Particulate phosphofructokinase of yeast: physiological studies

Nadkarni, Manasi; Lobo, Zita; Maitra, P. K.

doi:10.1016/0014-5793(82)81053-3

Cited by 14 publications

(5 citation statements)

References 8 publications

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“…Evidence has been reported for a particulate phosphofructokinase activity in pfk2 mutants, found in cells treated with toluene and dependent on PFKI (89,90). A similar activity is readily observed in a pfk2 mutant strain carrying P F Kl on a plasmid and, hence, having excess (X subunit (this lab, unpublished).…”

Section: Phosphofructokinase and Fructose Bisphosphatasementioning

confidence: 57%

Mutants in Glucose Metabolism

Fraenkel¹

1986

Annu. Rev. Biochem.

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Section: Phosphofructokinase and Fructose Bisphosphatasementioning

confidence: 57%

Mutants in Glucose Metabolism

Fraenkel¹

1986

Annu. Rev. Biochem.

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“…The lack of phosphofructokinase-1 activity in cell-free extracts of phosphofructokinase-1 singledeletion mutants has been a matter of controversial discussion in the past. Neither the 'by-pass' hypothesis, in which specific genes (BYP1-3) bypass the cytoplasmic phosphofructokinase-1 reaction, nor the assumption of the existence of a third gene (PFK3) encoding a 'particulate' phosphofructokinase-1 could be supported by recent yeast genome analysis (Breitenbach-Schmidt et al, 1984;Clifton and Fraenkel, 1982;Nadkarni et al, 1982).…”

Section: Glucose Consumption and Phosphofructokinase-1 Activitymentioning

confidence: 99%

Assembly of phosphofructokinase-1 fromSaccharomyces cerevisiae in extracts of single-deletion mutants

Klinder¹,

Kirchberger²,

Edelmann³

et al. 1998

Yeast

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Phosphofructokinase‐1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non‐identical subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell‐free extracts of single‐deletion mutants. In contrast to the previously described lack of phosphofructokinase‐1 activity in cell‐free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption. Upon mixing of separately prepared cell‐free extracts of both deletion mutants very low activity could be measured. About 40% of the wild‐type activity was regained when both mutants were mixed prior to disruption. The reactivation rate could be slightly increased by addition of ATP and fructose 6‐phosphate and was found to be a function of the growth state, particularly of the β‐subunit‐carrying cells. The individual subunits did not interact with Cibacron Blue F3G‐A, a biomimetic ligand of phosphofructokinase‐1. After reassembly of both subunits in vitro a strong affinity of the reconstituted phosphofructokinase‐1 to the dye‐ligand was observed. The inability of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following disruption. These changes give rise to induce an unproductive side reaction like self‐aggregation of the subunits. Because reconstitution of phosphofructokinase‐1 from S. cerevisiae behaves in a similar way to that of hemoglobin and luciferase, we would speculate a general mechanism for assembly of oligomeric proteins in vivo. © 1998 John Wiley & Sons, Ltd.

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“…Sedimentation experiments were done according to the procedure followed by Nadkarni et al [15]. The markers used were P-galactosidase and alkaline phosphatase of Escherichia coli and pyruvate kinase from rabbit muscle, corresponding to sZ0,,, values of 16.0 S [16] …”

Section: Other Proceduresmentioning

confidence: 99%

Complementation of soluble phosphofructokinase activity in yeast mutants

Gayatri

Maitra

1991

European Journal of Biochemistry

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We describe here the genetic and biochemical analyses of two classes of mutations in the soluble phosphofructokinase (PFK I) of Saccharomyces cerevisiae : those leading to the loss of activity and those giving rise to a kinetically altered enzyme. Complementation and allele-testing between these two classes of mutants show that loss of enzyme activity in vitro can come about not only by mutations in the catalytic subunit but also in the regulatory subunit. Also, a mutation in the catalytic subunit can give rise to an enzyme altered in its kinetic properties in a manner phenomenologically similar to that caused by a mutation in the regulatory subunit.The results of the complementation studies in diploids suggest that, in spite of their distinct functions, both the subunits are essential for activity to be detected in vitro. This is confirmed by the reconstitution of an active PFK I enzyme by mixing cell-free extracts of two complementing parents, each of which lacks the enzyme activity. PFK activity appears in the mixture, reaching a maximum value of 60-100% of that of the diploid in 15-30 min at 24°C. Unlike the catalytic subunit which exists in various multimeric states in cell-free extracts of the mutant bearing only this subunit, the regulatory subunit exists largely as a monomer in a mutant devoid of the catalytic subunit. The reconstituted enzyme, however, is indistinguishable from that of the wild type, as analysed by sedimentation studies and Western blot analysis, demonstrating that only the heteromeric complex of the two subunits is active, while neither of the individual subunits displays activity in vitro.The yeast, Saccharomyces cerevisiae, possesses two distinct phosphofructokinases: a soluble form (PFK I) and a particulate form (PFK 11) (for a recent review see [I]). The presence of either of these isozymes is sufficient for growth on glucose [2]. The soluble phosphofructokinase is an octamer with a native molecular mass of about 800 kDa and is composed of four CI subunits and four p subunits [l]. Analyses of mutants (reviewed in [3]) and molecular cloning of the genes involved [4] have shown that PFKI and PFK2 are the structural determinants of the p and CI subunits, respectively (for nomenclature see Materials and Methods). The gene PFK2 is known to be shared between the soluble and the particulate forms of phosphofructokinase [5].

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Particulate phosphofructokinase of yeast: physiological studies

Cited by 14 publications

References 8 publications

Mutants in Glucose Metabolism

Mutants in Glucose Metabolism

Assembly of phosphofructokinase-1 fromSaccharomyces cerevisiae in extracts of single-deletion mutants

Complementation of soluble phosphofructokinase activity in yeast mutants

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