John B. Allred

Direct enzymatic procedure for the determination of liver glycogen

Abstract A method is proposed to measure glycogen content in liver homogenates without extraction and acid hydrolysis of tissue glycogen. Homogenates were treated with amyloglucosidase, which degrades glycogen to glucose, and the glucose was the determined enzymatically by the use of glucose oxidase and peroxidase. The method was shown to yield nearly complete (99%) recoveries of standard glycogen, while 5 hr of acid hydrolysis of standard glycogen were required to obtain comparable recoveries. When compared to an acid hydrolysis method for liver, amyloglucosidase degradation of rat liver glycogen and subsequent determination of glucose resulted in higher values for glycogen content. The amyloglucosidase, glucose oxidase: peroxidase method has the advantage of rapidity, whereas the traditional method consisting of extraction, precipitation, and acid hydrolysis is not only time consuming, but may also be subject to losses of glycogen in each step.

Determination of coenzyme A and acetyl CoA in tissue extracts

Abstract A sensitive and specific method for the determination of coenzyme A and acetyl CoA in tissue extracts is described. CoA is recycled through a coupled enzyme system in which the rate of formation of NADH is proportional to CoA concentration when the necessary enzymes and substrates are present in excess. The same system can be used to measure acetyl CoA in tissue extracts by first binding free CoA irreversibly with N -ethylmaleimide. The method is convenient in that all of the required reagents are commercially available. The rate of NADH production can be followed either spectrophotometrically or, with greater sensitivity, fluorometrically. With the latter, concentrations of less than 40 picomoles per milliliter can be measured. This method was used to determine the CoA and acetyl CoA content of perchloric acid extracts of various rat tissues. Close agreement was found with literature values for liver acetyl CoA but CoA values were generally much lower than those previously reported, apparently because of increased specificity.

Inhibition of hepatic lipogenesis by cyclic-3′,5′-nucleotide monophosphates

Summary The incorporation of acetate-l-14C into lipids of liver slices from fed and fasted (3 hours) chickens was inhibited by the addition to the media of N6, O2 -dibutyryl cyclic-3′,5′-adenosine monophosphate (DiBu-c-AMP) or cyclic-3′,5′-guanosine monophosphate. Lipogenesis in liver slices from fed and fasted (6 hours) rats was also inhibited by DiBu-c-AMP. Short-term fasting caused a reduction in lipogenesis in liver slices from both species. It is suggested that very short-term effects of diet on hepatic fatty acid synthesis may be due to a change in hepatic cyclic-3′,5′-adenosine monophosphate content, which is known to be influenced by glucagon and insulin.

Advances in molecular biology: implications for the future of clinical nutrition practice

Advances in molecular biology during the past decade have substantially contributed to our understanding of how genes influence physiologic processes and, ultimately, our health. Genes associated with many nutrition-related chronic diseases are being identified and characterized. Nutrients may directly or indirectly influence the transcription and/or translation of specific gene products. Identifying genetic markers for specific diseases and exploring gene therapy will provide new opportunities and challenges for clinical nutrition practice in the 21st century. Nutrition practitioners must be cognizant of developments in molecular biology to meet the challenges of providing nutrition care in the future.

Copper deficiency alters isomyosin types and levels of laminin, fibronectin and cytochrome c oxidase subunits from rat hearts

The relative amounts of cardiac proteins such as laminin, fibronectin, cytochrome c oxidase, and isomyosin types were studied by gel electrophoresis and Western blotting in control and copper-deficient Sprague-Dawley rats of both sexes fed their respective diets from weanling for 3 weeks. Isomyosin types appeared to shift from V1 to greater levels of V3 in copper deficient rats for both genders. Male copper deficient rats had increased cardiac levels of fibronectin, decreased laminin levels, cardiac hypertrophy and anemia. Both male and female rats fed copper-deficient diet had lower levels of cytochrome c oxidase (CCO) subunit IV, and low liver copper, and high heart-to-body weight ratios compared with their respective controls.

Dietary dependent distribution of acetyl CoA carboxylase between cytoplasm and mitochondria of rat liver

Biotinyl proteins in cytoplasm and mitochondria of rat liver were examined by fluorography and the quantity of acetyl CoA carboxylase was determined after sodium dodecyl sulfate-denatured proteins were incubated with [14C] methyl avidin and separated by polyacrylamide gel electrophoresis. Results show that one-half of the total acetyl CoA carboxylase in liver of fed rats was associated with mitochondria in a relatively inactive form. Fasting shifted the distribution of the enzyme toward the mitochondrial fraction and refeeding previously fasted rats shifted the distribution towards cytoplasm. Thus, acetyl CoA carboxylase can be added to the list of ambiquitous enzymes whose subcellular distribution varies with physiological conditions.

Molecular weights of subunits of acetyl CoA carboxylase in rat liver cytoplasm.

Monomeric [14C] methyl avidin was shown to bind to sodium dodecyl sulfate-denatured biotinyl proteins and remain bound through polyacrylamide gel electrophoresis which allowed their detection by fluorography. This method was used to show that purified rat liver acetyl CoA carboxylase contained two high molecular weight forms of the enzyme (MR = 241,000 and 252,000) while rapidly prepared, crude rat liver cytoplasm contained two larger molecular weight (MR = 257,000 and 270,000) forms. Thus, the enzyme had undergone substantial proteolysis during purification. The crude enzyme preparation also contained a smaller biotinyl protein (MR = 141,000) which is likely a proteolytic product of the larger forms of acetyl CoA carboxylase.

Properties and subcellular distribution of enzymes required for acetoacetate biosynthesis in chicken liver

Abstract 1. 1. Evidence is presented which indicates that the rate-limiting enzyme of the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) pathway of acetoacetate synthesis in chicken liver is HMG-CoA synthase as it is in other species. 2. 2. Maximum rates of acetoacetate synthesis in the catalytic assay system, in which acetyl-CoA is generated from acetyl phosphate and coenzyme A in the presence of purified phosphotransacetylase, was observed when the reaction mixture contained 1 mM coenzyme A and was supplemented with equimolar amounts of both Mg 2+ and Mn 2+ . 3. 3. On the basis of studies on the subcellular distribution of HMG-CoA synthase and the other enzymes involved, it is estimated that acetoacetate synthesis capacity is about evenly divided between mitochondrial and cytoplasmic fractions.