Kenneth H. Ibsen

A comparison of kinetic parameters obtained with three major non-interconvertible isozymes of rat pyruvate kinase

Abstract The kinetic properties of partially purified kidney cortex, liver and muscle isozymes of rat pyruvate kinase (EC 2.7.1.40) were compared. The liver and kidney cortex enzymes were isolated in forms which were homotropically activated by phosphoenolpyruvate and heterotropically activated by fructose-1,6-diphosphate. In the absence of added modulators, the liver enzyme was less active, but both isozymes were fruther inactivated by l -alanine, l -phenylalanine or ATP. The liver enzyme was relatively more sensitive to ATP, but less sensitive to l -phenylalanine. The muscle enzyme, on the other hand, was isolated in a more active form which was insensitive to ATP or l -alanine inhibition and of intermediate sensitivity to l -phenylalanine inhibition. In the presence of l -phenylalanine, muscle enzyme also underwent homotropic and heterotropic activation. Not any of the isozymes were inhibited by NADH. All three isozymes were activated by K+ or NH4+. NH4+ was the more effective activator for the kidney cortex or liver enzymes, in the former case because of a greater affinity, the latter because of a higher catalytic efficiency. Of the divalent cations tested only Mg2+ and Mn2+ activated. All three isozymes had lower maximal rates when activated by Mn2+, but this ion also consistently acted as a typical K-type activator. Evidence also was obtained which suggested that the change from one conformational form to another might take minutes and therefore, measured kinetic parameters could reflect conformational as well as catalytic phenomena. This observation, plus suggested independent subunit interactions, were considered to be evidence favoring a sequential rather than a concerted mechanism of conformational transition.

Effects of amino acids on the kinetic properties of three noninterconvertible rat pyruvate kinases.

Abstract Three apparent isozymes of pyruvate kinase respond differentially to amino acids. The kidney cortex enzyme (K isozyme) was inhibited by the greatest number of amino acids, alanine and phenylalanine being the most effective. As the nonpolar side chain was increased in bulk relative to alanine or decreased relative to phenylalanine, the amino acids progressively became less effective inhibitors. Polarity also decreased inhibitory potential and serine (3-OH alanine) was a potent activator, able to increase substrate affinity and to reverse the inhibition induced by other amino acids. The amino acids having an effect on the muscle enzyme (M isozyme) show a similar relationship between structure and potential activity, except amino acids with less bulky side chains activate. Again hydroxylation appears to increase the potency of the amino acid as an activator. The liver enzyme (L isozyme) shows a pattern of inhibition similar to the kidney cortex enzyme except that it is much less sensitive to the amino acids with bulky side chains and serine remains an inhibitor, albeit less effective than alanine. These data, plus observed differences in the alanine and phenylalanine inhibition under varied conditions, support a hypothesis that there are two different types of amino acid binding sites with each isozyme having both. However, the response to the bound amino acid varies from isozyme to isozyme. It also was concluded that fatty acids were not modulators of pyruvate kinase activity.

Distribution of pyruvote kinase isozymes among rat organs

Abstract Pyruvate kinase isozymes were isolated in several variable forms using column isoelectric focusing techniques. Five separable forms of the kidney cortex (K-type) isozyme migrated toward the cathode when subjected to electrophoresis on cellulose acetate, but not necessarily with identical mobilities. The isolatedliver (L-type) isozyme migrated toward the anode, as a split activity band; whereas, the isolated muscle (M-type) isozyme remained at the origin. Homogenates from 11 rat organs also were subjected to cellulose acetate electrophoresis and then examined for pyruvate kinase activity. A total of at least five distinct activity bands were observed. However, comparison to the data obtained using enzymes previously separated showed that two of these could be variants of the K-type isozyme, and two could be L-type variants. The fifth band was M-type enzyme. These data thus support the concept that only three basic forms of pyruvate kinase exist in the rat. The K-type enzyme, in its variant forms, was the most widely distributed isozyme. Extracts of the rat organs also were subjected to gel electrofocusing. No evidence of isozymes other than K, L, or M types was obtained.

Human erythrocyte pyruvate kinase conformers obtained by electrofocusing

Abstract Conformers of pyruvate kinase (ATP: pyruvate-phosphotransferase, EC 2.7.1.40) could be consistently obtained with pI values of 5.28 and 6.70. These were converted to a form with a pI value of about 5.86 by (D)-1,6-fructose diphosphate. Still another conformer with a pI value of about 7.2 was obtained in some preparations electrofocused in the absence of 2-mercaptoethanol. Re-electrofocusing of the higher pI material resulted in conversion of some to the form with a pI value of 5.3. The forms with pI values of 5.3 and 6.7 were subjected to sedimentation and kinetic studies. The two conformers differed kinetically from each other and from the sample which had not yet been subjected to electrofocusing.

Control of glycolysis and respiration in substrate-depleted ehrlich ascites tumor cells.

Abstract A highly dampened train of oscillations may be obtained from aerobic suspensions of a hyperdiploid strain of Ehrlich ascites tumor cells, provided the cells are incubated until endogenous substrates just have been depleted. Several intermediates were measured in such oscillating systems and their interrelationships studied. These data were compatible with the concept of glycolytic control by intermediates at the levels of phosphofructokinase and hexokinase and incompatible with the concept of control of glycolysis by mitochondrial sequestering of ATP. On the other hand, they were consonant with the concept of respiratory control mediated by excess mitochondrial ATP, as well as with other possible mechanisms. They further suggested that citrate may act as a dampener, normally preventing oscillations of phosphofructokinase caused by rapid changes in mononucleotide levels; that hexokinase was inhibited by glucose-6-phosphate but ultimately controlled by some other factor; and that the α-GP oxidase was activated briefly but normally remains inactive in these cells.

PROPERTIES OF RAT PYRUVATE KINASE ISOZYMES

ABSTRACT. Data describing the relationship among six variant forms of the K-isozyme and at least three forms of the L-isozyme of the rat are reviewed. A few physical and kinetic parameters describing these variant forms as well as those of the M-isozyme are presented. It is shown that the pH 6.4 variant of the K-isozyme can exist in a higher and lower molecular weight form depending upon the presence of magnesium ion. Evidence is presented supporting the idea that the red cell enzyme is an L-isozyme variant rather than a fourth isozyme. The kinetic properties of what are believed to be the major in vivo forms of the three isozymes are briefly reviewed. Evidence is presented suggesting that K+ saturation follows a sequential pattern.

Stabilization, partial purification, and effects of activating cations, ADP, and phosphoenolpyruvate on the reaction rates of an erythrocyte pyruvate kinase

Abstract Stability of erythrocyte pyruvate kinase was found to be independently enhanced by inorganic ortho -phosphate, magnesium, and 2-mercaptoethanol. Phosphoenolpyruvate could substitute for and was more effective than phosphate. The enzyme was more stable below pH 7.4 than above. These data were utilized to help achieve a partial purification ( K m value for magnesium-ADP was found to be about 3.3 × 10 −4 m . High levels of ADP were found to inhibit reaction rates. This inhibition was released by addition of more magnesium, suggesting free ADP was the inhibitor. The K m value for phosphoenolpyruvate was found to be about 4.6 × 10 −5 m . Optimal assay conditions (TES buffer) were found to be at about: pH 7.0, 90 m m K; 6.4 m m Mg: 2 m m ADP, and 0.8 m m PEP.

Regulation of pyruvate kinase expression and growth in mastocytoma cells. I. Initial observations.

The specific activities of pyruvate kinase and phosphofructokinase but not lactate dehydrogenase increase as P-815 mastocytoma cells approach the stationary phase. During this growth period, the rates of uptake of labelled precursors into DNA, RNA and total protein decreases. On the other hand, the pyruvate kinase protein level changes in parallel with activity. Although the K-isozyme is the primary form of pyruvate kinase expressed, some M-type subunit is also present and both forms undergo an increase in specific activity. In addition, pyruvate kinase expression is also elevated by adding cAMP analogues with theophylline, butyrate or conditioned media. This increased level of expression is hypothesized to be a secondary event associated with a differentiation-like-induced expression of the mast cell phenotype.

Use of the gradient plate immunodiffusion technique

The use of the single dimensional gradient plate immunodiffusion technique as a qualitative and quantitative measure of antigen is described. This method gives results analogous to those obtained using the single radial immunodiffusion method, but it requires about 1/10 the antibody and it accentuates the visibility of multiple antibody—antigen systems at lower concentrations of antigen.

Distribution of pyruvate kinase isozymes in adult and developing Xenopus laevis

Abstract 1. 1. The pyruvate kinase (EC 2.7.1.40) isozyme composition of adult and developing Xenopus laevis tissues was investigated using isoelectrofocusing, DEAE-chromatographic, heat stability and kinetic studies. 2. 2. A total of nine different pI variants were identified. Evidence was presented supporting the concept that the lowest pI form (pH 5.0) was the L-isozyme, an intermediate pI form ( pH = 6.0) was the K-isozyme and the highest pI form ( pH = 7.0) the M-isozyme. The remaining forms were suspected KL and KM hybrids having pI values intermediate to the respective homotetrameric forms. 3. 3. Adduced evidence showed that in vitro incubation could convert the K-isozyme to forms having pI values of suspected KM hybrids indicating possible conversion of K- to M-type subunits. 4. 4. Skeletal muscle is characterized by expression of M-subunits at a high specific activity; heart muscle also has a relatively high specific activity which, however, is primarily due to K-type subunits. 5. 5. The developing embryo initially expresses both K and L type subunits but the specific activity decreases rapidly with development. By the neural crest stage, the expression of the L-subunit is essentially nil and some M-type subunits start to appear. It is suggested that the initial expression of K- and L-subunits is due to the presence of long-lived maternal m -RNA.