Publisher Summary The turbidity produced when protein is mixed with low concentrations of any of the common protein precipitants can be used as an index of protein concentration. The resulting turbidity is maximum after about 10 minutes and may be measured spectrophotometrically in the wavelength region of 600 m. Standardization may be effected by comparison with the turbidity produced by a suspension of a dried protein precipitate, or reference may be had to the methyl acrylate-styrene polymer. Turbidimetric techniques are rapid and convenient, but they yield different values with different proteins. They do not permit differentiation between protein and acid-insoluble compounds such as nucleic acids. Protein estimation with the Folin-Ciocalteu reagent include (1) biuret reaction of protein with copper ion in alkali, and (2) reduction of the phosphomolybdic-phosphotungstic reagent by the tyrosine and tryptophan present in the treated protein. Protein estimation by ultraviolet absorption takes advantage of the fact that nucleic acid, however, absorbs much more strongly at 260 mμ than at 280 mμ, whereas with protein the reverse is true. This advantage is used to eliminate, by calculation, the interference of nucleic acids in the estimation of protein.

Spectrophotometric and turbidimetric methods for measuring proteins

Apres l'ingestion de glucose, l'insulino-secretion du pancreas est stimulee et la combinaison de l'hyperglycemie et de l'hyperinsulinemie doit induire la captation de glucose dans les territoires splanchique (foie et tube digestif) et peripherique (muscles) et la suppression de la production hepatique du glucose. Le but de cette conference est de prouver que, bien que la perturbation du metabolisme hepatique du glucose joue un role dans le maintien de l'etat diabetique, le foie ne joue probablement pas de role majeur dans le developpement precoce de l'hyperglycemie a jeun des DNID

Lilly lecture 1987. The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM.

Non-insulin-dependent diabetes mellitus (NIDDM) results from an imbalance between insulin sensitivity and insulin secretion. Both longitudinal and cross-sectional studies have demonstrated that the earliest detectable abnormality in NIDDM is an impairment in the body's ability to respond to insulin. Because the pancreas is able to appropriately augment its secretion of insulin to offset the insulin resistance, glucose tolerance remains normal. With time, however, the beta-cell fails to maintain its high rate of insulin secretion and the relative insulinopenia (i.e., relative to the degree of insulin resistance) leads to the development of impaired glucose tolerance and eventually overt diabetes mellitus. The cause of pancreatic "exhaustion" remains unknown but may be related to the effect of glucose toxicity in a genetically predisposed beta-cell. Information concerning the loss of first-phase insulin secretion, altered pulsatility of insulin release, and enhanced proinsulin-insulin secretory ratio is discussed as it pertains to altered beta-cell function in NIDDM. Insulin resistance in NIDDM involves both hepatic and peripheral, muscle, tissues. In the postabsorptive state hepatic glucose output is normal or increased, despite the presence of fasting hyperinsulinemia, whereas the efficiency of tissue glucose uptake is reduced. In response to both endogenously secreted or exogenously administered insulin, hepatic glucose production fails to suppress normally and muscle glucose uptake is diminished. The accelerated rate of hepatic glucose output is due entirely to augmented gluconeogenesis. In muscle many cellular defects in insulin action have been described including impaired insulin-receptor tyrosine kinase activity, diminished glucose transport, and reduced glycogen synthase and pyruvate dehydrogenase. The abnormalities account for disturbances in the two major intracellular pathways of glucose disposal, glycogen synthesis, and glucose oxidation. In the earliest stages of NIDDM, the major defect involves the inability of insulin to promote glucose uptake and storage as glycogen. Other potential mechanisms that have been put forward to explain the insulin resistance, include increased lipid oxidation, altered skeletal muscle capillary density/fiber type/blood flow, impaired insulin transport across the vascular endothelium, increased amylin, calcitonin gene-related peptide levels, and glucose toxicity.

Pathogenesis of NIDDM: A balanced overview

http://www.jbc.org/content/250/14/5475.full.pdf

Purification and characterization of the flavoenzyme glutathione reductase from rat liver.

The function of cytidine coenzymes in the biosynthesis of phospholipides.

https://science.sciencemag.org/content/sci/125/3238/113.full.pdf

Defect in uptake of galactose-1-phosphate into liver nucleotides in congenital galactosemia.

Publisher Summary This chapter focuses on the enzymes of the leloir pathway, and describes the quantitative determination of enzymatic activity in broken cell preparations. Specific enzymatic assays designed especially for use with crude bacterial extracts are described. Galactokinase catalyzes the reaction ATP + Gal→ ADP +α -Gal-l-P. The Gal-1-P produced in a preincubation mixture is quantitatively determined. The Gal-l-P formed is determined in the proteinfree filtrate by an analytical incubation mixture. The absolute molar amount of TPNH formed is a direct measure of Gal-l-P and is thus a measure of galacto-kinase activity. The rate of G-1-P formation in Gal-l-P uridyl transferase is analyzed by adding the two indicator enzymes and coenzymes in excess, phosphoglueomutase (plus glucose-l,6-diphosphate) and G-6-P dehydrogenase plus TPN. In UDPGal-4-epimerase, UDPG formed is determined in a protein-free filtrate by means of a DPN-dependent specific dehydrogenase (UDPG dehydrogenase). The chapter also describes the purification of galactose-l-phosphate uridyl transferase, and purification of UDPgalactose-4-epimerase.

[20] Enzymes of the Leloir pathway

A specific high affinity galactose transport system called P(betag) can be induced by trace amounts of galactose in the medium by virtue of its own ability to capture and accumulate galactose The transport system is coregulated with the production of a high affinity periplasmic galactose binding protein, which constitutes but one part of the transport system Some transport negative mutants still remain producers of this binding protein A close correlation exists between production of the active binding protein and the presence of galactose chemotaxis The hypothesis, that this binding protein is a common element of the specific galactose transport system, P(betag), and of galactose chemotaxis is supported by observations on structural mutants, being defective in galactose binding protein as well as showing a lack of galactose chemotaxis The binding protein is a monomer with two binding sites for galactose Binding of one or two of the galactose molecules elicits specific conformational changes of the galactose binding protein (lowered affinity for galactose, increase of charges of the protein, increased fluorescence of tryptophan residues) The importance of these features for transport and for chemotaxis is discussed (70)

https://science.sciencemag.org/content/sci/174/4009/557.full.pdf

The Periplasmic Galactose Binding Protein of Escherichia coli

https://science.sciencemag.org/content/sci/125/3238/105.full.pdf

Herman M. Kalckar

Papers

Defect in uptake of galactose-1-phosphate into liver nucleotides in congenital galactosemia.

[20] Enzymes of the Leloir pathway

The Periplasmic Galactose Binding Protein of Escherichia coli

Biochemical Mutations in Man and Microorganisms

Galacto-waldenase and the enzymic incorporation of galactose 1-phosphate in mammalian tissues