A specific micromethod for the determination of acyl phosphates
01 Jun 1945-Journal of Biological Chemistry (American Society for Biochemistry and Molecular Biology)-Vol. 159, Iss: 1, pp 21-28
TL;DR: In this communication, a method is described which utilizes the reaction of acyl phosphates with hydroxylamine and the acyl part of the acid anhydride is converted into hydroxamic acid.
About: This article is published in Journal of Biological Chemistry.The article was published on 1945-06-01 and is currently open access. It has received 1373 citations till now.
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TL;DR: This chapter is dedicated to describing citrate synthase, which can be followed by measuring the appearance of the free SH group of the released CoASH through three methods discussed in the chapter.
Abstract: Publisher Summary This chapter is dedicated to describing citrate synthase. The assay of citrate synthase is performed by coupling it to the transacetylase reaction. The disappearance of acetyl phosphate is followed by a hydroxamate method and the formation of citrate by the pentabromoacetone method. The malate dehydrogenase catalyzed reaction is used to generate the oxaloacetate for the citrate synthase reaction. Another method for assaying citrate synthase uses 14 C-acetyl-CoA and measures its incorporation in 14 C-citrate, which is isolated as a silver salt. Citrate synthase can be followed by measuring the appearance of the free SH group of the released CoASH; three such methods are discussed in the chapter. One method is to measure the oxidation of the CoASH by dichlorophenol- indophenol, which is accompanied by a decrease in absorbancy at 578 mμ. Another method measures the CoASH polarographically. The third method measures SH by the use of 5, 5’-dithiobis-(2-nitrobenzoate) (DTNB) (Ellman's reagent).
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TL;DR: The present method, which is designed for use with short chain 0-acyl derivatives, is based on the finding that hydroxylamine at an alkaline pH in water rapidly converts acetylcholine stoichiometrically to hydroxamic acid throughout a wide range of ester concentration.
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TL;DR: A predictive algorithm is formulated in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures.
Abstract: Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)
1,128 citations
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...Acetate was measured by enzymatic conversion to acetyl phosphate and reaction with hydroxylamine (7, 8, 11)....
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TL;DR: It is found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase, which suggests that CPA interferes with the ATP-induced conformational changes related to Ca2- transport.
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TL;DR: In this paper, it was shown that the transfer of the phospholyl group between acetic and adenylic acid was reversible, and this transfer was found to be reversible through the use of acetyl phosphate as the intermediary.
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