Hector Carminatti

Biosynthesis of glycogen from uridine diphosphate glucose

Abstract An enzyme which leads to the formation of glycogen according to the equation: UDPG + primer → UDP + glucosyl α (1 → 4) primer has been studied. The optimal conditions for activity were determined with a partially purified preparation from rat muscle. The reaction requires the presence of a polysaccharide as primer and is strongly activated by hexose 6-phosphates. Using UDPG labeled in the glucose moiety, it was found that the radioactivity was transferred to the glycogen from which it could be removed as maltose with β-amylase or as G-1-P with phosphorylase. Thus it seems that the glucose residue becomes linked α (1 → 4) to the polysaccharide. Several inhibitors were tested as well as the occurrence of the enzyme in different organs.

Mannose transfer to lipid linked di-N-acetylchitobiose

Abstract Mannose was found to be transferred from guanosine diphosphate mannose to dolichol-P-P-di-N-acetylchitobiose when these substrates were incubated with hen oviduct or rat liver microsomes. The oligosaccharide moiety of the product appears to be the trisaccharide β-mannosyl-β-N-acetylglucosaminyl-(1→4)-N-acetylglucosamine as judged by the action of glycosydases. Under certain conditions further transfer of mannose occurs and larger oligosaccharides bound to lipid are formed.

SEPARATION OF SUGAR NUCLEOTIDES, PHOSPHORIC ESTERS AND FREE SUGARS BY PAPER CHROMATOGRAPHY WITH SOLVENTS CONTAINING BORATES OF ORGANIC BASES.

Abstract Four different chromatographic solvents containing borates of organic bases are described. Two of them permit the separation of sugar nucleotides differing only in the sugar moiety and give also a good resolution for some sugar 1-phosphates. The other two systems permit, in a single run, the separation of a group of sugars which is usually difficult to achieve.

Some kinetic differences between the M isoenzymes of pyruvate kinase from liver and muscle

Abstract The kinetic properties of type M pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) from rat liver have been studied. Similarly to the muscle enzyme, the plots of initial velocities versus phosphoenolpyruvate and ADP concentrations give hyperbolic curves. In contrast, the behavior of the liver enzyme toward amino acids differs from that of muscle. In a previous paper it has been found that the muscle enzyme is inhibited only by phenylalanine. This inhibition is pH dependent and is reversed by cysteine, serine or alanine. The liver enzyme is inhibited not only by phenylalanine but also by alanine, tryptophan, valine, tyrosine, proline and threonine. The inhibition by phenylalanine and alanine of the liver pyruvate kinase Type M is of the mixed type with respect to phosphoenol pyruvate and is only slightly dependent on pH. Thus on the basis of kinetic properties, at least two forms of pyruvate kinase Type M can be distinguished in rat tissues.

Further studies on a glycolipid formed from dolichyl-d-glucosyl monophosphate

Abstract Incubation of liver microsomes with dolichyl- d -glucosyl- 14 C monophosphate led to the labelling of an endogenous acceptor. This compound seems to be also a dolichol derivative. It contains a high-molecular weight oligosaccharide bound to dolichol through a phosphate or pyrophosphate bond. Various treatments of the labelled oligosaccharide afforded further information on its structure: Reduction with sodium borohydride, followed by acid hydrolysis gave only radioactive d -glucose indicating that the labelled d -glucose is not incorporated at the reducing end of the oligosaccharide. The percentage of radioactivity, liberated as formic acid after periodate oxidation, indicates that more than one molecule of d -glucose is incorporated and that at least one of them is inside the oligosaccharide chain. Alkaline treatment of the otherwise neutral oligosaccharide gave two positively charged derivatives which could be neutralized by N -acetylation, indicating the presence of two hexosamine residues. The oligosaccharides isolated from different tissues by the same method as that used for rat liver, were similar as judged by their migration in paper chromatography and by the pattern of products liberated by acetolysis.

A phosphatase acting on dolichyl phosphate in membranes from neuronal perikarya

Since the isolation of Del-P-Glc in 1970 [I] significant progress has been made in the elucidation ofhow the carbohydrate moieties of the asparagine-type glycoproteins are synthesized. Clycosylation occurs via sugar lipid intermediates [2]. Some of the properties of most of the enzymes in this pathway have been studied [2]. There is however scant amount of information* available about Dol-P and Dol-P-P phosphatases. The control mechanism operating in the dolichol pathway is yet to be elucidated. It has been proposed that the endogenous level of Dol-P could play a regulatory role in protein glycosylation [3]. By analogy with procaryotic systems [4-61, Dol-P, Dol.P-P phosphatases and dolichoi kinase may be implicated in the control bf the endogenous level of Dol-P. Enzymes that split the phosphate bonds from prenyl phosphates have been described in mammalian systems [7--91, although in these cases Dol-P was not used as substrate and Mgzf were always included in the incubation mixture. Here we report an enzymatic activity present in membranes from neuronal perikarya which removes the pl~osphate from Dol-P and requires no divalent cations.

The enzymic synthesis of yeast mannan

Abstract When guanosine diphosphate mannose (GDP-mannose) was first isolated from yeast, it was proposed that this compound might be the precursor of mannan ( Cabib and Leloir, 1954 ). Evidence supporting this hypothesis will be outlined in the present communication.

Estradiol induction of pyruvate kinase in the rat uterus

Abstract Estradiol-17β administered to ovariectomized rats increased the activity of uterine pyruvate kinase, while phosphopyruvate hydratase and phosphoglyceromutase remained unchanged. The effect of the hormone was inhibited by actinomycin D, 5-fluorouracil and cycloheximide. The increase in uterine pyruvate kinase was also partially blocked by the administration of progesterone to estradiol-treated rats. Uterine enzyme activity showed a cyclical variation in the normal animal and had the highest value at estrous. The behavior of hexokinase during the estrous cycle in the uterus of normal rats and after a chronic estradiol treatment was found to be similar to that of pyruvate kinase. However, the response of hexokinase activity to the hormone was much less marked than that of pyruvate kinase. The physiological significances of these findings are discussed.

Allosteric properties of the isoenzymes of pyruvate kinase from rat kidney cortex

Abstract The pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) isoenzymes from kidney cortex have been studied further. Pyruvate kinase type I which is the major component, shows homotropic cooperativity towards the substrate phosphoenol pyruvate, the effect is independent of pH. This isoenzyme is markedly inhibited by alanine and other amino acids but its activity is not modified by ATP or fructose 1,6-diphosphate. The kinetic behavior of this isoenzyme is then clearly different from that of the other pyruvate kinases from rat tissues. Pyruvate kinase type II exhibits sigmoid kinetics with respect to phosphoenolpyruvate. The enzyme is activated by fructose 1,6-diphosphate and inhibited by ATP, alanine and cysteine. The allosteric properties of the enzyme are strongly affected by changes in the pH. It is concluded that this kidney cortex isoenzyme is very similar to the pyruvate kinase type L from liver.

Two different forms of pyruvate kinase in rat kidney cortex.

The regulation of pyruvate kinase activity has an important role in gluconeogenic tissues [ I] . Liver and kidney, which are the main gluconeogenic organs in mammals, contain at least two different forms of pyruvate kinase [2-51 as shown by electrophoresis in different systems. In rat liver the level of the main component (type L) is under hormonal and dietary regulation and its activity is allosterically modulated by metabolites [2,4, 6-l l] The other hepatic isoenzyme (type M) appears to be formed constitutively [2] and its activity is not affected by fructose-l ,6-diphosphate (FDP) or ATP but is strongly inhibited by several amino acids [ 121. The kinetic behaviours of the different forms of kidney pyruvate kinase have not been investigated. Furthermore, previous studies, carried out with crude extracts, disagree regarding the activating effect of FDP [4,6, 131. This communication presents evidence showing that extracts from rat kidney cortex contain two forms of pyruvate kinase. One of them, pyruvate kinase type I (PK I) is the major component and displays a kinetic behaviour clearly different from that of all the other pyruvate kinases of rat tissues so far studied. The other, pyruvate kinase type 11 (PK II), has kinetic properties very similar to that of pyruvate kinase (type L) from liver.