The regulatory characteristics of yeast fructose-1,6-bisphosphatase confer only a small selective advantage.

The question of how the loss of regulatory mechanisms for a metabolic enzyme would affect the fitness of the corresponding organism has been addressed. For this, the fructose-1,6-bisphosphatase (FbPase) from Saccharomyces cerevisiae has been taken as a model. Yeast strains in which different controls on FbPase (catabolite repression and inactivation; inhibition by fructose-2,6-bisphosphate and AMP) have been removed have been constructed. These strains express during growth on glucose either the native yeast FbPase, the Escherichia coli FbPase which is insensitive to inhibition by fructose-2,6-bisphosphate, or a mutated E. coli FbPase with low sensitivity to AMP. Expression of the heterologous FbPases increases the fermentation rate of the yeast and its generation time, while it decreases its growth yield. In the strain containing high levels of an unregulated bacterial FbPase, cycling between fructose-6-phosphate and fructose-1,6-bisphosphate reaches 14%. It is shown that the regulatory mechanisms of FbPase provide a slight but definite competitive advantage during growth in mixed cultures.     

Spinach chloroplast fructose-1,6-bisphosphatase: identification of the subtilisin-sensitive region and of conserved histidines

Chloroplast fructose-1,6-bisphosphatase (FbPase) is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars. The properties of the chloroplast enzyme are clearly distinct from those of cytosolic gluconeogenic FbPases. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. However, preliminary amino acid sequence data (78 residues) have demonstrated that a significant degree of amino acid sequence similarity exists between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase [Harrsch, P.B., Kim, Y., Fox, J.L., & Marcus, F. (1985) Biochem. Biophys. Res. Commun. 133, 520-526]. In the present study, we have identified two structural features of spinach chloroplast FbPase that appear to be common to all FbPases. These include (a) the presence of a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FbPases and (b) the recognition of two conserved histidine residues, equivalent to histidines-253 and -311 of the mammalian enzymes. In addition, we have obtained sequence information accounting for more than three-fourths of the primary structure of spinach chloroplast FbPase. The high degree of homology observed between the chloroplast enzyme and gluconeogenic FbPases suggests a common evolutionary origin for all fructose-1,6-bisphosphatases in spite of their different functions and modes of regulation.     

Chicken liver fructose 1,6-bisphosphatase:purfication and some properties.

An improved procedure is described for the purification of fructose 1,6-bisphosphatase (FbPase) from chicken liver. The purified enzyme shows a single band in gel electrophoresis either in the presence or absence of sodium dodecyl sulfate. From 200 g of frozen liver, we have obtained about 29 mg of homogeneous enzyme, with the pH profile indistinguishable from that of the enzyme in crude extracts. The overall recovery of enzyme activity is about 71%. The FbPase protein was estimated to represent approximately 0.36% of the total soluble protein of crude liver extract. Treatment of purified enzyme with papain or subtilisin results in a rapid increase in activity at pH 9.2 and a gradual decrease at pH 7.5, while digestion with trypsin or chymotrypsin results in a concomitant decrease in activities at both pH 9.2 and 7.5. The rates of hydrolysis by these four proteases are all markedly decreased in the presence of AMP. Both AMP and fructose 1,6-bisphosphate increase the thermal stability of the enzyme, and their effects are additive. Attempts were made to investigate the structural requirements for histidine activation. The results suggest that activation by this amino acid involves not only the imidazole ring but also the α-amino and α-carboxyl groups.     

Heterogeneous cellular expression of creatine kinase isoenzyme during normal rat heart development

The degree to which developmentally related alterations in cardiac creatine kinase (CK) activity reflect modification of CK isoenzyme gene expression remains uncertain. The present studies addressed this question by assessing multiple aspects of CK in rat heart during the perinatal to adult transition. In addition to whole tissue, isolated and purified muscle and nonmuscle cells were studied, as well as myofibrillar, mitochondrial, and cytosolic subcellular fractions. Whole homogenate CK enzyme specific activity nearly doubled during the weanling to adult developmental period. Muscle cell CK activity increased by a similar magnitude. Nonmuscle cell activity decreased. In the adult heart, both myofibrillar and mitochondrial CK activities were augmented versus the weanling heart. The cytoplasmic fraction activity held constant during development. Electrophoretic isoenzyme analyses of both weanling and adult cardiac muscle cells indicated the presence of mitochondrial CK and MM-CK isoforms. Weanling heart nonmuscle cells contained mitochondrial, MM, MB, and BB isoforms; however, BB isoform was not detected in the adult heart nonmuscle cells. Arrhenius plots provided information regarding heart muscle and nonmuscle cell alterations during development. CK activation energies were also determined for whole tissue, muscle/nonmuscle cells, myofibrils, mitochondria, and cytosol. Results demonstrate that heterogeneous muscle/nonmuscle cellular composition and differential myofibrillar/mitochondrial subcellular composition account for normal, developmentally related changes in heart CK enzyme activity. CK isoenzyme gene expression changes were not detected in cardiac muscle cells, and transition of CK-B to CK-M gene expression is limited to nonmuscle cells during normal, weanling to adult development in the rat heart.     

Allosteric activation of brain hexokinase by magnesium ions and by magnesium-ion–adenosine triphosphate complex

1. The highest blood concentrations of ketone bodies were found at 5 days of age, after which time the concentration fell to reach the adult value by 30 days of age. 2. Both mitochondrial and cytoplasmic hydroxymethylglutaryl-CoA synthase activities were detected, with highest activities being found in the mitochondria at all stages of development. Activity of the mitochondrial enzyme increases rapidly immediately after birth, showing a maximum at 15 days of age, thereafter falling to adult values. The cytoplasmic enzyme, on the other hand, increased steadily in activity after birth to reach a maximum at 40 days of age, after which time activity fell to adult values. 3. Both mitochondrial and cytoplasmic aceto-acetyl-CoA thiolase activities were detected, with the mitochondrial enzyme having considerably higher activities at all stages of development. The developmental patterns for both enzymes were very similar to those for the corresponding hydroxymethylglutaryl-CoA synthases. 4. The activity of heart acetoacetyl-CoA transferase remains constant from late foetal life until the end of the suckling period, after which time there is a gradual threefold increase in activity to reach the adult values. The activity of brain 3-oxo acid CoA-transferase increases steadily after birth, reaching a maximum at 30 days of age, thereafter decreasing to adult values, which are similar to foetal activities. Although at all stages of development the specific activity of the heart enzyme is higher than that of brain, the total enzymic capacity of the brain is higher than that of the heart during the suckling period.     

p38α MAPK regulates myocardial regeneration in zebrafish

Although adult mammals are unable to significantly regenerate their heart, this is not the case for a number of other vertebrate species. In particular, zebrafish are able to fully regenerate their heart following amputation of up to 20% of the ventricle. Soon after amputation, cardiomyocytes dedifferentiate and proliferate to regenerate the missing tissue. More recently, identical results have also been obtained in neonatal mice. Ventricular amputation of neonates leads to a robust regenerative response driven by the proliferation of existing cardiomyocytes in a similar manner to zebrafish. However, this ability is progressively lost during the first week of birth. The fact that adult zebrafish retain the capacity to regenerate their heart suggests that they either possess a unique regenerative mechanism, or that adult mammals lose/ inhibit this process. p38α ΜAPK has previously been shown to negatively regulate the proliferation of adult mammalian cardiomyocytes. We sought to determine whether a similar mechanism exists in adult zebrafish, and whether this needs to be overcome to allow regeneration to proceed. To determine whether p38α ΜAPK also regulates zebrafish cardiomyocytes in a similar manner, we generated conditional transgenic zebrafish in which either dominant-negative or active p38α ΜAPK are specifically expressed in cardiomyocytes. We found that active p38α ΜAPK but not dominantnegative p38α ΜAPK blocks proliferation of adult zebrafish cardiomyocytes and, consequently, heart regeneration as well. It appears that adult zebrafish cardiomyocytes share many characteristics with adult mammalian cardiomyocytes, including p38α MAPK-mediated cell cycle inhibition. These findings raise the possibility that zebrafish-like heart regeneration could be achieved in adult mammals.     

Subcellular distribution of the enzymes of the malate-aspartate shuttle in rat heart and effect of experimental cardiac hypertrophy

The effect of experimental cardiac hypertrophy on the enzymes of the malate - aspartate shuttle aspartate aminotransferase (AAT) and malate dehydrogenase (MDH) was studied. ( l ) Aortic constriction in adult rats resulted in 25% cardiac hypertrophy in 2 1/2-3 weeks. Total DNA (mg per heart) did not change. ( 2 ) The proportions of mitochondrial and cytosolic isozymes of AAT and MDH did not change as a result of cardiac h y p e r t r o p h y . About two-thirds of each enzyme occurred in the mitochondrial form and one-third in the cytosolic form. ( 3 ) Total AAT in hypertrophic hearts, in enzyme units per mg DNA, increased by 24% compared to AAT content in the hearts of sham-operated animals . Total MDH did not change. SoIubilized protein increased by 20%. Normal hearts contained 10 times more enzyme units of MDH than of AAT. (4) Cardiac growth stimulation induced in newborn rats did not result in specific changes of either enzyme. It is suggested that true cardiac hypertrophy acts as a specific stimulus for the possibly rate-limiting enzyme AAT of the shuttle.     

Developmental changes of the content of acetyl-CoA carboxylase mRNA in chicken liver

Utilizing RNA blot hybridization and immunoblotting techniques, the changes of the hepatic contents of acetyl-CoA carboxylase mRNA and of the enzyme protein in growing chicks have been investigated. In the post-hatching period, the hepatic mRNA level markedly increased at least 70-fold when compared to that before hatching. This increase was not observed in chicks receiving no diet. These changes were closely paralleled with the rise of the hepatic content of acetyl-CoA carboxylase protein in chicks up to 10 days old. Neither the acetyl-CoA carboxylase mRNA level nor the enzyme quantity significantly changed in heart. It is concluded from these results that the developmental regulation of acetyl-CoA carboxylase in the post-hatching period of chicks is tissue specific and occurs primarily at a pretranslational step. The content of acetyl-CoA carboxylase mRNA in adult chicken liver was low, which is comparable to those in embryos at 3 days before hatching and chicks at hatching day. Although acetyl-CoA carboxylase mRNA was detected in adult chicken brain, heart, lung, kidney, uropygial gland, spleen, testis, and chest muscle as well as liver, the mRNA level in these tissues was much lower than that in liver of growing chicks.     

Regulatory properties of 14 day embryo and adult hen heart AMP-deaminase

Chromatography on phosphocellulose column revealed changes in the elution profile of chicken heart AMP-deaminase during ontogenesis. The extracts from the heart of adult hen and 14 day-old embryo displayed a single peak of the enzyme activity at a slightly different elution volume, whereas in the heart extract of 1 day-old chicken two molecular forms of adenylate deaminase have been eluted. The kinetic and regulatory properties of the purified adult hen heart AMP-deaminase were studied and compared with those of the corresponding enzyme from 14 day-old embryo heart. Both enzymes exhibited a slightly sigmoid-shaped plot of the reaction rate versus substrate concentration, which shifted to hyperbolic form when ATP or ADP were added into the incubation medium. The enzymes were strongly activated by ATP, less efficiently by ADP and the activatory effect was enhanced at low substrate concentration. Orthophosphate inhibited both enzymes but this inhibition was more potent for the embryo heart enzyme. Palmitoyl-CoA inhibited adult hen but not the embryo heart AMP-deaminase. The data presented indicate that the differences also in the regulatory properties of the molecular forms studied do exist and correspond with the ontogenetic differences observed previously (Kaletha and Skladanowski (1981) Experientia 37, 232-234) concerning the effect of temperature on the chicken heart adenylate deaminase.     

A COMPARATIVE STUDY OF L-GLUTAMATE DECARBOXYLASE FROM MOUSE BRAIN AND BOVINE HEART WITH PURIFIED PREPARATIONS1

Abstract— L-Glutamate decarboxylase (EC 4.1.1.15) (GAD), the enzyme responsible for the formation of GABA, has been purified to homogeneity from mouse brain (Wu et at., 1973) and antibodies specific for neuronal GAD have been obtained (SAITO et al., 1974a). The present report describes the purification of GAD from bovine heart more than 2000-fold over the homogenate by initial solubilization with Triton X-100. subsequent fractionation with ammonium sulfate, column chromatography on DEAE cellulose, calcium phosphate gel, and DEAE-Sephadex, and gel filtration. At least two forms of GAD have been observed in bovine heart preparations; one of them appears as a high molecular weight form (Peak I, MW 360,000) and the other one as a low molecular weight form (Peak II, MW 105,000). Cysteine sulfinic acid and cysteic acid, both precursors of taurine, had no effect on the purified heart enzyme or on neuronal GAD at 10 mM, suggesting that cysteine sulfinic acid and cysteic acid probably are not substrates for any species of GAD described above. The heart enzyme and neuronal GAD differ in several respects. First, they are different immunochemically as judged by the lack of cross reactivity between the purified heart enzyme and the antibody against purified neuronal GAD. Second, they are different biochemically. 5,5′-Dithiobis[2-nitrobenzoic acid] (DTNB). one of the most potent inhibitors of neuronal GAD [K_i= 1.0 × 10^?8M] inhibits the heart enzyme only to a small extent at 1 mM. On the other hand, pyruvic acid, which inhibits the heart enzyme to an extent of 90% at 10 mM, only inhibits the neuronal enzyme slightly. Third, they are different in their substrate specificity. The neuronal enzyme can catalyze α-decarboxylation of both L-glutamate and L-aspartate while the heart enzyme can use only L-glutamate as substrate. Moreover, an unidentified product probably derived from L-glutamate is obtained in the reaction mixture of the heart enzyme but is not observed with the brain enzyme, suggesting that the heart enzyme may catalyze a reaction converting L-glutamate to products other than GABA. It is therefore concluded that heart GAD and neuronal GAD are two different entities. Work is in progress to determine whether the heart enzyme is related to the glial enzyme. Should the antibody against the heart enzyme cross-react with the glial enzyme, the role of the glial enzyme in GABA function can then be studied by immunochemical and immunocytochemical methods.     

Troponin T switching in the developing rat heart

A monoclonal antibody specific for cardiac troponin T has been used to investigate troponin changes during development in the rat heart. Specificity of the antibody was determined by immunoblot analysis with purified bovine cardiac troponin. In the rat heart, immunoblot analysis shows that anticardiac troponin T reacts with a 42.5-kDa band in fetal ventricles and with a 41-kDa band in adult ventricles. The faster migrating troponin T is present in traces in the fetal heart and increases markedly during the first 2 weeks after birth, concomitantly with the progressive decrease of the slower migrating form that is no longer detectable in the adult. The pattern of reactivity of the monoclonal antibody is not modified by alkaline phosphatase pretreatment, suggesting that the antibody is not specific for a phosphorylated epitope. Conditions known to affect cardiac myosin composition, such as hypothyroidism and hypertrophy secondary to systemic hypertension, do not change the troponin T isoform profile of adult rat ventricles. The expression and accumulation of the adult isoforms of troponin T are not suppressed by propylthiouracil treatment of pregnant and nursing rats.     

Tissue distribution and molecular heterogeneity of bovine thiol:protein-disulphide oxidoreductase (disulphide interchange enzyme)

1. The distribution of thiol:protein-disulphide oxidoreductase (disulphide interchange enzyme) in 17 bovine tissue extracts was determined by rocket immunoelectrophoresis and by measuring the reductive cleavage of insulin. 2. The relative concentration (per mg total protein) was found to be in the order: Pancreas greater than liver greater than lymph node greater than testes, fat tissue greater than parotid gland, brain, spleen, lung greater than small intestine, spinal cord, large intestine, kidney greater than paunch, aorta greater than skeletal muscle greater than heart. 3. The distribution of specific activity showed a similar pattern, irrespectively of whether glutathione or L-cysteine was used as cosubstrate. 4. The concentration varied 200-fold and the specific activity 400-fold between pancreas and heart muscle, respectively. 5. Crossed immunoelectrophoresis demonstrated that a fast-migrating form of the enzyme was the only one present in almost all tissues, but 15% of the enzyme in liver was a slow-migrating form and 50% in heart muscle a medium-migrating form. 6. The lung contains a species having partial immunological identity to the enzyme. 7. Purified enzyme from bovine liver has a somewhat lower mobility than the fast-migrating form in extract. 8. The results seem to support the general view that the enzyme is involved in synthesis of disulphide-bonded extracellular proteins, although the presence of the enzyme in tissues like fat, brain, spinal cord, skeletal muscle and heart indicates other cellular functions as well.     

Phosphorylcreatine shuttle enzymes during perinatal heart development

Mammalian heart development, from the time of weaning until adulthood, is characterized by progressive and significant enhancement in functional performance. Aerobic metabolism and contractile protein ATPase activity increase in parallel with augmented cardiac function. The present studies examined the potential contribution of phosphorylcreatine shuttle enzymes to the developmentally linked alterations in heart performance. Mitochondrial ATPase specific activity was not altered between weanling and adult heart; however, creatine kinase activity was enhanced approximately threefold. Myofibrillar ATPase activity doubled over the developmental time course, while creatine kinase activity increased to an even greater extent. Enhanced myofibrillar ATPase activity was not due to alterations in either calcium sensitivity or ATPase activity measured in purified myosin. Both the mitochondrial and myofibrillar creatine kinase enzyme activities are enhanced during normal heart growth; however, relatively greater enhancement of the myofibrillar component occurs. Thus, enzymatic reactions comprising the phosphorylcreatine shuttle system are dramatically increased during normal heart development. This mechanism deserves consideration as a potentially powerful contributor to enhanced cardiac function during the perinatal period.     

p38α MAPK regulates myocardial regeneration in zebrafish

Although adult mammals are unable to significantly regenerate their heart, this is not the case for a number of other vertebrate species. In particular, zebrafish are able to fully regenerate their heart following amputation of up to 20% of the ventricle. Soon after amputation, cardiomyocytes dedifferentiate and proliferate to regenerate the missing tissue. More recently, identical results have also been obtained in neonatal mice. Ventricular amputation of neonates leads to a robust regenerative response driven by the proliferation of existing cardiomyocytes in a similar manner to zebrafish. However, this ability is progressively lost during the first week of birth. The fact that adult zebrafish retain the capacity to regenerate their heart suggests that they either possess a unique regenerative mechanism, or that adult mammals lose/ inhibit this process. p38α ΜAPK has previously been shown to negatively regulate the proliferation of adult mammalian cardiomyocytes. We sought to determine whether a similar mechanism exists in adult zebrafish, and whether this needs to be overcome to allow regeneration to proceed. To determine whether p38α ΜAPK also regulates zebrafish cardiomyocytes in a similar manner, we generated conditional transgenic zebrafish in which either dominant-negative or active p38α ΜAPK are specifically expressed in cardiomyocytes. We found that active p38α ΜAPK but not dominantnegative p38α ΜAPK blocks proliferation of adult zebrafish cardiomyocytes and, consequently, heart regeneration as well. It appears that adult zebrafish cardiomyocytes share many characteristics with adult mammalian cardiomyocytes, including p38α MAPK-mediated cell cycle inhibition. These findings raise the possibility that zebrafish-like heart regeneration could be achieved in adult mammals.     

Uncoupling protein 3 transcription is regulated by peroxisome proliferator-activated receptor (alpha) in the adult rodent heart.

Relatively little is known concerning the regulation of uncoupling proteins (UCPs) in the heart. We investigated in the adult rodent heart 1) whether changes in workload, substrate supply, or cytokine (TNF-alpha) administration affect UCP-2 and UCP-3 expression, and 2) whether peroxisome proliferator-activated receptor alpha (PPARalpha) regulates the expression of either UCP-2 or UCP-3. Direct comparisons were made between cardiac and skeletal muscle. UCP-2, UCP-3, and PPARalpha expression were reduced when cardiac workload was either increased (pressure overload by aortic constriction) or decreased (mechanical unloading by heterotopic transplantation). Similar results were observed during cytokine administration. Reduced dietary fatty acid availability resulted in decreased expression of both cardiac UCP-2 and UCP-3. However, when fatty acid (the natural ligand for PPARalpha) supply was increased (high-fat feeding, fasting, and STZ-induced diabetes), cardiac UCP-3 but not UCP-2 expression increased. Comparable results were observed in rats treated with the specific PPARalpha agonist WY-14,643. The level of cardiac UCP-3 but not UCP-2 expression was severely reduced (20-fold) in PPARalpha-/- mice compared to wild-type mice. These results suggest that in the adult rodent heart, UCP-3 expression is regulated by PPARalpha. In contrast, cardiac UCP-2 expression is regulated in part by a fatty acid-dependent, PPARalpha-independent mechanism.