The preparation and properties of pyruvate kinase from yeast

Abstract

A new method is described for the preparation of pyruvate kinase from yeast. This eliminates proteolysis during the preparation. The molecular weight of yeast pyruvate kinase is 215000, and it is composed of four subunits. Such properties of the enzyme as its extinction coefficient, cold-lability, thiol-group reactivity and binding of Mn(2+) ions are compared with those previously reported for yeast pyruvate kinase prepared by different methods. The specific activity is significantly higher than previously observed, but otherwise the enzyme is similar, apart from its molecular weight and Mn(2+)-binding characteristics, to preparations from Saccharomyces cerevisiae obtained in this laboratory (e.g. Fell et al., 1972, and references therein) and that of C. H. Suelter (e.g. Kuczenski & Suelter, 1971, and references therein), and is different from the enzyme isolated from Saccharomyces carlsbergensis by B. Hess and his co-workers (e.g. Wieker & Hess, 1972, and references therein).

Metal-Ion-Mediated Allosteric Triggering of Yeast Pyruvate Kinase 2. A Multidimensional Thermodynamic Linked-Function Analysis

“…The enzyme was over 95% pure as judged by SDS-PAGE (data not shown). The percent yield of total PK activity from this procedure is comparable with other purification methods (Yun et al, 1976;Fell et al, 1974). The total yield of PK per gram of cells from the recombinant yeast is 10-50 times higher than the yields for wild-type PK reported in those previous studies.…”

“…rectangular hyperbola that can be fit to eq 1 with a dissociation constant of 7.2 ( 0.9 mM and a Q max value of 9.3 ( 0.5%. The K D value for Mn 2+ binding to free YPK has been previously estimated from EPR studies to be > 1 mM (Fell et al, 1974;Nowak, T., unpublished data). The K D value of Mn 2+ to free YPK is approximately 130 times larger than the K D value measured for Mn 2+ binding to the rabbit muscle PK-K + complex (Nowak & Lee, 1977).…”

Proton nuclear magnetic resonance study of the effect of pH on tRNA structure

“…[3H] Val-tRNAVali was prepared according to the method of Lagerkvist and Waldenstrom (1964) using a partially purified mixture of E. coli tRNA synthetases (Pillinger et al, 1969). Aminoacylation of yeast tRNAPhe was achieved using purified yeast phenylalanyl-tRNA synthetase prepared according to the method of Schmidt et al (1971) utilizing toluene lysis at -20 °C (Fell et al, 1974). tRNA was recovered by ethanol precipitation, suspended in 0.01 M cacodylate buffer (pH 5.7), 0.01 M MgC^, and 0.1 M NaCl, and dialyzed for 3 h. NMR experiments were conducted immediately after dialysis.…”

Chance and Necessity in Arthur Peacocke's Scientific Work

“…For example, his experiments with various proteins (Williams and Peacocke 1965;Foord, Jakeman, et al 1970;Lloyd and Peacocke 1970;Ashton and Peacocke 1971;Oliver, Pike, et al 1971;Cleave, Kent, et al 1972;Fell, Liddle, et al 1974;McVittie, Esnouf, et al 1977) that were of biological significance provided important contributions to the field. Of particular interest were studies on some proteins that bound to nucleic acids (Diggle and Peacocke 1968;Haydon and Peacocke 1968;Diggle, McVittie, et al 1975).…”