A rapid purification method for rat liver pyruvate carboxylase and amino acid sequence analyses of NH2-terminal and biotin peptide.
A rapid method for the purification of pyruvate carboxylase from rat liver has been developed. The method involves extraction of the enzyme from frozen liver powder followed by polyethylene glycol fractionation and avidin-affinity chromatography. The purified enzyme has a specific activity of 9-10 mumol/min/mg protein when assayed at 22 degrees C in the presence of acetyl-CoA. Polyacrylamide gel electrophoresis of the preparation in the presence of sodium dodecyl sulfate showed the presence of one protein band with an estimated Mr 125,000 and no significant contamination by other biotin-containing enzymes. In addition to being rapid, the method is advantageous because prior isolation of mitochondria is not necessary. Using these preparations we have determined the sequence of the first 15 amino acids from the NH2-terminal end of the molecule to be Ser-Gly-Pro-Val-Ala-Pro-Leu-Asn-Val-Leu-Leu-Leu-Glu-Tyr-Pro. The sequence of the 24 amino acid residues around the biotin site was determined to be Gly-Ala-Pro-Leu-Val-Leu-Ser-Ala-Met-biocytin-Met-Glu-Thr-Val-Val-Thr-Ser -Pro- Thr-Glu-Gly-Thr-Ile-Arg.
Thampy
K G
KG
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Huang
W Y
WY
Wakil
S J
SJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001710:Biotin; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011766:Pyruvate Carboxylase / Q000302:isolation & purification*; D051381:Rats
D000596:Amino Acids; D001710:Biotin; D011766:Pyruvate Carboxylase