Studies on avian heart pyruvate kinase during development.

The cytokinin-active nucleoside 6-(4-hydroxy-3-methyl-$\text{cis}$-2-butenylamino)-9-β-$\text{D}$-ribofuranosylpurine, i.e. ribosyl-$\text{cis}$-zeatin, has been isolated from an hydrolysate of tRNA from $\text{Corynebacterium fascians}$. The identification of ribosyl-$\text{cis}$-zeatin is based on biological activity, liquid chromatographic mobility and uv spectrum of the purified material as well as the mass spectrum and gas chromatographic mobility of its trimethylsilylated derivative.     

Evolution of the functional properties of pyruvate kinase isozymes: pyruvate kinase L fromRana pipiens

Summary The regulatory properties of type L pyruvate kinase fromRana pipiens are intermediate between those of the mammalian K and L isozymes. As with mammalian type L, the levels of the frog isozyme are affected by the animal's nutritional state. The mammalian and amphibian isozymes show similar sensitivities to fructose 1,6-bisphosphate activation and amino acid inhibition. By contrast, the frog L isozyme shares several properties of the K class: ie. irreversible inactivation by oxidized glutathione and lack of response to a cyclic AMP stimulated phosphorylation. Furthermore, as for some mammalian K isozymes, frog type L shows a high PEP affinity and a low cooperativity of PEP binding.Insofar as the properties of this present day enzyme reflect those of its counterpart in the amphibian ancestor of higher vertebrates, our results suggest that at its first expression, the type L resembled the type K. Many important regulatory properties of the L isozyme, especially the sensitivity to phosphorylation, were acquired more recently perhaps in association with an increased importance of constant blood glucose.Abbreviations DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis(2-aminoethylether)-N,N-tetraacetic acid - FB fructose-1,6-bisphosphate - PEP phosphoenolpyruvate - PK pyruvate kinase     

Postnatal changes in canine erythrocyte pyruvate kinase isozymes

The isozyme pattern of pyruvate kinase in canine erythrocytes changes following birth. These changes have been followed by electrophoretic, immunologic, and kinetic measurements of the isozymes. At birth, a mixture of isozymes is present consisting of the M₂ isozyme and hybrid molecules containing M₂ and R subunits. With increasing animal age, the content of M₂ subunits decreases and the content of R subunits increases. At 6 months of age, the isozyme pattern is indistinguishable from that of adult erythrocytes which contain only the R tetramer. We conclude that there is a switch in erythrocyte pyruvate kinase gene expression during the first 6 months of postnatal life. The existence of hybrid molecules during the switch indicates that both M₂ and R genes are expressed within each erythroid precursor cell. The developmental changes in erythrocyte pyruvate kinase are consistent with the role of this enzyme in the regulation of the oxygen-transport function of canine hemoglobin by 2,3-diphosphoglycerate in the postnatal period.This research was supported by Public Health Service Grant HD-10595.     

Hybrid isozymes of rat pyruvate kinase. Their subunit structure and developmental changes in the liver

Thin-layer polyacrylamide gel electrophoresis of various rat tissues revealed three major isozymes (types L, M1 and M2) and various intermediate forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40). In vitro dissociation and reassociation of purified enzymes showed that the three major isozymes had homotetrameric structures. L.M2 hybrids and M1.M2 hybrids closely resembled some naturally occurring intermediates; the subunit structure of intermediates isolated from the small intestine (form 3 or form 4) were estimated to be (L)2(M2)2 and (L)(M2)3, respectively. Pyruvate kinase activity after electrophoresis could be estimated quantitatively from densitometric measurements of the electrophoretic pattern. Type L activity in fetal liver was separated from type R activity derived from intrahepatic erythropoietic cells. It changes in three distinct steps during development: it increased during the late fetal period, remained steady during the neonatal period and increased again after weaning. Some of the intermediates found in extracts of early fetal iver were shown to cross-react with both anti-L and anti-M1 serum, suggesting that they might be L.M2 or R.M2 hybrids. These hybrid enzymes were shown to appear only during early fetal and neonatal periods.     

Bovine pyruvate kinase isozymes and hybrid isozymes. Electrophoretic studies and tissue distribution.

Electrophoresis of various bovine tissue extracts revealed, in addition to the three major homotetrameric isozymes of pyruvate kinase (K₄, L₄, and M₄), 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 K₂L₂ and K₃M, 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 K₄ 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 M₄. 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 M₄ with a little KM₃. Connective tissue from heart valves, on the other hand, has a five-membered set dominated by K₃M, while Purkinje fibers have a five-membered set dominated by KM₃. 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.     

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.     

Differential regulation of diacylglycerol kinase isozymes in cardiac hypertrophy

To examine the involvement of diacylglycerol kinase (DGK) and phosphatidic acid phosphatase (PAP) in pressure overloaded cardiac hypertrophy, rats were subjected to either ascending aortic banding for 3, 7, and 28 days or sham operation. In comparison with sham-operated rats, the left ventricular (LV) weight of the aortic-banded rats increased progressively. At 28 days after surgery, the expression of DGKepsilon mRNA but not DGKzeta or PAP2b mRNA in the LV myocardium significantly decreased in the aortic-banded rats compared with the sham-operated rats. DGKzeta protein in the LV myocardium translocated from the particulate to the cytosolic compartment in the aortic-banded rats. Furthermore, the myocardial content of 1,2-diacylglycerol and PKCdelta protein expression in the particulate fraction of the LV myocardium significantly increased in aortic-banded rats compared with sham-operated rats. These results suggest that DGKepsilon and DGKzeta play distinct roles in the development of pressure overloaded cardiac hypertrophy and that the two isozymes are differentially regulated.     

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.     

Purification of four pyruvate kinase isozymes of rats by affinity elution chromatography.

1. Purification of four isozymes of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) L, M1, M2 and R was much improved to give good yields by affinity elution chromatography. The enzyme was eluted from a phosphocellulose column with 0.5 mM phosphoenolpyruvate. Types L, M2 and R were stabilized with fructose 1,6-diphosphate throughout the purification procedures. 2. The isozymes were crystallized under various conditions: types L and R were readily crystallized from medium of low ionic strength, types L, M1, and M2 were crystallized from ammonium sulfate solution in different forms in the presence and absence of phosphoenolpyruvate. Type M1 was also crystallized in different forms in the presence and absence of fructose 1,6-diphosphate. 3. Amino acid analyses showed that the compositions of types L and R, and of types M1 and M2, respectively, were very similar.     

Purification and properties of two isozymes of pyruvate kinase from Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was found to contain up to five electrophoretic forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) depending on growth conditions. M. racemosus hyphal cells grown on glutamic acid as the carbon source contained only the fastest electrophoretic form, designated PK1, while yeast cells grown on glucose contained only the slowest electrophoretic form, PK5. Intermediate electrophoretic forms PK2, PK3, and PK4 as well as PK1 and PK5 were found in hyphal cells grown on media containing fructose or cellibiose. All five electrophoretic forms had molecular weights of ca. 230,000 as determined from plots of log Rm versus acrylamide gel concentration. Both PK1 and PK5 were purified to homogeneity and determined to be homotetramers, with subunit molecular weights of 54,000 and 58,100, respectively. The amino acid content of PK1 and PK5 was determined and found to be similar but not identical. Analysis of limited tryptic digests and cyanogen bromide cleavage fragments of PK1 and PK5 indicate that the subunits of the two isozymes are significantly different.     

Formation of functional heterodimers by isozymes 1 and 2 of pyruvate dehydrogenase kinase

Pyruvate dehydrogenase kinase (PDK) is a mitochondrial enzyme responsible for regulation of the pyruvate dehydrogenase complex and, consequently, aerobic oxidation of carbohydrate fuels in general. In mammals, there are four genetically and biochemically distinct forms of PDK that are expressed in a tissue-specific manner (PDK1, PDK2, PDK3, and PDK4). These protein kinases have been shown to function as dimers, but the possibility of heterodimerization between various isozyme subunits has not yet been investigated. Here, we demonstrate that two members of the PDK family, PDK1 and PDK2, form heterodimeric species when coexpressed in the same Escherichia coli cell. The heterodimeric kinase produced in vivo was purified to near homogeneity by affinity chromatography. The purified kinase was stable and was not subjected to reassortment of the subunits. The heterodimeric kinase was catalytically active and was clearly distinct from homodimeric PDK1 or PDK2 with respect to kinetic parameters, site specificity and regulation. These data strongly suggest that heterodimerization between PDK1 and PDK2 adds another level of diversity to this protein family in addition to that which arises from gene multiplicity.     

Inherited persistence of immature type pyruvate kinase and hexokinase isozymes in dog erythrocytes

1. Red cell pyruvate kinase (EC 2.7.1.40) and hexokinase (EC 2.7.1.1) in high and low potassium (K) dogs were shown to exist as multiple forms which were separable by electrophoresis and ion-exchange chromatography. The R2-type pyruvate kinase, which was determined to be a young type enzyme in canine red cells, was shown to be the predominant form of pyruvate kinase in high K cells. 2. The M2-type pyruvate kinase, a prototype isozyme in erythroid cells, existed in high K dog erythrocytes as well as in high K and low K dog reticulocytes. 3. Isozyme analysis of high K red cell hexokinase also showed a profile similar to that obtained for low K reticulocytes. 4. These results seem to reflect the immaturity of high K erythrocytes, which suggest that an abnormal cell differentiation or maturation may occur at an early stage of erythroid cell proliferation in high K dogs.     

Multienzymic nature of pyruvate kinase during development of Hymenolepis diminuta (Cestoda).

H. diminuta at different stages of development contained as many as five pyruvate kinase isozymes. Four of these were unusually sensitive to allosteric activation by fructose-1,6-P2. One isozyme which occurred only in adults or near-adults was insensitive but had a relatively low Km. All were inhibited by ATP and Ca2+, none by alanine, and the pH optimum was unaffected by fructose-1,6-P2. The five isozymes were present in gravid or reproductively active proglottids. Two of them occurred after eight days growth in the rat intestine, and three after four days. These three were also present in the immature, anterior proglottids of adult parasites. Hexacanth larvae from gravid proglottids, as well as cysticercoids developing from these larvae in Tenebrio molitor, possessed only two isozymes. It was inferred from information on tissue concentrations of ADP, ATP, phosphoenolypyruvate (PEP) and on K0.5S and Km that competition between pyruvate kinase and PEP carboxykinase is probably controlled by fructose-1,6-P2 concentrations. Since H. diminuta is an obligatory fermenter in which gluconeogenesis is minimal, the probable function of its L-type pyruvate kinases is to control the specific composition of lactic, acetic and succinic acid mixtures that are excreted at different stages of development.     

Modification of pyruvate kinase isozymes in prolonged primary cultures of adult rat hepatocytes.

Pyruvate kinase isozymic changes were studied in the adult hepatocyte cultures, by electrophoretic, kinetic and immunological methods. We were able to maintain parenchymal cells from normal adult rat liver in non-proliferating monolayer cultures up to 10 days. Hepatocytes appeared to contain a dominant PK I type up to 4-5 days of culture. After day 5, PK III type was regularly present with PK I and after 7 days PK III type was always the only isozyme detected in culture. It must be pointed out that, by the Ouchterlony method and sometimes by electrophoresis, concentrated extracts from freshly isolated hepatocytes or starting hepatocyte cultures did also contain Pyruvate kinase PK III type. These results suggest that Pyruvate kinase III is present but partly repressed in the adult parenchymal cells and becomes derepressed in culture.     

Effector analogues detect varied allosteric roles for conserved protein-effector interactions in pyruvate kinase isozymes

The binding site for allosteric inhibitor (amino acid) is highly conserved between human liver pyruvate kinase (hL-PYK) and the rabbit muscle isozyme (rM(1)-PYK). To detail similarities/differences in the allosteric function of these two homologues, we quantified the binding of 45 amino acid analogues to hL-PYK and their allosteric impact on affinity for the substrate, phosphoenolpyruvate (PEP). This complements a similar study previously completed for rM(1)-PYK. In hL-PYK, the minimum chemical requirements for effector binding are the same as those identified for rM(1)-PYK (i.e., the l-2-aminopropanaldehyde substructure of the effector is primarily responsible for binding). However, different regions of the effector determine the magnitude of the allosteric response in hL-PYK vs rM(1)-PYK. This finding is inconsistent with the idea that allosteric pathways are conserved between homologues of a protein family.