Cleland, W. W.; Porter, John W.

doi:10.1016/s0021-9258(18)62775-9

Cited by 58 publications

(15 citation statements)

References 24 publications

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“…The DEAE-cellulose step gives a high increase in specific activity and a phosphatase-free preparation. The Bio-Gel P-150 column separates mevalonate kinase, because of its higher molecular weight [98 000 (Beytia et al, 1970)]. This preparation was used in many experiments not requiring a homogeneous enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…The lactone of mevalonic acid was converted previously to the potassium salt with KOH. Mevalonate kinase was partially purified according to the method of Beytia et al (1970) up to the DEAE-cellulose step, obtaining a preparation with a specific activity of 0.35 unit/mg, free of phosphatases and phosphomevalonate kinase. In later experiments, a mevalonate kinase preparation, obtained as the byproduct in the purification of phosphomevalonate kinase at the Bio-Gel P-150 step (see below), was employed.…”

mentioning

confidence: 99%

“…Cytochrome c was determined spectrophotometrically at 412 nm, the other standards were determined at 280 nm, and phosphomevalonate kinase was determined by using the usual assay. (2) The second was sucrose density gradient centrifugation according to Beytia et al (1970). Gradients were prepared from 5 to 20% sucrose in 10 mM phosphate buffer, pH 7.5, containing 10 mM 2-mercaptoethanol.…”

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confidence: 99%

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Pig liver phosphomevalonate kinase. 1. Purification and properties

Bazaes¹,

Beytía²,

Jabalquinto³

et al. 1980

Biochemistry

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Pig liver phosphomevalonate kinase (EC 2.7.4.2) has been purified to homogeneity as shown by polyacrylamide gel electrophoresis. The molecular weight estimates range from 21,000 to 22,500. Each molecule is composed of one polypeptide chain. The presence of SH-containing reagents is essential for the preservation of enzymes activity at all steps in the purification. The enzyme shows absolute specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 7.5 to over 9.5. Kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.075 mM and 0.46 mM have been obtained for phosphomevalonate and ATP, respectively. Amino acid composition shows a high content of acid amino acids, one cysteine residue per molecule of enzyme, and the absence of methionine. The results obtained suggest that the enzyme plays no regulatory function in cholesterol biosynthesis in pig liver, although a variable enzyme content was detected in different livers.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Pig liver phosphomevalonate kinase. 1. Purification and properties

Bazaes¹,

Beytía²,

Jabalquinto³

et al. 1980

Biochemistry

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“…75 Kinetic studies suggest that the enzyme catalyzes an ordered sequential reaction, with mevalonic acid binding prior to the enzyme in relation to ATP and the phosphomevalonate being the first product of the reaction. 77 Site-specific mutagenesis studies have been employed to identify catalytic residues and to investigate the mechanism of the enzyme. Asp204 has been suggested as the catalytic base that abstracts the proton from the C5 OH group of mevalonic acid.…”

Section: Biochemistrymentioning

confidence: 99%

Cholesterol Biosynthesis: A Mechanistic Overview

et al. 2016

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Cholesterol is an essential component of cell membranes and the precursor for the synthesis of steroid hormones and bile acids. The synthesis of this molecule occurs partially in a membranous world (especially the last steps), where the enzymes, substrates, and products involved tend to be extremely hydrophobic. The importance of cholesterol has increased in the past half-century because of its association with cardiovascular diseases, which are considered one of the leading causes of death worldwide. In light of the current need for new drugs capable of controlling the levels of cholesterol in the bloodstream, it is important to understand how cholesterol is synthesized in the organism and identify the main enzymes involved in this process. Taking this into account, this review presents a detailed description of several enzymes involved in the biosynthesis of cholesterol. In this regard, the structure and catalytic mechanism of the enzymes involved in cholesterol biosynthesis, from the initial two-carbon acetyl-CoA building block, will be reviewed and their current pharmacological importance discussed. We believe that this review may contribute to a deeper level of understanding of cholesterol metabolism and that it will serve as a useful resource for future studies of the cholesterol biosynthesis pathway.

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“…Previously, Beytia and coworkers (37) reported that MVK loses its activity when stored at 4ЊC in the absence of ␤mercaptoethanol or DTT, most probably because of oxidation of an active site cysteine. Furthermore, they showed that partial restoration of enzyme activity may occur on incubation with 10 mM DTT at 38ЊC for 1 h. To study whether the deficient activity in the ZS liver samples is at least partly due to oxidation of MVK and PMK, we tried to reactivate the enzyme activities by incubation at 38ЊC in the presence of DTT.…”

Section: Activities and Levels Of Selected Enzymes In Livers Of Human Zellweger Patientsmentioning

confidence: 99%

Absence of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis

Hogenboom

Romeijn

Houten

et al. 2002

Journal of Lipid Research

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To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on functional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for human Zellweger syndrome. We found that all enzymes measured, including putative peroxisomal enzymes, are at least as active in the peroxisome-deficient Zellweger mice as in control mice, indicating that mislocalization of enzymes to the cytosol does not lead to decreased activity or degradation. Prompted by these results, we re-examined this aspect in human subjects by specific enzyme activity measurements and immunoblotting with highly specific antisera. Our results show that the previously reported deficiencies of mevalonate kinase and phosphomevalonate kinase activity in livers from human Zellweger patients reflect the bad condition of the livers, rather than mislocalization to the cytosol. Our data provide an explanation for the conflicting findings in the literature and show that great care should be taken in the interpretation of data obtained in postmortem material.

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Purification and Mechanism of Action of Hog Liver Mevalonic Kinase

Cited by 58 publications

References 24 publications

Pig liver phosphomevalonate kinase. 1. Purification and properties

Pig liver phosphomevalonate kinase. 1. Purification and properties

Cholesterol Biosynthesis: A Mechanistic Overview

Absence of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis

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