Mammalian pyruvate kinase hybrid isozymes: tissue distribution and physiological significance.

The purpose of this study was to examine the pyruvate kinase isozymic patterns of a wide variety of tissues from rats and mice, particularly regarding hybrid isozymes. For these studies, we employed longer electrophoresis times than used in most earlier studies in order to improve the resolution of closely spaced bands. The tissue distributions of types K, L, and M pyruvate kinases were found to be approximately the same as those reported earlier for rats and other mammals. In addition, K-M hybrids could be detected in most tissues examined in relative quantities which differed from one tissue to another in the same organism, in corresponding tissues from different species, and within a single tissue during development. Hybrid isozymes containing type L subunits occur in only a few tissues of either the fetus or the adult of either animal. In earlier studies utilizing L-M hybrid isozymes produced in vitro, we showed that the kinetic properties of a given subunit are profoundly affected by the nature of its neighbors within the tetramer (Dyson and Cardenas, ['73] J. Biol. Chem., 248: 8482-8488). Based on these altered kinetic properties, we suggest that there is little need for anorganism to suppress completely the gene activity for one subunit type of pyruvate kinase during the synthesis of larger quantities of a second subunit type.

Pyruvate kinase isozymes in cells isolated from fetal and regenerating rat liver.

There are at least three major mammalian isozymes of pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40), designated K4, L4, and M4. Whereas parenchymal cells from adult rat liver contain only the type L isozyme, parenchymal cells isolated from fetal and regenerating liver were found to synthesize both the K4 and L4 isozymes. A small amount of K-M hybrid was seen in regenerating liver, but there were no detectable M-L or K-L hybrids. Thus, it appears that type L pyruvate kinase is not synthesized at the same time in the same liver cell with either of the other two isozymes. The intermediate electrophoretic bands seen with homogenates of whole fetal liver, and in some earlier work attributed to either hybrid isozymes or to the presence of M4, are contributed by nonparenchymal cells which, in the fetus, are largely hemopoietic. These additional bands of pyruvate kinase are electrophoretically and immunologically similar to the pyruvate kinase isozymes found in adult erythrocytes. The results reported here suggest a very rigorous control in the synthesis of K4 and L4 isozymes in parenchymal cells of both fetal and regenerating liver as opposed to developing neurons and glia, where the shift from synthesis of type K to type M subunits appears to occur gradually and results in the production of substantial amounts of hybrid isozymes.

The reversibility of skeletal muscle pyruvate kinase and an assessment of its capacity to support glyconeogenesis.

The kinetics of pyruvate phosphorylation by rabbit skeletal muscle pyruvate kinase (EC 2.7.1.40) has been studied with a coupled assay using P-enolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). The reaction sequence is (See journal for formula). Although the equilibrium of the pyruvate kinase reaction by itself strongly favors pyruvate production, the over-all equilibrium of this coupled system favors the depletion of pyruvate, thus greatly reducing the problem of back reaction during the assay. In addition, the oxidation of NADH by malate dehydrogenase makes it possible to monitor the system with a spectrophotometer. The Michaelis constant of pyruvate kinase was found to be 0.9 mM for ATP and 7 mM for pyruvate, values that agree reasonably well with earlier studies using direct assays. However, the maximum velocity is about 6 mumol of pyruvate phosphorylated/min/mg of enzyme, which is very much faster than that indicated by earlier studies. These results suggest that the metabolic significance of the reverse reaction of muscle pyruvate kinase may have been underestimated. In particular, the data given here suggest that its rate in vivo is probably comparable to the observed rate of glycogen synthesis from lactate, making possible glyconeogenesis in muscle by pyruvate kinase reversal without the need for an enzymatic bypass of the kind employed by liver and kidney.

Pyruvate kinase isozymes in adult and fetal tissues of chicken.

Tissues of fetal and adult chickens were examined for pyruvate kinase activity. Two electrophoretically distinguishable and noninterconvertible isozymes were found. One of these, designated as type K (for kidney), is the sole pyruvate kinase in the early fetus and is found in appreciable quantities in all adult tissues except striated muscle. The second isozyme, type M, appears shortly before hatching in striated muscle and brain. These two isozymes correspond in their developmental pattern, tissue distribution, electrophoretic, immunological, and kinetic propertiesto similarly designated mammalian pyruvate kinases. However, no kinetic, immunological, or electrophoretic evidence could be found for a chicken isozyme corresponding to the mammalian type L pyruvate kinase. As the latter isozyme seems to be limited in its distribution mostly to highly differentiated gluconeogenic tissues (notable liver, kidney, and small intestine), our results support the proposition that the mammalian type L pyruvate kinase is a specilized isozyme that is present in mammals but not in birds.

Properties of chicken skeletal muscle pyruvate kinase and a proposal for its evolutionary relationship to the other avian and mammalian isozymes.

Pyruvate kinase (EC 2.7.1.40) was isolated and purified from chicken and turkey breast muscle with a purification procedure very similar to that used for the bovine skeletal muscle isozyme (Cardenas, J., Dyson, R., and strandholm, J. (1973), J. Biol. Chem. 248,6931). A study of the chemical and physical properties of the chicken enzyme revealed that it is a tetramer of four apparently identical subunits, closely resembling in this and most other respects the mamalian type 7 isozyme. The properties of these two enzymes are similar enough to permit subunits of chicken type M pyruvate kinase to combine with subunits of mammalian type L (one of the three mammalian isozymes) to form interspecies tetrameric hybrid isozymes in relative quantities that do not differ makedly from those formed when both the M and L isozymes are of mammalian origin. The similarity between the mammalian and avian type M pyruvates kinases suggests a close evolutionary relationship. Further comparisons among the three mammalian and two avian isozymes of pyruvate kinase are consistent with a common evolutionary origin, perhaps from an ancestral form of the type K isozyme, which is the only pyruvate kinase identified in mammalian and avian embryos.

Studies on the analysis of fluorescence decay data by the method of moments.

The method of moments, as presented by Isenberg and Dyson (1969; Biophys. J. 9:1337) has been shown to be a reliable way of obtaining the amplitudes and time constants of several simultaneously emitting species, even in the presence of an overlapping excitation. Recent improvements in the method include (a) a component incrementation test for determining the number of relaxations, (b) a procedure, which we call exponential depression, for dramatically improving convergence, and (c) a new algorithm for implementing the method of moments on a digital computer with a high degree of flexibility and efficiency. These improvements, as well as new general theory, are described and tested using both synthetic and real experimental data. Component incrementation consists of examining models with increasing numbers of exponential terms. Given adequate precision, we find that an analysis for N + 1 components, of data that are actually represented by N components, provides the correct amplitudes and time constants plus an N + 1 term with an insignificant amplitude. Exponential depression is a transformation in which the original excitation and fluorescence, E(t) and F(t), are multiplied by exp (-lambdat), where lambda is an arbitrary parameter. While the convolution is invariant to this transformation, the proper choice of lambda greatly reduces the number of iterations necessary to obtain the amplitudes and time constants and may even improve their accuracy. In addition, an appendix by John P. Mullooly presents a statistical analysis of the effect of counting error on the method of moments estimates of fluorescence decay parameters, applicable when data are obtained by the monophoton technique. Formulas are derived that give the approximate precision of the decay parameters for the general case of N exponential components, with calculational details for one and two component systems.

The analysis of fluorescence decay by a method of moments.

The fluorescence decay of the excited state of most biopolymers, and biopolymer conjugates and complexes, is not, in general, a simple exponential. The method of moments is used to establish a means of analyzing such multi-exponential decays. The method is tested by the use of computer simulated data, assuming that the limiting error is determined by noise generated by a pseudorandom number generator. Multi-exponential systems with relatively closely spaced decay constants may be successfully analyzed. The analyses show the requirements, in terms of precision, that data must meet. The results may be used both as an aid in the design of equipment and in the analysis of data subsequently obtained.

An investigation of bacteriophage lambda, its protein ghosts and subunits.

The sedimentation coefficients and molecular weights of bacteriophage lambda and its ghost were determined as So(20,w) = 416 x 10(-13) second and M = 57 x 10(6) for the virus, and So(20,w) = 141 x 10(-13) second and M = 21 x 10(6) for the ghost. The protein portion of the virus consists of at least two distinct types of subunits, with molecular weights of 55,000 and 110,000, which differ markedly in their amino acid content.