Janet M. Cardenas

PYRUVATE KINASE ISOZYMES IN NEURONS, GLIA, NEUROBLASTOMA, AND GLIOBLASTOMA

Abstract– The distribution of pyruvate kinase isozymes (EC 2.7.1.40) was examined in cells and tissues from the central and peripheral nervous system of the rat. Most tissues contain significant quantities of both the K4 (fetal type) and M4 (skeletal muscle type) isozymes plus tetrameric hybrids comprised of various combination of the type M and type K subunits. Retina, for example, contains a five‐mem‐bered hybrid set weighted toward K4, while sciatic nerve and spinal cord have patterns very similar to that of adult brain, consisting predominantly of M4 with small amounts of K4 and K‐M hybrids. This adult pattern is achieved by a gradual shift from a hybrid set dominated by K4 in fetal life, to the pattern at birth at which time the two most prominent bands were M4 and K2M2, and finally to the adult pattern by about 28 days after birth.

Bovine pyruvate kinase isozymes and hybrid isozymes: Electrophoretic studies and tissue distribution☆

Abstract Electrophoresis of various bovine tissue extracts revealed, in addition to the three major homotetrameric isozymes of pyruvate kinase (K 4 , L 4 , and M 4 ), numerous intermediate bands that behave electrophoretically as hybrid isozymes. Kidney, for example, contains both K-L and K-M hybrid sets. Representative hybrids from each set, tentatively identified as K 2 L 2 and K 3 M, were isolated from kidney by ionexchange chromatography and their subunit compositions were confirmed by dissociation and subsequent reassociation into new hybrid sets. All of the tissues examined that contain type K 4 also have substantial quantities of K-M hybrids, establishing the presence of the type M isozyme in a great many tissues other than striated muscle and brain, where it is most abundant. In addition, small quantities of K subunits apparently are produced even in striated muscle, which previously had been thought to contain only M 4 . The pattern of hybrids and enzyme specific activities differ markedly within tissues from the same organ, as shown by dissection of the heart and great vessels. Aortic smooth muscle has a fairly uniform distribution of K-M hybrids, while cardiac muscle has mostly M 4 with a little KM 3 . Connective tissue from heart valves, on the other hand, has a five-membered set dominated by K 3 M, while Purkinje fibers have a five-membered set dominated by KM 3 . The occurrence of K-M hybrids in these and many other tissues indicates that the distribution of mammalian pyruvate kinase isozymes is much more complex than previously reported.

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 detection of proteins in unstained gels by the use of phosphorescence.

Abstract The intrinsic phosphorescence of unstained proteins may be used to locate their position in gels. The method is especially useful for preparative procedures but can be used analytically.

Pyruvate kinase isozymic shifts of differentiating chick myogenic cells in vivo and in culture

Abstract Developing chick skeletal muscle undergoes an isozymic shift from type K pyruvate kinase to type M during development. A major increase in pyruvate kinase activity follows the isozymic shift, resulting in at least 40-fold higher specific activities by adulthood. Similar isozymic changes occur in primary cultures of myogenic cells from 12-day-old chick embryos. Cultures initially contain only type K pyruvate kinase. Type M appears by the fourth day of culture and accounts for 80–90% of the activity by the eleventh day. Type M did not accumulate when cell fusion was prevented by removing Ca2+ from the growth medium or when protein synthesis was inhibited by cycloheximide.

BOVINE AND CHICKEN PYRUVATE KINASE ISOZYMES INTRASPECIES AND INTERSPECIES HYBRIDS

ABSTRACT. Bovine tissues, like those of other mammals, contain at least three non-interconvertible pyruvate kinase isozymes, designated type K or K 4 , type L or L 4 , and type M or M 4 . The K- and L-type isozymes have sigmoidal kinetics with P-enolpyruvate and are activated by fructose 1,6-diphos-phate, while type M pyruvate kinase has hyperbolic kinetics even in the absence of fructose diphosphate.

Enolase isozymes in Coho salmon.

1. Analysis of the enolase isozymic distribution has been performed in tissues of the Coho salmon, using electrophoretic separation on cellulose acetate strips followed by localization of enzymatic activity. 2. A total of six electrophoretically distinct forms are seen in Coho salmon in patterns that differ both qualitatively and quantitatively from one tissue to another. 3. The isozymes in skeletal muscle and liver are sufficiently similar to one another that a purification procedure previously developed for trout muscle enolase by Cory & Wold (1966) can be used to partially purify enolase from either of the above-mentioned Coho tissues. The main form of enolase in Coho muscle has an isoelectric point of 7.57. 4. Both liver and skeletal muscle enolases can be reversibly denatured in guanidine HCl and subsequently renatured. Liver enolase appeared to renature somewhat faster than muscle enolase under the same conditions. 5. While polyploidy among salmonids may contribute to the complexity of enolase patterns in fish, the differences in isozymic patterns seen from one tissue to another indicate the presence of distinct, nonallelic genes, probably arising through gene duplication.