Glycogen precipitability

Recover the DNA by phenol chloroform exttaction followed by isopropanol/ sodium acetate plus glycogen precipitation. 

In nature, amylases act hand in hand with each other and with other carbohydrases in bringing about the rapid breakdown of starches and glycogens to sugars which can be utilized readily by the living cell. Much important and practical information has been accumulated by the use of extracts and crude precipitates in which more or less natural mixtures of enzymes have been studied in the laboratory. However, the... 

A well-established inhibitor of gluconeogenesis is ethyl alcohol. This can cause problems in at least two situations. When alcoholic patients enter an alcoholic binge , they do not eat, so that liver glycogen is soon depleted. Since gluconeogenesis is inhibited, both hypoglycaemia and, as indicated below, lactic acidosis can develop. Indeed they may be the two most important factors that precipitate coma and collapse in the alcoholic patient. 

Incorporation of [14C]-glucose into glycogen by the reverse reaction of the hydrolase amylo-l,6-glucosidase. Measurement of the incorporated radioactivity in the precipitated glycogen [20, 39]. This assay can only test the enzymes function of the hydrolysis of the 1-6 bond (see section 4.6.16.1). 

A polymaltose prepared by Ricketts and Rowe140 reacted with pneumococcus Type XII antiserum more extensively than did a polyglucose prepared by the same authors, but both polysaccharides left behind some antibody reactive with glycogen. Neither polymer reacted with Type II antiserum. Polygalactose gave a slight precipitate with Type IV antiserum, the capsular polysaccharide of which is known to contain D-galactose. 

Concanavalin A is an antibody-like protein isolated from the jack bean. 82 It was shown to form a precipitate with glycogen and with yeast mannan, and was later also used to differentiate between glycogen-like polysaccharides from various sources.283 284 It has now been shown that concanavalin A forms a precipitate only with branched polysaccharides which contain terminal, nonreducing a-D-glucopyranosyl or a-D-manno-pyranosyl groups.18 The combining sites of the protein appear to be directed against the 2-deoxy-a-D-aratano-hexopyranosyl system.2 ... 

The selected RNA must be reverse-transcribed into cDNA for further amplification by PCR. Therefore, the RNA is precipitated and subjected to a reverse transcription reaction according to the protocol of the reverse transcriptase supplier. If the RNA concentration is very low (as in the first selection rounds) a coprecipitant like glycogen has to be added to recover the RNA quantitatively. 

This method precipitates also the small quantity of glycogen in the meat as a rule this does not influence the results appreciably, but when the separation of starch from glycogen is necessary, especially in presence of horseflesh, Mayr-hofer s modified method is used (see later). 

The hot liquid is mixed with 100 c.c. of 50% alcohol, the impure glycogen being filtered off after cooling. The precipitate is washed with 30 c.c. of hot alcoholic potash and then with cold 90% alcohol until the filtrate is no longer rendered turbid by addition of a few drops of dilute hydrochloric acid it is then heated in a no c,c. flask with 50 c.c. of normal potash on a water-bath for half an hour to dissolve the glycogen. When cold the liquid is acidified with concentrated acetic acid, made up to no c.c. with water and filtered. 

To 100 c.c. of the filtrate are added 150 c.c. of absolute alcohol and after 12 hours the precipitate of pure glycogen is collected on a tared filter, washed successively with about 70% alcohol, absolute alcohol and ether, dried at 40° and finally at 1000 to constant weight. The weight found, multiplied by 2-2, gives the percentage of glycogen. 

Only lysine residues on the exterior of the protein molecule are thought to react and these usually account for 40-60% of the total lysyl residues.29 Formalin does not precipitate proteins and only slightly precipitates other components of the cell it is also a good fixative for complex lipids but has no effect on neutral fats and neither preserves nor destroys adipose tissue. Formalin also traps carbohydrates because it preserves proteins so that they hold glycogen, which is otherwise readily leached from the cell. Another advantage of the compound is that it does not produce large bands that obscure the major bands associated with the macromolecules in tissue samples. Furthermore formalin is inexpensive and available in most laboratories. 

Recovery of DNA from dilute solutions (<10 pg DNA/mL) can be enhanced by the addition of an appropriate carrier substance before ethanol precipitation. Molecular biology grade glycogen, which is available commercially, is added to the sample at a concentration of 20 to 40 pg/mL before the addition of ethanol. The dilute DNA solutions can also be concentrated by repeated extraction with. vec-butanol (mixing the solution by inverting several times) followed by centrifugation at 3000 x g for 5 minutes at room temperature, discarding the butanol phase each time. Each extraction will extract water out of the solution and hence concentrate the DNA. Finally, DNA can be ethanol precipitated. 

Starch synthase or glycogen synthase activity can be measured by transfer of [14C]glucose from ADPGlc into an appropriate primer, such as amylopectin or rabbit glycogen, followed by precipitation of the labeled polymer.171172... 

The first molecular weight measurements appear to be the osmotic pressure determinations carried out by Oakley and Young32 on unsubstituted rabbit liver and muscle glycogens in 0.1 A calcium chloride solution. They claimed that products obtained by alkaline extraction, and by water extraction followed by precipitation by acetic acid, had the same molecular size. The molecular weight of rabbit liver glycogen was found to be (1.2-2.2)10°, and that of muscle glycogen (0.7-1.8)10°. 

A molecular weight of 6.1(10)6 has been recorded for the acetate of mollusc muscle glycogen by Meyer and Jeanloz48 on the basis of osmotic pressure determinations. Fractions obtained from this glycogen by precipitation with methanol possessed values foi the molecular weight of 6.1(10), 2.1(10)6, and 3.0(10). It was suggested that these represented minimum values because of the effect of impurities of small molecular weight. 

---