Clara R. Krisman

A method for the colorimetric estimation of glycogen with lodine

Abstract A rapid, simple, and specific method for the colorimetric estimation of glycogen in concentrations varying from 0.15 to 1 mg/ml with an iodine-iodide reagent in the presence of salts has been studied. It eliminates interference by polysaccharides, which do not develop color in the presence of iodine. This colorimetric procedure can be applied to the estimation of tissue glycogen.

The role of dolichol monophosphate in sugar transfer.

Abstract The specificity of the transfer of monosaccharides from sugar nucleotides to dolichol monophosphate catalyzed by liver microsomes was studied. Besides uridine diphosphate glucose, uridine diphosphate-N-acetylglucosamine and guanosine diphosphate mannose were found to act as donors for the formation of the respective dolichol monophosphate sugars. Uridine diphosphate galactose and uridine diphosphate- N -acetylgalactosamine gave negative results. The optimal conditions for transfer from dolichol monophosphate glucose to endogenous acceptor was determined. Studies were carried out on the glucosylation of ceramide by brain extracts and of collagen by skin enzymes in order to find out if dolichol monophosphate glucose is an intermediate in these reactions. The results, while not definite, were not in favor of this assumption.

Further studies on high molecular weight liver glycogen

Abstract A simple method for the extraction of undegraded liver glycogen is described. The molecular weight distribution curve as measured by gradient centrifugation was quite variable and was found to be unrelated to glycogen content. The concentration of malto-oligosaccharides in liver was estimated by a new method and was found to remain constant under conditions in which glycogen content varied considerably. Native glycogen was compared with that prepared in vitro with purified enzymes. Although both preparations were similar in molecular weight and in their appearance under the electron microscope, they differed in the type of breakdown produced by acid, alkali, heat, and ultrasonic vibrations. When glycogen prepared in vitro was submitted to these procedures, its sedimentation coefficient decreased progressively, while native glycogen was broken down preferentially to a lighter population of about S = 100.

A possible intermediate in the initiation of glycogen biosynthesis

Summary A partially purified glycogen synthase (UDPG: α-1,4-glucanα-4-glucosyltransferase, EC 2.4.1.11) preparation catalyzes the transfer of glucose to a trichloroacetic acid insoluble fraction. The reaction occurs in the absence of added glycogen and requires high salt concentration. The reaction product is acid labile and contains α 1–4 linked glucosyl residues.

The initiation of glycogen biosynthesis in Escherichia coli

Previous work from this laboratory has shown that a protein acts as the initial acceptor of glucose from UDPglucose for glycogen biosynthesis in rat liver [ 1,2]. Because of the different solubility of protein and glycogen in trichloroacetic acid, it has been possible to determine differentially the incorporation of radioactivity from labelled sugar nucleotide into protein or into glycogen. Based on results obtained with rat liver preparations [3], we have proposed the participation of two enzymes and an acceptor protein in the initiation of glycogen biosynthesis. We have applied the same approach to the study of glycogen initiation in bacteria. In this paper we report the isolation from Escherichia cofi of an enzyme complex which synthesizes a 1,4-ol-glucoprotein not only from ADPglucose, which is considered to be the specific donor in bacterial glycogen biosynthesis but also from UDPglucose.

a-1,4-glucan: a-1,4-glucan 6-glycosyltransferase from liver

Abstract a -1,4-glucan: a -1,4-glucan 6-glycosyltransferase was purified about 35-fold from rat-liver extracts. Removal of α-amylase could be accomplished by high-speed centrifugation of extracts from livers with high glycogen content. Enzyme action on amylopectin is optimum at pH 6.4 in 0.3 M citrate buffer. It requires salts for maximal activity, and is inhibited by molybdate, Mn 2+ , Mg 2+ and sodium p -chloromercuribenzoate. Amylose and amylopectin s-limit dextrin were also substrates for the liver branching enzyme. A method is described for the assay of the enzyme in the presence of α-amylase.

In vitro synthesis of particulate glycogen from uridine diphosphate glucose

Abstract High molecular weight glycogen has been prepared in vitro with liver glycogen synthetase (uridine diphosphate glucose: α-1,4-glucan α-4-glucosyltransferase, EC 2.4.1.11) and branching enzyme (α-1,4-glucan: α-1,4-glucan 6-glycosyltransferase, EC 2.4.1.18) and uridine diphosphate glucose as glucose donor. The product obtained did not differ significantly from the native glycogen as judged by iodine spectrum, sedimentation coefficient in sucrose gradients, and by the effect of treatment with acid or alkali. Glycogen obtained from uridine diphosphate glucose differed from that prepared with glucose 1-phosphate as glucosyl donor.

A POSSIBLE INTERMEDIATE IN THE INITIATION OF GLYCOGEN BIOSYNTHESIS

Leloir and Cardini found in 1957 that partially purified preparations from rat liver catalyze the synthesis of glycogen from UDPG. Glycogen phosphorylase is also able to carry out the in vitro synthesis of the a-l,4 glucosidic bonds from G-1-P; however, experimental findings are consistent with the view that there are two different pathways of glycogen metabolism in the cells. The synthetic route is catalyzed by glycogen synthetase, and phosphorylase is involved in the degradation.2 Although the progress in the field of glycogen metabolism has been enormous since 1957, information on how the molecule starts to grow is scarce. Most of the glycogen synthetases studied have been shown to be inactive in the absence of glycogen or oligosa~charides.~-~ From the available data, it seems that fetal livers are devoid of glycogen until a certain time in the gestation period.6 This indicates that there must be some mechanism for the initiation of the biosynthesis of glycogen.

Some properties of rat liver amylase

Abstract Some properties of rat liver amylase were studied. It was confirmed that this enzyme is located mainly in the microsomal fraction and that it is activated by detergents. The effects of digitonin, Triton X–100 and sodium deoxycholate on the amylase were compared. It was observed that amylase requires a higher concentration of Triton X–100 for maximal activation than lysosomal acid phosphatase. When lysosomes and microsomes were submitted to a detergent treatment capable of activating completely acid phosphatase and α-amylase, the former was solubilized, while the second remained particulate. The activation of α-amylase was found to be reversible in the first stages. The microsomal α-amylase is not bound to glycogen in fed rats. It was observed that the free α-amylase did not change when the liver was induced to accumulate different amounts of glycogen by administration of sugar, but that latent amylase was doubled when large amounts of glycogen accumulated. The K m of liver amylase for glycogen is 2.5 mg/ml as compared with 0.4 mg/ml for serum amylase. The significance of these observations is discussed.

Properties of synthetic and native liver glycogen

Abstract The properties of high molecular weight glycogen extracted from rat liver and of that prepared in vitro with muscle phosphorylase and liver branching enzyme have been compared. The stability at different pH values was measured spectrophotometrically for liver, corn, and synthetic glycogen. The former is more labile, but the shape of the pH-stability curve is very similar for all of them. Borate, copper, and iron accelerate the decomposition of the three types of glycogen. Sonication produces breakdown but affects in the same way synthetic and liver glycogen. After shortening the outer chains with β-amylase, native liver glycogen becomes slightly more stable to acid treatment and decomposes giving smaller molecules than the untreated glycogen. Glycogen extracted from livers of toad and pigeon was similar in molecular weight distribution and acid lability to that of rat liver. Rat muscle glycogen had a molecular weight of about 8 million.