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1.
Pyruvate kinase isozyme patterns in the ventricle of developing chicks shift gradually from one dominated by type K at ten days of embryonic development to the adult pattern, which is dominated by type M. Hybrid isozymes are apparent throughout development and are most prominent from two days before hatching until at least 14 days after hatching. These hybrid isozymes indicate simultaneous synthesis of the two subunit types in the same cells.The complex isozyme patterns of the chick heart probably limit the usefulness of simple kinetic analyses on tissue extracts for determing isozymic compositions during development.  相似文献   

2.
The L- and M2-type pyruvate kinase from the liver of 1-day old rats demonstrated no significant activation nor inhibition by treatment with cyclic AMP, glucagon or insulin. Neither was there any change in their isozymic composition. By means of incorporation with [3H]leucine followed by immunoprecipitation, the rates of synthesis of both the L- and M2-type pyruvate kinase were not considerably affected by all three modulators. Insulin and glucagon do not direct an immediate change in the synthesis of liver pyruvate kinase and a fluctuation in the insulin/glucagon ratio is not a probable signal for regulating the isozymic expression in the neonatal period.  相似文献   

3.
PYRUVATE KINASE ISOZYMES IN NEURONS, GLIA, NEUROBLASTOMA, AND GLIOBLASTOMA   总被引:2,自引:2,他引:0  
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. Neurons and glial cells were isolated from rat and mouse brains at the various developmental levels. The pyruvate kinase isozyme patterns in the two cell types were similar to each other and to the patterns seen in whole brain homogenates at all ages, indicating similar rates of isozymic maturation in the two cell types. The correlation of maturation with pyruvate kinase isozyme patterns was further tested in cultures of malignant cell lines. A K-M hybrid set, weighted toward K4, was seen in two clonal lines of mouse neuroblastoma under normal culture conditions. However, lowering the serum concentration in the culture medium or adding bromodeoxyuridine caused a shift in the patterns toward type M as the cells differentiated, mimicking in part the in vivo maturation of normal cells. On the other hand, a rapidly growing and poorly differentiated line of rat glioblastoma had only K4 under all conditions examined.  相似文献   

4.
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.  相似文献   

5.
We here studied the protein kinase activity and in vitro phosphorylable sites of non-histone nuclear proteins, 0.4 M NaCl extracts (mostly chromosomal proteins) from chick embryo fibroblasts (CEF), infected or not with a Schmidt Ruppin strain subgroup A of Rous sarcoma virus (RSV).The infection and transformation of chick fibroblasts by RSV induced an increase in kinase activity and endogenous phosphorylation of non-histone chromosomal (NHC) proteins. The stimulation, by a change of medium, of the proliferation of dense cultures of normal chick fibroblasts also induced an increase in the kinase activity and endogenous phosphorylation of NHC proteins.However, two-dimensional gel electrophoresis of the 32P-phosphorylated proteins showed that stimulation due to a change of medium and that due to the expression of transformation were very different. The stimulation by a change of medium increased to a greater or lesser extent the phosphorylation of the different NHC proteins, with no fundamental variations in the pattern of protein phosphorylation. In contrast, RSV infection induced significant changes in the pattern of protein phosphorylation. One of the most striking feature was the large increase of amount and phosphorylation of high molecular weight (HMW) proteins in particular of phosphoproteins having an evaluated molecular weight (MW) of 78 K and 82 K and pI>8.2.The percent of phosphotyrosine residues in NHC proteins was clearly increased when the proteins were extracted from transformed cells instead of normal cells. But the alkaline treatment of two-dimensional gel electrophoresis indicated that the 80 K phosphoproteins did not contain phosphotyrosine residues, and thus cannot be considered as substrates for pp60src kinase.  相似文献   

6.
Pyruvate kinase requires K+ for maximal activity; the enzyme exhibits 0.02% of maximal activity in its absence [Kayne, F. J. (1971) Arch. Biochem. Biophys. 143, 232-239]. However, pyruvate kinase entrapped in reverse micelles exhibits an important K+-independent activity [Ramírez-Silva, L., Tuena de Gómez-Puyou, M., & Gómez-Puyou, A. (1993) Biochemistry 32, 5332-5338]. It is possible that the amount of water, as well as interactions of the protein with the micelles, can account for this behavior. We therefore explored the solvent effects on the catalytic properties of muscle pyruvate kinase. The enzyme exhibited an activity of 19.4 micromol x min(-1) x mg(-1) in 40% dimethylsulfoxide, compared with 280 and 0.023 micromol x min(1) x mg(-1) observed with and without K+ in water, respectively. pH activity profiles and kinetic constants for the substrates of pyruvate kinase in dimethylsulfoxide without K+ were similar to those in 100% water with K+, and differed from those in water without K+. The spectral center of mass of the emission spectrum of pyruvate kinase in 100% water exhibited a blue shift of 3.5 nm in the presence of Mg(2+), phosphenolpyruvate, and K+, ligands that induce the active conformation of the enzyme. The spectral center of mass of the apoenzyme in 30-40% dimethylsulfoxide coincided with that of the enzyme-Mg(2+)-phosphenolpyruvate-K+ complex in 100% water. The water relaxation rate enhancement factor and binding of phosphenolpyruvate to the pyruvate kinase-Mn(2+)-(CH3)4N+ complex in 30-40% dimethylsulfoxide were similar to those of the pyruvate kinase-Mn(2+)-K+ complex in water. The aforementioned results indicate that when muscle pyruvate kinase is without K+, 30-40% dimethylsulfoxide induces its active conformation.  相似文献   

7.
Thymidine kinase in chick embryo retina reaches its highest values on the 8–10th day of development, then declines reaching the lowest value at hatching. The rate of DNA synthesis essentially follows this activity while, in contrast, nucleoside phosphotransferase increases progressively during development. Glucocorticoids at 5 × 10?6M lower the level of thymidine kinase in isolated retinas of chick embryo. The most effective steroid was hydrocortisone. The effect was observed in retinas from 8–18-day-old chick embryo and, except on the 18th day, was always of the same magnitude. We suggest that a glucocorticoid can be the natural factor responsible for the marked fall in thymidine kinase during development. Brief periods of exposure to steriods increase nucleoside phosphotransferase activity in isolated chick embryo retinas. When the exposure was longer than 3 h this activity was also clearly decreased. We conclude that other factors are responsible for the natural increment which occurs for this activity during development.  相似文献   

8.
The addition of glucagon (10?6 M) to an incubation mixture containing 32Pi and hepatocytes isolated from livers of rats fed ad libitum results in both a 3-fold increased incorporation of 32P into L-type pyruvate kinase and a decreased catalytic activity. The 32P incorporated into pyruvate kinase was covalently bound to the enzyme as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In addition, exogenous cyclic AMP (10?3 M) stimulated the phosphorylation and the suppression of catalytic activity to a similar extent. On the other hand, insulin (10?7 M) had essentially no effect on the incorporation of 32P into pyruvate kinase or on its catalytic activity under the conditions used in this study. These results suggest that phosphorylation of pyruvate kinase invivo is stimulated by glucagon via cyclic AMP and cyclic AMP-dependent protein kinase and that the activity of the enzyme is, at least in part, regulated by a phosphorylation-dephosphorylation mechanism.  相似文献   

9.
Bovine muscle pyruvate kinase was inactivated by treatment with trinitrobenzenesulfonic acid; approximately one trinitrophenyl group was incorporated per subunit. ADP or Mg-ADP decreased the rate of inactivation but Mg++ alone or phosphoenolpyruvate had no effect. The inactivated protein was treated with trypsin and the trinitrophenylated peptide isolated by gel filtration. Homogeneity of the isolated peptide was shown by high voltage electrophoresis and high pressure liquid chromatography. Amino acid analysis and sequence determination revealed the presence of an acidic peptide 34 amino acids long and containing ?-trinitrophenylated lysine.  相似文献   

10.
A calcium-sensitive, phospholipid-dependent protein kinase (protein kinase C) and its three isozymes were purified from rat heart cytosolic fractions utilizing a rapid purification method. The purified protein kinase C enzyme showed a single polypeptide band of 80 KDa on SDS-polyacrylamide gel electrophoresis, and was totally dependent on the presence of Ca2+ and phospholipid for activity. Diacylglycerol was also found to stimulate enzymatic activity. Autophosphorylation of the purified PKC showed an 80 KDa polypeptide. The identity of the purified protein was also verified with monoclonal antibodies specific for PKC. Further fractionation of the purified PKC on a hydroxylapatite column yielded three distinct peaks of enzyme activity, corresponding to type I, II and III based on similar chromatographic behaviour as the rat brain enzyme. All three forms were entirely Ca2– and phosphatidylserine dependent. Type II was found to be the most abundant. Type I was found to be highly unstable. PKC activity studies demonstrate that types II and III isozymic forms are different with respect to their sensitivity to Ca2+.Abbreviations PKC Protein Kinase C - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis - Km Michaelis constant - NBT Nitro-Blue Tetrazolium - BCIP 5-Bromo-4-Chloro-3-Indolyl Phosphate  相似文献   

11.
A cAMP-independent protein kinase from chicken liver phosphorylated and inactivated pyruvate kinase type M2 from the same tissue. Complete inactivation was reached when 4 mol of phosphate were incorporated/mol of tetrameric pyruvate kinase. The protein kinase bound with high affinity to pyruvate kinase type M2 (Km value for pyruvate kinase = 6 X 10(-10)M; it phosphorylated phosvitin and casein but not histones, ATP and GTP were substrates. The differences between the properties of this protein kinase in the interconversion of pyruvate kinase and that described previously are discussed.  相似文献   

12.
The effect of glucagon on the phosphorylation of pyruvate kinase in 32P-labelled slices from rat liver was investigated. Pyruvate kinase was isolated by immunoadsorbent chromatography. The enzyme was partially phosphorylated in the absence of added hormone (0.2 mol of phosphate/mol of enzyme subunit). Upon incubation with 10?7 M glucagon, the incorporation of [32P]phosphate was 0.6–0.7 mol/mol of enzyme subunit. Concomitantly, the concentration of intracellular cyclic 3′,5′-AMP increased from 0.3 to 3.2 μM. The phosphorylation inhibited the enzyme activity at low concentrations of phosphoenolpyruvate (60% at 0.5 mM). Almost maximal phosphorylation of the enzyme was reached within 2 min after the addition of glucagon. The concentration of hormone giving half maximal effect on the pyruvate kinase phosphorylation was about 7×10?9M. The inactivation of the enzyme paralleled the increase in phosphorylation. It is concluded that pyruvate kinase is phosphorylated in the intact liver cell.  相似文献   

13.
Prothymosin α (ProTα) is a multifunctional protein that, in mammalian cells, is involved in nuclear metabolism through its interaction with histones and that also has a cytosolic role as an apoptotic inhibitor. ProTα is phosphorylated by a protein kinase (ProTαK), the activity of which is dependent on phosphorylation. ProTα phosphorylation also correlates with cell proliferation. Mass spectrometric analysis of ProTαK purified from human tumor lymphocytes (NC37 cells) enabled us to identify this enzyme as the M2-type isoenzyme of pyruvate kinase. A study on the relationship between ProTαK activity and pyruvate kinase isoforms in NC37 cells and in other cell types confirmed that the M2 isoform is the enzyme responsible for ProTαK activity in proliferating cells. Yet, about 10% of the cellular pool of the M2 isoform shows specific affinity for ProTα and is responsible for ProTαK activity. This pool of M2 protein possesses no observable pyruvate kinase activity and changes its responses to various effectors of pyruvate kinase activity; however, these responses to PK effectors are maintained by the main cellular fraction containing the M2 isoform. Acquisition of ProTαK activity by M2 seems to be due to the phosphorylation of serine and threonine residues, which, besides being essential for its catalytic activity, induces a trimeric association of ProTαK. This association can be shifted to a tetrameric form by fructose 1, 6-bisphosphate, which results in a decrease in ProTαK activity.  相似文献   

14.
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.  相似文献   

15.
In the absence of glycolytic intermediate, fructose-1,6-bisphosphate, pyruvate kinase type K exists in the dimeric form and is readily phosphorylated, whereas in the same sample and the same conditions pyruvate kinase type M is present as a tetramer and is not phosphorylated. Addition of fructose-1,6-bisphosphate results in the association of dimeric K2 molecules to a tetrameric K4 enzyme as determined by gel filtration and cellulose acetate electrophoresis, with concomitant loss of the capacity of the K isozyme to become phosphorylated. Phosphorylated K2 dimers can also tetramerize, but with a low recovery of the radiolabel, suggesting a fructose-1,6-bisphosphate induced dephosphorylation or selective degradation. The dimeric K isozyme is enzymatically active; inactive K-type monomers can be detected by immunoblot analysis in the absence of fructose-1,6-bisphosphate, but no phosphorylated pyruvate kinase is present in this fraction. The formation of K4 tetramers can not be accomplished by the substrate phosphoenolpyruvate. Fructose-1,6-bisphosphate is an allosteric activator of pyruvate kinase type K and induces hyperbolic saturation curves for phosphoenolpyruvate. In contrast, in the absence of effectors, pyruvate kinase type M exhibits Michaelis-Menten kinetics, but sigmoidal curves can be induced by the amino acid phenylalanine. However, even in the presence of phenylalanine, the M-type maintained its tetrameric configuration and did not serve as a substrate in the phosphorylation reaction. These findings argue for the importance of subunit interaction in the regulation of phosphorylation of pyruvate kinase.  相似文献   

16.
Preparations of the 90K and 110K components of the chick oviduct progesterone receptor (PR) purified to near homogeneity were tested for protein kinase activity. The 90K component was shown to incorporate radioactive phosphate from [γ-32P]-ATP in the presence of Ca2+ but not of Mg2+ ions, while the 110K component was phosphorylated in the presence of Mg2+, but not of Ca2+. The enzymatic activity of the 90K polypeptide appeared selective, since added proteins (histones) did not become phosphorylated. However, all proteins present in the 110K preparations were phosphorylated in the presence of Mg2+. These data suggest that components of the chick oviduct PR display protein kinase activity.  相似文献   

17.
ACETYLCHOLINESTERASE IN DEVELOPING CHICK EMBRYO BRAIN   总被引:1,自引:0,他引:1  
–Acetylcholinesterase has been assayed at different stages of development to see whether changes in the activity of this enzyme are correlated in any way with the ontogenesis of electrical activity in the brain of growing chick embryo. The specific activity of the enzyme was highest in the synaptosomal fraction of the brain. The activity of the enzyme increased progressively with the age of the embryo. There were three isozymic forms of the enzyme in the 6-day-old embryo brain. A new isozyme appeared around the 9th day. The Km values of the enzyme for acetylthiocholine from 6- and 20-day-old embryo brains were 6.5 ± 10-5m and 3.3 ± 10-5m respectively. Enzyme preparations from 6-day-old embryos were found to lose 50 per cent of their activity when heated at 50°C for 10 min. Under similar conditions the loss in activity in 18-day-old embryo brain enzyme was 22 per cent.  相似文献   

18.
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.  相似文献   

19.
Anti human M2 type and anti human L type pyruvate kinase sera allowed us to distinguish two groups of pyruvate kinase in man. Erythrocyte and liver (L type) enzymes on the one hand were inhibited by anti L and not all by anti M2 serum; pyruvate kinase from all the other tissues on the other hand were inhibited by anti M2 and not at all by anti L serum. This latter group represent the M type pyruvate kinase isozymes. The M type isozymes have been studied by electrofocusing in thin layer acrylamide-ampholine gel. In adult tissues 4 types of isozymes were found, designated, from acid to alkaline pH, as M2 (predominant form in spleen, leukocytes, lung...), M3, M4 and M1 (predominant form in muscle and brain). In foetal tissues an extra band M2, called M2f, more anodic than M2, was added to the previously described isozymes. Except in brain (in which the isozymes M2, M3, M4 and M1 were found), the most anodic bands (M2f, M2 and M3) were predominant in all the foetal tissues. The isozymes M2f and M2 seem therefore to be the original M type pyruvate kinase forms from which the other isozymes issue. The rate of each isozyme seems to depend on tissue factors characterizing the state of differentiation of some tissues, as indicated by the ability of adult muscle extracts to change the isozymes M2 and M3 into more cathodic forms.  相似文献   

20.
In hepatocyte cultures derived from 15-day-old foetal rats, the appearance of the liver (L) form of pyruvate kinase is blocked when cytosine arabinoside is added on the 2nd day of culture. When added on the 3rd day of culture, the inhibitor of DNA synthesis does not prevent the appearance of the enzyme. If cytosine arabinoside is added on the 2nd day of culture and removed on the 4th day, the enzyme is detected by the 6th day of culture. The specificity of the action of cytosine arabinoside for the L form of pyruvate kinase is in contrast with the lack of effect observed on total protein synthesis and the activity of the embryonic (M2) form of the enzyme.  相似文献   

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