Erythrocyte Lii-Nao countertransport system Inhibition by N-ethylmaleimide probes for a conformational change of the transport system

Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Li_i-Na_o countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Li_i-Na_o countertransport. In Na- and Li-free medium, inhibition of Li_i-Na_o countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent K_m  12 mM) is higher than the affinity of Na to its external countertransport site (apparent K_m  25 mM), as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249–265). Reactivity of $\text{N-}\text{ethyl}\text{[}{}^{14}\text{C}\text{]}\text{maleimide}$ was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes.     

Reduced maximum capacity of glycolysis in brown adipose tissue of genetically obese, diabetic (db/db) mice and its restoration following treatment with a thermogenic beta-adrenoceptor agonist

The maximal activities of the key glycolytic enzymes hexokinase and 6-phosphofructokinase, were reduced in brown adipose tissue in db/db mice compared to their lean littermates. Treatment of db/db mice with the thermogenic beta-adrenoceptor agonist, BRL 26830, restored normoglycaemia. The only significant increase in activity of hexokinase and 6-phosphofructokinase in the BRL 26830-treated db/db mice occurred in brown adipose tissue where the total tissue activity increased 10- and 11-fold respectively. These changes together with increased 2-deoxyglucose uptake in vivo suggest that brown adipose tissue can play a quantitatively important role in the removal of glucose from the blood.     

The influence of glycyl residues on the flexibility of peptide hormones in solution. A 13C-nuclear-magnetic-resonance study of luteinizing hormone-releasing hormone (luliberin) and its des-glycinamide10 N-ethylamide analog.

13C nuclear magnetic resonance spectroscopy in used to gain information on the flexibility of the backbone in peptide hormones and peptide hormone analogs. 13C spin-lattice relaxation times (T1) were measured on luliberin, the luteinizing-hormone-releasing hormone and des(Gly-NH2)10-luliberin-N-ethylamide in aqueous solution at 25.2 and 67.9 MHz at temperatures of 32 degrees, 40 degrees and 55 degrees C. The 13C spin-lattice relaxation times indicate increased flexibility of the peptide backbone in the immediate environment of glycyl residues in luliberin (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and the hormone analog des(Gly-NH2)10-luliberin-N-ethylamide (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NH-CH2CH3) in aqueous solutions. 13 C NMR spectroscopy is shown to be a sensitive technique for monitoring the time-averaged conformational flexibility of peptides in solution. Activation energies (Ea) of about 25 kJ/mol were obtained for rotational reorientation of non-terminal alpha-carbons in the peptide backbone. Rotation of methyl groups was characterized by an Ea of 9.6 kJ/mol whereas reorientation of the N-terminal pyroglutamyl residue showed an Ea value of 14.6 kJ/mol. The Ea values of individual carbons in the side-chains of prolyl, arginyl and leucyl residues in the peptides were similar to those obtained for the alpha-carbon of the same amino acid residue in the peptide backbone of the hormones.     

Effects of ambient and local cutaneous temperatures on hand blood flow

The hand blood flow ( ) was investigated in response to a wide range of general and local cutaneous thermal stimuli (0–36°C and 4–42°C respectively), the local stimulus consisting of a thermostatically controlled water bath for the right hand (T_w), and the general stimulus, the ambient room temperature (T_a). was measured at the right wrist by strain gauge plethysmography; it was seen to respond more significantly to variations in T_w than to those in T_a at cold to comfortable ambient temperatures (T_a<22°C). A paradoxical vasodilatation was observed at T_w=4°C (Lewis' hunting phenomenon). The graphs of versus T at average to high local cutaneous temperatures (T_w > 33°C) are remarkably similar, except for an upward shift at successively higher values of T_w. The slope (or vasomotor reactivity) is interpreted as being controlled by variations in T_a. The curves exhibited maximum values at T_a = 31°C. Their subsequent decrease could represent a thermoregulatory adaptation to environment-organism heat transfer, the relative vasoconstriction tending to reduce the transfer. Although the qualitative response was the same for both sexes, the absolute value of was generally greater in male than in female subjects.     

Labeling of glucagon binding components in hepatocyte plasma membranes.

A photosensitive derivative of glucagon, ¹²⁵I-N^?-4-azido-2-nitrophenyl-glucagon, has been synthesized and used to specifically label glucagon binding proteins in hepatocyte plasma membranes. Photolysis of the derivative in the presence of a membrane suspension results in the incorporation of radioactivity primarily into membrane components with a molecular weight range of 23,000–25,000. The binding properties of the derivative are essentially identical to that observed for glucagon. The binding of ¹²⁵I-NAP-glucagon was completely inhibited in the presence of glucagon (3 μM) while greater than 90% of the covalent labeling was also inhibited in the presence of glucagon. These studies suggest that the labeled membrane protein may be a component of the glucagon receptor.