Allosteric Properties of a Pyruvate Kinase Isoenzyme from Rat Liver Cells in Culture

• Published in: The Journal of biological chemistry 1973-07, Vol.248 (13), p.4610-4616
• Main Authors: Walker, P. Roy, Potter, Van R.
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate; Adenosine Triphosphate; Adipose Tissue - enzymology; Amino Acids; Animals; Binding Sites; Cell Line; Culture Media; Electrophoresis, Starch Gel; Enzyme Activation; Fructosephosphates; Glucose; Isoenzymes - antagonists & inhibitors; Isoenzymes - isolation & purification; Kinetics; Liver - cytology; Liver - enzymology; Magnesium; Male; Phosphoenolpyruvate; Pyruvate Kinase - analysis; Pyruvate Kinase - antagonists & inhibitors; Rats; Structure-Activity Relationship
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)43707-1

A cell-line in culture derived from adult rat liver was used as a source of a pyruvate kinase isoenzyme (PK-III) for kinetic studies. The culture conditions of the cells were found to affect the kinetic properties of the enzyme in a crude preparation. When the enzyme was extracted from cells maintained without glucose in the medium the substrate-velocity curve with phosphoenolpyruvate was sigmoidal in shape with an apparent Km of 0.33 mm. However, when the enzyme was extracted from cells maintained in the presence of glucose up to the time of harvest the substrate-velocity curve was hyperbolic with an apparent Km of 0.05 mm. These two forms have been designated PK-IIIa and PK-IIIb, respectively, and appear to be the same as two similar forms known to exist in adipose tissue. Differences in inhibition by ATP and the amino acids l-alanine and l-phenylalanine were found. The IIIa form of the enzyme was more susceptible to the inhibitors than the IIIb form. Fructose diphosphate was able to relieve amino acid inhibition of PK-IIIb but not of PK-IIIa. The two forms of the enzyme were found to be interconvertible in vitro. The A to B transition could be mediated by fructose-P2 or by magnesium ions, whilst the B to A transition was mediated by EDTA, ATP, or citrate. The possible role of these two forms of the enzyme in metabolic regulation is discussed with the proposal that PK-IIIa is the inactive form of the enzyme and must be converted to PK-IIIb for enzyme activity.