Mammalian folylpoly-. gamma. -glutamate synthetase. 1. Purification and general properties of the hog liver enzyme

Journal Article · Tue Jan 27 04:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00376a024· OSTI ID:6620094

Cichowicz, D J; Shane, B

Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of M/sub r/ 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K/sup +/ was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the K/sub m/ value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied.

• Research Organization: Johns Hopkins Univ., Baltimore, MD
• OSTI ID: 6620094
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:2; ISSN BICHA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
BODY
CARBON 14 COMPOUNDS
CATIONS
CHARGED PARTICLES
CHEMISTRY
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
GLANDS
ION EXCHANGE CHROMATOGRAPHY
IONS
ISOTOPES
LABELLED COMPOUNDS
LIGASES
LIGHT NUCLEI
LIVER
MAGNESIUM COMPOUNDS
MAMMALS
NUCLEI
ODD-ODD NUCLEI
ORGANS
PH VALUE
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PURIFICATION
RADIOASSAY
RADIOISOTOPES
SEPARATION PROCESSES
SWINE
TRITIUM COMPOUNDS
VERTEBRATES