Phosphorylase assay

Would you expect the glycogen phosphorylase assay data from phosphorylase kinase reaction 3 to be any different if the concentration of the kinase used in this reaction was increased by a factor of 5 Explain. 

Enzymes have evolved such that their values (or A o.s values) for substrate(s) are roughly equal to the in vivo concentration(s) of the substrate (s). Assume that glycogen phosphorylase is assayed at [P[] = A o.s in the absence and presence of AMP or ATP. Estimate from Figure 15.15 the relative glycogen phosphorylase activity when (a) neither AMP or ATP is present, (b) AMP is present, and (c) ATP is present. 

The synthesis of linear 4 —> 1-a-D-glucans from D-glucopyranosyl phosphate by the action of phosphorylases has been shown by comparison of results of methylation and end-group assay and viscosity determination,209 and by potentiometric, iodine titrations82 on the product. The chain length of the enzymic product (100 to 200 D-glucose units) is less than that of the natural component. Whether this is due to impure enzymes cannot yet... 

In early work on glycogen phosphorylase, in the early 1940s and 1950s, the activity was assayed by measnring the release of phosphate from glucose 1-phosphate in the reaction... 

The existence of two forms of phosphorylase with different catalytic activities, which were capable of being enzymatically interconverted, suggested that the two forms might be involved in regulation of glycogenolysis in muscle tissue. However, in the early studies, whenever phosphorylase was assayed in extracts of muscle it was always found to be in the a form. This was the case even in extracts prepared from... 

SELENOPHOSPHATE SYNTHETASE STARCH PHOSPHORYLASE SUCCINYL-CoA SYNTHETASE SUCROSE PHOSPHORYLASE TUBULIN.TYROSINE LIGASE ORTHOPHOSPHATE CONTINUOUS ASSAY Orthovanadate,... 

The dialkoxyphosphinydifluoromethyllithium reagent has also been used in the preparation of bioactive compounds such as 2-amino-7,7-difluoro-7-phos-phonoheptanoic acid for evaluation in the -methyl-D-aspartic acid binding assay [265], 9-(5,5-difluoro-5-phosphonopentyl)quanine as a multisubstrate analog inhibitor of purine nucleoside phosphorylase [266], fluorinated phosphoserine analog [267, 268] (Scheme 91) and nucleoside 5 -deoxy-5 -di-fluoromethylphosphonates [267,269] (Scheme 92). 

The produced glucose from phosphorylase limit dextrin is determined as a measure of the debranching enzyme activity. This assay method is used for the detection of GSD Hid, a subtype in which only the translocase activity of the enzyme (see section 4.6.17.2) is affected [9,12, 20]. 

Photometric determination of P( production. One colorimetric unit of phosphorylase a is the amount of enzyme that produces 1 pmol Pi/min under assay conditions [3, 22]. 

Table 4.6.19 Enzyme activities for phosphorylase b-kinase assay... 

Besley GTN (1987) Phosphorylase b kinase deficiency in glycogenosis type VIII differentiation of different phenotypes and heterozygotes by erythrocyte enzyme assay. J Inherit Metab Dis 10 115-118... 

Uyttenhove K, Bollen M, Stalmans W (1991) An optimized assay of phosphorylase kinase in crude liver preparations. Biochem J 278 899-904... 

The activity thymidine phosphorylase can be detected in leukocytes using a non-radiochemical assay in which thymine is detected at 265 nm after separation with reverse-phase HPLC [9]. 

The activity of hypoxanthine-guanine phosphoribosyltransferase, adenine phos-phoribosyltransferase, adenosine deaminase, and purine nucleoside phosphorylase can be determined in dried blood spots using an HPLC-linked assay [3]. 

Van Kuilenburg ABP, Zoetekouw L (2005) Determination of thymidine phosphorylase activity by a non-radiochemical assay using reversed-phase high-performance liquid chromatography. J Chromatogr Biomed Sci Appl 820 271-275... 

Melki, R., Fievez, S., and Carlier, M.-F. (1996). Continuous monitoring of Pi release following nucleotide hydrolysis in actin or tubulin assembly using 2-amino-6-mer-capto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase as an enzyme-linked assay. Biochemistry 35, 12038-12045. 

In this laboratory exercise, you will study the effects of phosphorylation and allosteric regulation on the activity of glycogen phosphorylase. In the first experiment, you will phosphorylate glycogen phosphorylase b in vitro using y-[32P]ATP and glycogen phosphorylase kinase. In the second experiment, you will study the effect of phosphorylation on glycogen phosphorylase activity, as well as the effect of AMP on glycogen phosphorylase b activity with the use of a coupled enzymatic (kinetic) assay (Fig. 15-3). 

Figure 15-3 Coupled enzyme assay to determine glycogen phosphorylase activity. As long as the activity of phosphoglucomutase and glucose-6-phosphate dehydrogenase are not rate-limiting, the AA340/At is directly proportional to the A[glucose-l -phosphate]/Atime.

Day 2 In Vitro Assay for Phosphorylase Kinase and Glycogen Phosphorylase Activity... 

To start the phosphorylase kinase assay, add 25 pi of 30 mM ATP solution to each tube and pipette rapidly up and down several times to mix. All three reactions can be run simultaneously, staggering the start of each reaction by about 30 sec. At exactly 5 min and 10 min, remove 100 pi from each reaction and stop it by adding it to 1.9 ml of 40 mM glycerophosphate,... 

Perform the same procedure using tubes B to G by adding 100 /A of your different phosphorylase kinase assay reactions to these tubes. Be sure that you know which phosphorylase kinase reaction corresponds to which test tube (e.g., tube B is phosphorylase kinase reaction 1—5 min, tube C is phosphorylase kinase reaction 1—10 min, etc). Measure and record the A340 of each reaction at 30-sec intervals for at least 5 min, or until each reaction achieves a linear change in A340 versus time. 

NOTE No dilution factor (20) is required for the calculation of glycogen phosphorylase activity in tubes H to J, since the enzyme stock solution was added directly to the assay. Otherwise, the same formula can be used for tubes H to J. 

After you have completed these calculations, you will have obtained two values for the concentration of glycogen phosphorylase kinase present in this assay, one based on the 5-min time point and one based on the 10-min time point. From these data, determine the average value of glycogen phosphorylase kinase present in this assay (units/ml of enzyme). 

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