Accumulation of cystine from glutathione-cysteine mixed disulfide in cystinotic fibroblasts; blockade by an inhibitor of gamma-glutamyl transpeptidase

Cystinotic and normal skin fibroblasts in tissue culture were treated with varying concentrations of reduced glutathione, oxidized glutathione and glutathione-cysteine mixed disulfide, substrates of gamma-glutamyl transpeptidase, the catabolic enzyme of the gamma-glutamyl cycle. Cystine accumulated more rapidly and to a greater extent from the glutathione-cysteine mixed disulfide in cystinotic than in normal cells. Inhibition of gamma-glutamyl transpeptidase activity by serine in a borate buffer partially blocked this accumulation of cystine. Reduced glutathione and oxidized glutathione have lesser effects on cystine accumulation. Stored cystine in cystinotic tissues may derive in part from glutathione-cysteine mixed disulfide via transpeptidation.     

Photooxidation by Photosystem II of Tris-washed Chloroplasts

Irradiation of tris-washed chloroplasts with moderate intensities of red light caused a partial bleaching of chloroplast pigments and an inhibition of the hydroquinone-supported photoreduction of NADP. The presence of an electron donor for photosystem 2 (PS2) during the irradiation prevented the bleaching and inhibition. It is concluded that the strong oxidant produced by PS2 accumulates in tris-washed chloroplasts during irradiation and an electron donor for PS2 protects against the photooxidation reactions.     

A protein and peptide monitor with a hollow-cathode light source.

This communication describes an inexpensive system that will monitor protein and peptide concentration in chromatogram eluates by light absorbance at an adjustable wavelength.Proteins in chromatogram eluate streams are commonly metered for concentration by absorbance measurement at 280 nm. Besides a range of commercially manufactured monitors, there is the apparatus described by Bennett et al. (1) which uses a selectively modulated magnesium lamp. Measurements in the region of 280 nm are of no value, however, when the material does not contain aromatic amino acids. Monitoring then becomes necessary at 230 nm in the region of absorption due to the peptide bond. The common resort in such a case is the standard ultraviolet-visible spectrophotometer, which has the disadvantage of being both unnecessarily elaborate and expensive for the purpose required. The deuterium lamps in these instruments require frequent replacement because of the extended periods of operation, adding to the cost factor.We have investigated the use of a hollow-cathode lamp of the type manufactured for use in an atomic absorption spectrophotometer. These lamps have high stability and a long working life, due to the considerably lower level of power dissipation compared with deuterium lamps. Their emission spectra are discontinuous, but the lamp for the element iron provides adequately strong lines at both 229.5 and 279.2 nm, suitable for a protein monitor.     

Chemical energy usage and myosin light chain phosphorylation in mammalian smooth muscle

Experiments have been done to determine the relationships among active force output, average rate of high-energy phosphate utilization, and the degree of phosphorylation of the 20,000-dalton myosin light chain in the rabbit tenia coli at 18 C. During an isometric tetanus at l0 the degree of light chain phosphorylation increases to a maximum of 30-40% before maximum force is developed, and then phosphorylation slowly decreases while active force is maintained. During the period when there is a small decrease in degree of phosphorylation, the average rate of chemical energy usage falls by fourfold. In contrast, when the calcium concentration of the bathing medium is lowered from 1.9 to 1.0 mM a very large decrease in degree of phosphorylation is associated with only a small decrease in both energy usage and active force. At lower calcium levels both force and chemical energy usage decrease proportionately with little further decrease in degree of phosphorylation. We conclude that under isometric conditions there is no consistent relationship between degree of myosin light chain phosphorylation and the rate of cross-bridge cycling as measured by the rate of high-energy phosphate usage in this mammalian smooth muscle.