Jasna Radojković

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.

Comparative studies on glucose phosphorylating isoenzymes of vertebrates—VII. Mammalian hexokinases☆

1. 1. The glucose-phosphorylating isoenzymes from the liver of animals belonging to 25 mammalian species were separated by DEAE-cellulose chromatography and characterized with respect to Michaelis constants for glucose and other sugars and substrate specificities. 2. 2. The full complement of four hexokinases (A, B, C and D) was observed in laboratory rodents only. A marsupial, several wild rodents and dog, presented hexokinases A, B and D. Monkey, rabbit, pig and a wild rodent (Octodon degus) displayed hexokinases A, C and D. A pattern of hexokinases A and C was shared by horse, alpaca, cow, sheep and goat. The pattern consisting of hexokinases A and B was found in bat and cat. 3. 3. The hexokinases from skeletal muscle of a few mammals (bat, rabbit, rat, long-haired mouse, guinea pig and sheep) were also studied. In all these species hexokinase B was the predominant form.

The influence of diet on liver phosphorylase: III. Role of inactivation on the decrease of phosphorylase under certain dietary conditions☆☆☆

Abstract The addition of epinephrine to the incubation medium did not modify significantly and regularly the phosphorylase activity of liver slices prepared from well-fed or fasted rats. Liver phosphorylase was rapidly inactivated in homogenates prepared in KCl. The rate of inactivation was lower in fasted animals or in those fed a high-fat, carbohydrate-free diet than in rats fed equilibrated or carbohydrate diets. The addition of an activating mixture composed of ATP, magnesium, fluoride, and caffeine promoted the reactivation of the enzyme. Phosphorylase activity was well maintained in homogenates prepared in KF and did not change in the presence of the activating mixture. Homogenates obtained from fasted rats or from animals fed equilibrated or high-fat, carbohydrate-free diets behaved similarly. These results are interpreted as an indication that the low phosphorylase activity observed during fasting or when animals are fed a high-fat, carbohydrate-free diet is not the consequence of a higher proportion of the inactive form with respect to the total enzyme.

The influence of diet on liver phosphorylase. II. Effect of different proportions of carbohydrates, proteins and fats☆☆☆

Abstract Liver phosphorylase activity was found to be proportional to the caloric intake of the animal. Rats fed for 6 or 10 days an exclusively carbohydrate diet showed a phosphorylase content of the liver similar to animals fed isocaloric amounts of equilibrated diet, in spite of the protein depletion. The same results were observed with fat-free carbohydrate diets containing variable proportions of casein. The phosphorylase activity decreased markedly in rats kept on carbohydrate-free, high-fat diets, containing as much protein as 25% of the total calories. The diminution of the enzyme content was observed even after only 1 day on the high-fat diet. The rate of increase in liver phosphorylase, after a decrease provoked by a 48-hr, fasting, was the same in animals subsequently fed an equilibrated diet or carbohydrates alone. In the latter conditions the liver proteins remained at the same low levels as during fasting.

Chromatographic and electrophoretic evidence for several sugar dehydrogenases in mammalian liver

Some enzymes catalyzing the pyridine nucleotidelinked oxidation of free monosaccharides have been described in the mammalian liver. These include the microsomal glucose dehydrogenase first described by Harrison [1 ] later purified and characterized by several investigators [2 -5 ] , and the soluble galactose dehydrogenase reported by Cuatrecasas and Segal [6, 7]. In bacteria, several dehydrogenases acting on free pentoses have been described, for instance L-arabinose dehydrogenase [8], D-arabinose dehydrogenase [9 11 ] , and an aldose dehydrogenase with broad specificity [9-11 ]. To our knowledge none of these enzymes have been described so far in the mammalian liver, with the possible exception of L-fucose dehydrogenase from pork liver, acting also on D-arabinose [12]. This communication presents evidence for the presence in pig liver of several enzymes catalyzing the general reaction

Hexokinase-coupled radioassays: Glyceraldehyde-3-phosphate dehydrogenase and enolase☆

Abstract A general method for the assay of enzymes which produce ATP, or are susceptible to be coupled to ATP-producing enzymes, is described. We have applied it to the assay of glyceraldehyde-3-phosphate dehydrogenase and enolase. For these enzymes, the product of the reaction, 1,3-bisphosphoglycerate or phosphoenolpyruvate, were coupled to ADP and either phosphoglycerate kinase or pyruvate kinase, respectively. The ATP formed in both cases is used by hexokinase plus labeled glucose to produce labeled glucose 6-phosphate which is quantitatively separated in small Dowex 1 columns and measured by liquid scintillation spectrometry. The conditions described permitted the detection of 0.1 mU of glyceraldehyde-3-phosphate dehydrogenase or enolase. As a further example of the sensitivity of the radioassay, effluents of a CM-cellulose column charged with an extract prepared from one single frog oocyte were analyzed and shown to contain a single enolase and two glyceraldehyde-3-phosphate dehydrogenase fractions.

Organization of Glucose Metabolism: A Model of Compartments by Poly-Isozymic Complexes

A metabolite being released from the active site of an enzyme may find itself in the predicament of deciding which pathway to choose among several possibilities. For instance, once glucose-6-P has been synthesized several alternative paths are possible, e.g., glycogen synthesis via phosphoglucomutase, glycolysis via phosphoglucose- isomerase, pentose-P pathway via glucose-6-P dehydrogenase, or back again to glucose through glucose-6-phosphatase (Fig. 1). Furthermore, the same situation will happen again at every branch-point along the metabolic maze until a committed step is reached.