Spin-exchange NMR spectroscopy in studies of the kinetics of enzymes and membrane transport

Spin-exchange NMR techniques enable the measurement of the rates of exchange of solutes between chemically or physically distinct sites in reactions taking place at chemical equilibrium. The time scale of the events that are able to be investigated lies in the neighbourhood of 1 s. The earliest studies in this area of NMR spectroscopy involved chemical reactions in vitro but the procedures have been adapted to the study of enzyme-catalysed reactions both in vitro and in vivo, and more recently to transmembrane exchange processes. The emphasis in this review is on the various types of spin-exchange experiments, the analysis of data derived from them, estimates of uncertainty in measured rate constants, and their shortcomings. Those methods given special attention are saturation transfer, two-dimensional exchange spectroscopy (2D EXSY), the 'accordion' experiment and 'overdetermined' one-dimensional exchange spectroscopy.     

Regeneration of phosphorylated metabolites in stored erythrocytes in an open perfusion system: studies using 31P NMR spectroscopy

Human erythrocytes were maintained at high haematocrit in a metabolically functional state for several hours in a thermodynamically open perfusion apparatus. The concentrations of ATP and 2,3-bisphosphoglycerate (2,3-DPG) and pH were continuously monitored before and after metabolic perturbations by using 31P NMR; the monitoring was achieved with a 31P flow-through probe. Methylphosphonate was added to plasma perfusion medium as a phosphorus concentration standard and as a 31P NMR pH probe molecule. The rates of decline of ATP and 2,3-DPG levels in fresh cells in a glucose-free medium were measured as were the rates of reformation in response to a 'rejuvenation' medium. Also, rates of ATP and 2,3-DPG synthesis during perfusion with Krebs bicarbonate-0.5 mmol/l glucose and perfusion with pooled plasma were measured in cells that had been previously stored at 4 degrees C for 5 weeks.     

Decreased deformability of erythrocytes from smokers

The deformability of erythrocytes from smoking and non-smoking human subjects was examined by filtration through 3-mu pores and capillary viscometry of cell suspensions. In both cases, small but significant differences were found between the two groups that are consistent with a reduction in the surface area-to-volume ratio and/or a diminished membrane flexibility in erythrocyte from smokers. Additional evidence suggests that these findings represent a chronic rather than an acute effect of smoking on erythrocyte deformability.     

Effects of beta-adrenergic blockade on plasma cyclic AMP and blood sugar responses to glucagon and isoproterenol in man.

In healthy humans glucagon infusion resulted in a significant increase in blood sugar and in plasma cyclic AMP. No discernible hemodynamic effects were found. Isoproterenol infusion on a mole per mole basis in the same subjects induced a significant, although less pronounced rise in plasma cyclic AMP, heart rate, and a fall in diastolic blood pressure but had no effect on blood sugar. Propranolol administration abolished the hemodynamic effects of isoproterenol and significantly decreased the response of plasma cyclic AMP; the same blocking dosage had little effect on plasma cyclic AMP changes induced by glucagon wheras the response in blood sugar was significantly reduced. These data in vivo are compatible with the in vitro demonstration of separate receptors for glucagon and isoproterenol.     

Effects of ACTH on steroid metabolism.

Metabolic clearance rates (MCR) of aldosterone, cortisol, DOC, corticosterone, and progesterone were simultaneously measured by constant infusion in 8 control subjects before and during an ACTH infusion (12.5 U/4 h). Plasma levels of aldosterone and cortisol, the heat-labile plasma protein-bound fraction of aldosterone, and hepatic blood flow as estimated by the fractional clearance of indocyanine green, were concomitantly determined. The mean MCR of aldosterone, cortisol, and DOC increased to a peak value of 26%, 23%, and 22%, respectively, above baseline values. The increase was already significant (P is less than 0.001) 30 min after the beginning of the ACTH infusion. In contrast, no consistent changes occurred in the MCR of corticosterone and progesterone or in hepatic blood flow. ACTH induced a marked fall in the heat-labile plasma protein-bound fraction of aldosterone. Plasma levels of aldosterone and corsitol increased by 190 and 250%, respectively. A significant negative correlation (r=-0.423, P is less than 0.01) between the MCR and the heat-labile protein-bound fraction of aldosterone was observed. We suggest that competitive interactions in the protein binding properties of various steroids account for the selective effect of ACTH on the MCR of aldosterone, cortisol, and DOC.     

A new method for the simultaneous analysis of free and sulfoconjugated normetanephrine, metanephrine, and 3-methoxytyramine in human urine by HPLC with electrochemical detection

A sensitive and specific assay for free and sulfoconjugated normetanephrine, metanephrine, and 3-methoxytyramine was achieved by tetraphenylboron complexing and by high-performance liquid chromatography with electrochemical detection. Sulfoconjugated metabolites were hydrolyzed by sulfatase. Complexing the 3-O-methylated catecholamines with tetraphenylboron followed by their extraction in either and reextraction in dilute hydrochloric acid succeeded in partially purifying the sample without the use of a prepacked column. Detection by electrochemical technique additionally eliminates unoxidable components in the urine. The relatively high sensitivity of this method permits measurement of metabolite concentrations as low as 10 ng/ml, allowing for the first time the determination of free 3-O-methylated catecholamines in human urine. Hydrolysis by sulfatase specifically identifies the form of the conjugated metabolite. The mean total (free plus sulfoconjugated) values of normetanephrine, metanephrine, and 3-methoxytyramine obtained from 25 normal volunteers were 214, 102, and 227 micrograms/day, respectively. Values were greatly increased in patients with pheochromocytoma. The degree of sulfoconjugation varied, and each individual metabolite was highest with normetanephrine (86%). This method is sensitive, rapid, simple, and can be easily standardized for clinical investigation of pheochromocytoma.     

Atrial natriuretic factor in sympathetic ganglia of the rat: dependence on cholinergic innervation

Atrial natriuretic factor is detectable in the peripheral autonomic ganglia of the rat by radioimmunoassay and immunohistochemistry. In the present study, surgical and neurochemical methods were used to evaluate the source of this peptide in sympathetic ganglia. Decentralization of the ganglia and/or central administration of colchicine diminished the atrial natriuretic factor content in para- and prevertebral ganglia. Axotomy did not affect levels of ganglionic atrial natriuretic factor. A messenger ribonucleic acid species hybridizing with rat atrial natriuretic factor complementary deoxyribonucleic acid was not found within the total ribonucleic acid extracted from superior cervical ganglia. These results indicate a direct dependence of ganglionic atrial natriuretic factor on cholinergic innervation.     

Minoxidil in severe hypertension

Summary Minoxidil is a potent anti-hypertensive drug which acts on the peripheral arteriolar smooth muscle. It was administered to 14 patients with severe hypertension where standard therapy failed to control the blood pressure. The mean initial blood pressure was 209/129 when receiving treatment. Minoxidil was given in doses varying from 7.5 to 40 mg (mean = 24 mg) and was associated with propranolol (mean dose = 170 mg) and furosemide (mean dose = 120 mg). Twelve patients had an adequate therapeutic response. The systolic blood pressure was reduced by 44 mm Hg which represents a 23±3% reduction and the diastolic blood pressure was reduced by 33 mm Hg which represents a 26±3% reduction. Two patients were considered treatment failures and two patients died during Minoxidil treatment. Three patients showed electrocardiographic changes caracterized by T wave inversion at the onset of Minoxidil treatment and these changes were reversible in two. All patients except two had hirsutism and two women developped polymenorrhea. Other side effects consisted of tachycardia and weight gains. One patient with lupus erythematodes had an exacerbation of her disease while on Minoxidil. Minoxidil represents a useful and effective adjuvant to the treatment of resistant hypertension. It can delay bilateral nephrectomy in patients with terminal renal failure and high blood pressure.