Tito Ureta

The comparative isozymology of vertebrate hexokinases.

1. Multiple hexokinase isozymes have been found in most vertebrates. Since each isozyme displays distinctive structural, kinetic and regulatory characteristics, the system qualifies as a useful probe for studies on molecular evolution. 2. At least seven types of chromatographic patterns of liver hexokinases have been observed in mammals. In contrast, each Class of lower vertebrates present only two or three distinct profiles. 3. Aves and higher Reptiles do not have the same hexokinase isozymes as other vertebrates. The nature of the differences is poorly understood. 4. Ontogenetic changes of liver hexokinase profiles are quite different in rat, chick and frog. 5. Structural comparisons of three vertebrate hexokinases having a molecular weight of approximately 100,000 suggest that those isozymes originated from a pre-vertebrate ancestor through gene duplication followed by fusion and further duplication events. Another hexokinase (the so-called glucokinase), with half the molecular weight, may have arisen either as the result of subsequent even splitting of the fused gene or, less probably, by divergence from a duplicated gene before the fusion event.

The role of isozymes in metabolism: a model of metabolic pathways as the basis for the biological role of isozymes.

Publisher Summary This chapter discusses the role of isozymes in metabolism. Isozymes are the multiple forms of enzymes catalyzing the same reaction in the same cell or organism. If several forms of the same enzymic activity exist, they perform a function in a distinct manner, and the difference is not the product of the reaction. The difference must then reside in the manner in which the product is formed. In fact, the components of some isozymic systems have been shown to differ, inter alia , in their affinity for substrates or cofactors, substrate or cofactor specificities, response to allosteric effectors, subcellular localization, susceptibility to dietary and/or hormonal treatments, or time of appearance during differentiation. This chapter discusses the involvement of isozymes in metabolic regulation and the role of compartmentation in metabolic regulation. A hypothetical model of metabolism and polyisozymic complexes in glucose utilization are also described in the chapter.

Adaptive character of liver glucokinase

Summary1.Glucokinase is one of four glucose phos-phorylating enzymes present in rat liver. Its distinctive features are a high Km for glucose (high-Km isozyme) and a rather narrow substrate specificity. In contrast, the other three enzymes, collectively called hexokinases or low-Km isozymes, exhibit low Km values for glucose and a wider substrate specificity.2.Glucokinase is present in the liver of mammals (with some exceptions), amphibians and lower reptiles. It is absent from higher reptiles and birds. The presence or absence of glucokinase may represent an evolutionary adaptation to feeding habits and other physiological peculiarities. Differences in the immunological behavior and in the kinetic parameters of glucokinases from different taxa suggest the operation of divergent evolution.3.The levels of glucokinase in rat liver depend strictly on the supply of carbohydrate in the diet. Glycogen phosphorylase and glycogen synthetase behave similarly, whereas other carbohydrate-metabolizing enzymes depend on the provision of either protein or protein plus carbohydrate. Glucokinase decays with a half-life of 33 hr when rats are starved or fed a carbohydrate-free diet, and is induced by the administration of glucose. The adaptive character is not exhibited by all mammals, indicating evolutionary discrimination within the same class and even within the same single order Rodentia. Enzyme adaptation in the liver may partially explain the condition known as ‘hunger diabetes’.4.The endocrine system plays a paramount role in glucokinase adaptation, since insulin is essential for glucose-dependent glucokinase induction and, on the other hand, glucagon, catecholamines and cyclic AMP prevent the induction. Glucocorticoids and some pituitary hormones modulate the rate of induction. The mechanisms underlying the hormonal regulation of glucokinase levels are not well known.5.The variations in liver glucokinase correspond to changes in the amount of enzyme protein as assessed by immunochemical titration. This fact agrees with the effects of inhibitors of protein synthesis on glucokinase induction.6.An antiserum against rat glucokinase reacts with the enzyme from mammals and turtles but not with the amphibian enzyme. It does not react with low-Km hexokinases from different sources.7.The saturation function for glucose is sigmoidal in mammalian and amphibian glucokinases but not in glucokinase from lower reptiles. The Hills coefficient is very constant with values about 1.6. The K0.5 (concentration for half saturation) values in the different species studied vary between 1.5 and 8mm. These kinetic parameters may be considered as another adaptive feature aimed to give maximal efficiency to the liver uptake of glucose at the changeable concentrations in the blood resulting from variations in the amount of dietary glucose.

Comparative studies on glucose phosphorylating isoenzymes of vertebrates—I. The influence of fasting and the nature of the diet on liver glucokinase and hexokinases of rodents☆

Abstract 1. 1. Glucose phosphorylating activities were measured in liver extracts from rat, mouse, hamster guinea pig and degu. The high- K m isoenzyme (glucokinase) was distinguished from the low- K m isoenzymes (hexokinases) by performing the assays at two levels of glucose. 2. 2. Total glucose phosphorylating activities varied widely in the various species. However, glucokinase was always the predominant form. The ratio glucokinase/hexokinases was the highest in the hamster, due to a very low level of hexokinases. The lowest ratio was found in the guinea pig, because of a low level of glucokinase. 3. 3. Fasting induced a decrease of glucokinase in the rat, mouse and hamster, but not in the guinea pig and degu. Hexokinase levels on the contrary were not significantly modified by this treatment. 4. 4. Feeding a carbohydrate-free diet (high fat, high protein) did not affect the glucokinase level in BALB mice, and moderately lowered the levels in the C 57 BL strain. Feeding a carbohydrate diet (carbohydrate as the sole source of calories) reduced glucokinase in both strains. The administration of glucose, however, restored totally (BALB mice) or partially (C 57 BL mice) the enzyme activities lowered by fasting. Glucose also restored normal levels of glucokinase in C 57 BL mice fed a carbohydrate-free diet. 5. 5. Feeding the carbohydrate diet caused a significant increase of the levels of hexokinases in both strains of mice. A similar effect was observed in C 57 BL mice fed the carbohydrate-free diet.

PHYLOGENY, ONTOGENY, AND PROPERTIES OF THE HEXOKINASES FROM VERTEBRATES

ABSTRACT. Four hexokinases (A, B, C, and D) are present in rat tissues. Isozymes A, B, and C have low Km glucose values (~10- 5 M), molecular weights of 100,000 daltons, and broad sugar specificities and tissue distributions. Isozyme D has a high Km for glucose (~10- 2 M), a molecular weight of about 55,000 daltons, a narrow substrate specificity, and is restricted to the liver. The isozymes seem to be products of independent genes.

Comparative studies on glucose phosphorylating isoenzymes of vertebrates—II. Chromatographic patterns of glucokinase and hexokinases in the liver of rodents

Abstract 1. 1. Four isoenzymes (A, B, C, D) catalyzing the phosphorylation of glucose were resolved by DEAE-cellulose column chromatography from the liver of rat, mouse, hamster, guinea pig and degu. The chromatographic mobility of peak D (glucokinase) was rather constant within different species. The mobilities of peaks A, B and C (hexokinases) varied widely. 2. 2. According to the order of elution, two patterns were distinguished. The rat type, defined by the elution sequence A, B, C, D, was present in the rat and degu. The mouse type, with the elution sequence A,C, B, D, was found in the mouse, hamster and guinea pig. The identification of the chromatograhic peaks was made mainly through the K m glucose values and the typical inhibition by excess substrate of isoenzyme C. 3. 3. Glucokinase was the predominant isoenzyme in all the species studied. It was markedly decreased by fasting in the rat, hamster and mouse, whereas apparently it was not affected in the guinea pig and degu. 4. ]4. Fasting did not change significantly the amount of the hexokinase isoenzymes in the animals studied. In the fasted guinea pig isoenzyme C was not inhibited by excess substrate.

Comparative studies on glucose phosphorylating isoenzymes of vertebrates. III: Isolation and properties of two hexokinases from chick liver

Abstract 1. 1. Only two ATP: d -hexose 6-phosphotransferase isoenzymes (A and B) were resolved when liver extracts from adult male well fed chickens were chromatographed in DEAE cellulose columns. The other two phosphotransferases usually found in rodent liver i.e., isoenzyme C and the high- K m glucokinase (isoenzyme D), were not detected in chick liver. 2. 2. Both isoenzymes were found to be typical hexokinases on the basis of their low K m glucose values, broad sugar specificities, and molecular weights of about 100,000 daltons.

Comparative studies on glucose phosphorylating isoenzymes of vertebrates—IV. Chromatographic profiles of hexokinases from the liver of several avian species

Abstract 1. 1. ATP : d -hexose 6-phosphotransferase activity was measured in the liver of thirteen species belonging to six avian orders. Total enzyme levels ranged from 0·2 (yellow finch) to 2·7 units (goose) per g of liver, very similar to those found in mammalian liver. 2. 2. Chromatographic fractionation of liver extracts on DEAE-cellulose columns separates two low- K m phosphotransferase isoenzymes, called A and B according to their order of elution. In some species a small third peak was observed. None of the isoenzymes were inhibited by excess of glucose and no high- K m isoenzyme fraction could be detected.

Comparative studies on glucose phosphorylating isoenzymes of vertebrates: Identification and characterization of amphibian liver hexokinases☆

Abstract Glucose phosphorylating activities were measured in liver extracts from two urodeles and twenty-six anurans. Fractionation on diethylaminoethyl-cellulose columns of liver extracts from these amphibians permitted the recognition of four hexokinases which are called A, B, C, and D. However, any given amphibian displays only three liver hexokinases and the profiles so far observed are either of the type A-B-D or C-B-D. The distribution of the amphibians in either type of pattern does not show any simple taxonomic relationship. A wide generic and specific, but not individual, variation of the relative proportion of each isoenzyme was observed. Hexokinases A and B were shown to be low Km glucose isoenzymes (0.06 and 0.15 m m glucose, respectively) with normal hyperbolic kinetics. Hexokinase C, also a low Km isoenzyme (0.05 m m ) was found to be inhibited by excess substrate at physiological levels of glucose. Hexokinases A, B, and C were able to phosphorylate fructose, mannose, and 2-deoxyglucose at equal or higher rates than glucose when assayed at saturating sugar levels. Hexokinase D was found to be a high Km isoenzyme ( K 0.5 ⋍ 2 mM ) with sigmoidal saturation curves for glucose (Hill coefficient ⋍ 1.6). Fructose and mannose were also phosphorylated by this isoenzyme at about 70% of the glucose rate when studied at saturating sugar concentrations. The properties of the amphibian hexokinases are thus similar, although not identical, to those of mammalian hexokinases.

Comparative studies on glucose phosphorylating isoenzymes of vertebrates. V. Glucose phosphotransferases in the liver of reptiles.

1. 1. ATP: d-hexose 6-phosphotransferase activity was measured in the liver of several reptiles. Total values ranged from 0.20 to 1.20 units/g of liver, i.e. similar to those found in mammalian or avian liver. 2. 2. Chromatography of liver extracts on DEAE-cellulose columns revealed several hexose phosphotransferases in all animals studied. 3. 3. Four isoenzymes were found in adult turtle liver, one of them a typical high-Km phosphotransferase. 4. 4. This isoenzyme was not detected in seven lacertids, one amphisbenid and two colubrid snakes. 5. 5. Three low-Km hexokinases were found in lacertid liver and only two in Amphisbena and the snakes.