Hsien-Gieh Sie

ENZYMORPHOLOGIC DEMONSTRATION OF GLUCOSE 6-PHOSPHATE-DEPENDENT GLYCOGEN SYNTHETASE IN MOUSE LIVER:

Mouse liver possesses G-6-P-dependent glycogen synthetase and not the G-6-P-independent variety. This fact explains the higher requirement (450mg per 25 ml) for G-6-P in the incubation mixture for staining mouse liver glycogen synthetase than that present in the Takeuchi and Glenner substrate mixture. The higher G-6-P content, as well as other considerations, necessitated the establishment of new conditions for the staining reaction for glycogen synthetase, i.e., pH 9.4, UDPG concentration 200 mg per 25 ml, etc. The presensce of NaF (4.8 x 10–2 M) prevents thermal inactivation of glycogen synthetase at 37°, which is the temperature now recommended for the staining reaction. Postfixation and dehydration makes the sections more readily interpretable and prolongs markedly the life of the iodine staining reaction. The new procedure is reproducible and consistent in serial sections of the same animal and among different animals of like glycogen content. The enzyme appears to be located on membranes as well as in granules. Some similarities between the characteristics of the dependent and independent forms of the enzyme, as we and others have observed them by both histochemical and biochemical techniques, are discussed.

Capacity of rat tissues to form heptulose phosphates in thiamine deficiency

1. 1. The heptulose phosphate formed from nucleoside and from sugar phosphates (glucose 6-phosphate, mannose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate and ribose 5-phosphate) by normal and thiamine-deficient rat tissues was measured under standard conditions. 2. 2. The thiamine-deficient rat exhibited a loss of activity of the enzymes synthesizing heptulose phosphate, particularly in brain, heart, kidney, spleen, lung and hemolysate. 3. 3. Addition of thiamine hydrochloride and thiamine pyrophosphate in vitro did not change the activity of tissue preparations from thiamine-deficient animals. 4. 4. In the presence of triose phosphate, supernatant preparations of thiamine-deficient hemolysate, kidney, liver and lung underwent as much as a three-fold increase in activity (substrates: glucose 6-phosphate, mannose 6-phosphate). The optimum concentration of triose phosphate was 2.5·10−4.

Glycogen Synthetase: Its Response to Cortisol

Glycogen synthetase activity in the livers of starved mice is stimulated by the glucocorticoid, cortisol.

Labeling of rat liver glucose-1-phosphate, glucose-6-phosphate, uridine diphosphate glucose, and glycogen during glycogen synthesis

Abstract 14 C-Glucose was injected intravenously into rats and the livers were rapidly removed at intervals from 2 to 30 min and homogenized in perchloric acid. Fractionation was carried out on Dowex-1-formate resin and glucose, glucose-1-phosphate, glucose-6-phosphate, uridine diphosphate glucose, and glycogen were isolated by repeated chromatography. Both a comparatively high specific radioactivity and total radioactivity of glucose-1-phosphate as compared to glucose-6-phosphate persisted from the earliest 2-min point to 10 min of incorporation. This indicated that formation of glucose-1-phosphate may be an early product of glucose phosphorylation during glycogen synthesis in vivo from exogenous glucose units. It may also be an early product during hydrocortisone-induced glycogenesis in animals receiving 14 CO 2 on the basis of a second series of experiments. The observations are explained on the basis of two separate pools of glucose-6-P in metabolism.

A TECHNIQUE FOR PREPARING PERMANENT HISTOCHEMICAL PREPARATIONS OF LIVER PHOSPHORYLASE

at sites of kusowus endogemsous peroxidase activity, suuch as leuncocytes, mast cells and Kupifer cells. However, if the imscuhatiuug mediusm contained inssuuffi cieuut exogeuiouns peroxidase activity, some react ioms product was observed at the endogenouns sites. Presumably, this was the result of inefficienicy of the peroxidatic capture reaction, with resunltintg diffusion of the liberated H202 to the sites of eusdogenous peroxidase activity. This problem could be obviated by increasing the concenut ration of exogenouss peroxidase. Sections of rat and guinuea pig liver and kidney, both with and without acetone pretreatment, were iuscunbated for monoamine oxidase activity according to the method of Glenner et al. (J. Histochem. Cytochem. 5: 591, 1957). The general localizatioms of activity was similar to that obtained with the coupled oxidation method, except that the reactiout produnct in the coupled oxidation method was particulate, whereas that obtained by ums with the tetrazolitnm method was more diff muse inn both rat amid guuintea pig tissues. The reasons for this differeusce are umtkmtowmt. The mousoarninie oxidase of rat liver is relatively iutsoluble and is associated with the mitochomudnial fractious (Cotzias amid Dole, Proc. Soc. Exp. Biol. Med. 78: 157, 1951). Ins guinea pig liver amid kidney, although the buulk of monoamine oxidase activity is presemst as the insoluble mitochonidnial emszyme, appreciable activity is present ims soluble form (Weissbach, et al., J. Biol. C/rem. 214: 267, 1955). It is likely that this solumble fractious is nuot detectable with the coupled oxidations method. In our opiution, the tetrazolium method remaiuss the most satisfactory method for general unse in the histochemical demonstration of mousoamine oxidase activity. Its reaction product is easier to see amid to photograph. The particulate bocalizations obtained with the coupled oxidations method

Primary incorporation of U-14C-glucose into glucose-1-phosphate and glucose-6-phosphate by incubated pigeon liver homogenate

That glucose-6-P is a necessary step in the transformation of glucose to glycogen has been questioned in several reports using homogenates and slices from rat liver [l-3] , or pigeon liver homogenate [4] .The work of Pocchiari [S] on the incubated rat diaphram has also led others [6] to suggest that glucosed-P might not be the only immediate product of glucose phosphorylation. Glycogen synthesis from glucose was shown by pigeon liver homogenate [4, ‘71. This provides an ideal, cell-free system for following the incorporation of “C-glucose into glucose-6-P and glucose-l-P, which is described in the present communication. The preparation of pigeon liver homogenate and of the incubation mixture was as described by Nigam and Fridland [7]. A total of 24 flasks were incubated for 5 min at 37”, each flask containing 1 &i of radioactivity and 100 @moles of glucose in 10 ml. The control flasks were incubated similarly without glucose. The reaction was stopped by the addition of an equal vol of 1.2 M perchloric acid. Following the enzymatic dete~ination of the amount of glucose, glucose-6-P and glucose-l-P in neutralized perchloric acid supernate of both the experiments and controls [8], a known amount of glucose-6-P and glucose-l-P as carrier was added to the experimental perchloric acid supernate for further purification of the sugar phosphates.

Conversion of glucose 6-phosphate to ketoheptose phosphate by calf-lung enzyme.

Abstract The calf-lung enzyme which catalyzes the conversion of glucose 6-phosphate to ketoheptose phosphate was purified and concentrated using ammonium sulfate fractionation. The protein collected within the limits of 0.6 to 0.7 saturation with respect to ammonium sulfate was extracted with 0.65 saturated ammonium sulfate to dissolve the enzyme. The product after repeating this process represents a 20-fold increase in specific activity over the starting material. Also, it is much less capable of producing heptose phosphate from ribose 5-phosphate. Terminal glycol linkage present in 3-ketoheptose phosphate was determined by its cleavage with periodate to yield formaldehyde which is measured with the chromotropic acid procedure Two pH optima were exhibited, the one at 5.5 showed equivalence of terminal glycol and heptose and that at 7.5 more heptose than terminal glycol.

14C labeling of glucosyl oligosaccharides during starvation and during hydrocortisone-induced glycogenesis

Abstract 14C-Glucose was administered intravenously into starved anesthetized male rats, and at intervals of 2, 5, 10, and 30 min afterwards, the animals were killed and the liver homogenates were processed for perchloric acid-soluble and -insoluble glycogen, UDP-glucose, and glucosyl oligosaccharides. In every case the order of 14C-glucose incorporation is UDPG >glucosyl oligosaccharides >glycogen. The same pattern of labeling was obtained when 14CO2 was injected into rats undergoing hydrocortisone-stimulated glycogenesis. During active glycogen synthesis 14C-glucose incorporation into glycogen and oligosaccharides increased while the specific radioactivity of UDP-glucose declined. The results signify that glycogen and glucosyl oligosaccharides are products evolving simultaneously from the same transfer reaction catalyzed by UDP-glucose-glucosyl transferase.

Heptulose phosphate formation in thiamine deficiency and other physiological states.

Summary In the rat the systems responsible for heptulose phosphate formation from sugar phosphate and nucleoside were completely developed at birth with the exception of low activity in the liver towards nucleoside. The thiamine-deficient adult rat exhibited a reduction of 50 to 95% of enzyme activity in heptulose phosphate formation in lung, spleen, intestine and pancreas. Subsequently, a supplement of thiamine HCl to the deficient animal restored the enzyme level to normal. The effect on heptulose phosphate formation was less extensive in mice made Vit. B1 deficient by feeding pyrithiamine hydrobromide than it was in rats, but there was a marked decrease in activity towards adenosine. Neither the absence of insulin nor of adrenocorticoid hormone had any effect on heptulose phosphate formation from sugar phosphate and nucleoside. In normal rat and mouse tissues 20 to 45% of heptulose phosphate was due to 3-ketoheptose phosphate, the formation of which was also depressed in Vit. B1 deficient tissue.