Abstract
T-protein, a component of the glycine cleavage system, has been purified to apparent homogeneity from chicken liver mitochondria. The molecular weight was 37,000 by sedimentation equilibrium centrifugation and 38,000 by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a molecular weight of 41,000, indicating that the protein is composed of a single polypeptide. A partial specific volume of 0.73 ml/g was obtained from the amino acid composition. The absorbance coefficient, A1%280nm, is 6.63 in 50 mM potassium phosphate buffer, pH 7.0. T-protein is a basic protein having a pI value of 9.8 and relatively rich in arginine rather than lysine. The protein catalyzed the degradation of the protein-bound intermediate (-CH2NH2 moiety of glycine) to a 1-carbon unit and NH3. The reaction is dependent on tetrahydrofolate (H4-folate). Apparent Km values for the intermediate and H4-folate were 3.7 microM and 0.17 mM, respectively. T-protein associated with H-protein forming a complex which was composed of one molecule each of T-protein and H-protein. Formation of the complex was not affected by the modification of the cysteinyl residues on H-protein with N-ethylmaleimide.
Highlights
T-protein, a component of the glycine cleavage sys- P2).Titration of the sulfhydryl groups on H-protein ledto the tem, has been purified to apparent homogeneityfrom conclusion that the site of accepting -CHzNHz fragment is one chicken liver mitochondriaT. he molecularweight was of the sulfhydryl groups of lipoic acid [3]
We have purified T-protein from chicken liver mitochondria to apparent homogeneity
Two critical aspects of o w preparation were the elimination of the acetone treatment of mitochondria from the purificationprotocol previously described for rat liver [2] and the addition of glycerol to all buffer solutions
Summary
T-protein, a component of the glycine cleavage sys- P2).Titration of the sulfhydryl groups on H-protein ledto the tem, has been purified to apparent homogeneityfrom conclusion that the site of accepting -CHzNHz fragment is one chicken liver mitochondriaT. he molecularweight was of the sulfhydryl groups of lipoic acid [3]. The describes the purification and properties of T-protein from absorbance coefficient,A$&-, is 6.63 in 50 m~ potassiumphosphatebuffer, pH 7.0.T-protein is abasic protein having a PI value of9.8 and relatively rich in arginineratherthan lysine. The chicken liver T-protein is a basic protein having a PI value of 9.8 and readily associates with H-protein. M~terials--[2-'~C]Glyciwneas purchased from Amersham/Searla Sepharose 4B with covalently bound bovine serum albumin was prepared according to Brandt et al [11].H-protein was purified from chicken liver as described previously [3], and P-protein was isolated associated with H-protein forming a complex which from Arthrobacter globiformis [8].The sources of all other chemicals was composed of one molecule each of T-protein and and reagents are identical with earlier publications [2, 3]. Formationof the complex was not affected Assays-H-protein was assayed photometrically as described in a by the modificationof the cysteinyl residueosn H-pro- previous paper (I).A unit of H-protein activity represented 1nmol of tein with N-ethylmaleimide
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