Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

요약

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

초록

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of α1,4-linked glucose residues with branches introduced through the addition of α1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

서문

Glycogen is widely distributed in nature, with the compound being found in bacteria, many protists, fungi, and animals. In microorganisms, glycogen is important for cell survival when nutrients are limiting and, in higher organisms such as mammals, synthesis and degradation of glycogen serve to buffer blood glucose levels^1,2,3. The study of glycogen metabolism is, therefore, of importance to such diverse fields as microbiology and mammalian physiology. Understanding glycogen metabolism requires studying the key enzymes of glycogen synthesis (glycogen synthase and the branchin....

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프로토콜

1. Determination of glycogen synthase activity

1. Prepare stock solutions of required reagents as indicated in Table 1 (prior to the experimental day).

Component	Directions
50 mM Tris pH 8.0	Dissolve 0.61 g of Tris base in ~ 80 mL of water.  Chill to 4 °C.  Adjust the pH to 8.0 with HCl and make the volume up to 100 mL with water.
20 mM HEPES buffer

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결과

Determination of glycogen synthase activity
Figure 1 shows representative results from glycogen synthase assays using purified enzymes. In panel A, following a slight lag, there was a linear decrease in the absorption at 340 nm over time for a period of around 12 min. The rate of change in absorption in Figure 1A was ~0.12 absorbance units/min. A rate of change in absorbance between ~0.010 and ~0.20 absorbance units/min is optimal and the .......

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토론

In general, the key advantages of all of the methods presented are their low cost, ease, speed, and lack of reliance upon specialized equipment. The major disadvantage that they all share is sensitivity compared to other available methods. The sensitivity of the procedures that involve production or consumption of NADH/NADPH are easy to estimate. Given that the extinction coefficient of NADH/NADPH is 6.22 M^-1 cm^-1, simple arithmetic indicates that ~10-20 µM changes in concentration can be readi.......

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자료

Name	Company	Catalog Number	Comments
Amylopectin (amylose free) from waxy corn	Fisher Scientific	A0456
Amylose	Biosynth Carbosynth	YA10257
ATP, disodium salt	MilliporeSigma	A3377
D-Glucose-1,6-bisphosphate, potassium salt	MilliporeSigma	G6893
D-glucose-6-phosphate, sodium salt	MilliporeSigma	G7879
Glucose-6-phosphate dehydrogenase, Grade I, from yeast	MilliporeSigma	10127655001
Glycogen, Type II from oyster	MilliporeSigma	G8751
Hexokinase	MilliporeSigma	11426362001
Methacrylate cuvettes, 1.5 mL	Fisher Scientific	14-955-128	Methacrylate is required since some procedures are conducted at 340 nm or below
β-Nicotinamide adenine dinucleotide phosphate sodium salt	MilliporeSigma	N0505
β-Nicotinamide adenine dinucleotide, reduced disodium salt	MilliporeSigma	43420
Nucleoside 5'-diphosphate kinase	MilliporeSigma	N0379
Phosphoenolpyruvate, monopotassium salt	MilliporeSigma	P7127
Phosphoglucomutase from rabbit muscle	MilliporeSigma	P3397
Phosphorylase A from rabbit muscle	MilliporeSigma	P1261
Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle	MilliporeSigma	P0294
UDP-glucose, disodium salt	MilliporeSigma	U4625