A new major transmembrane glycoprotein, gp155, in goat erythrocytes. Isolation and characterization of its association to cytoskeleton through binding with band 3-ankyrin complex

Erythrocyte membranes from goat contain a considerable amount, more than 10% of the total amount, of a glycoprotein with Mr = 155,000 (gp155) on sodium dodecyl sulfate-polyacrylamide electrophoresis gel. This report describes the first isolation and characterization of gp155. This gp155 has major trypsin-sensitive sites at each side of the plasma membrane to generate membrane-bound fragments, indicating that the gp155 spans the lipid bilayer several times. This protein consists of a single polypeptide containing about 1,200 amino acid residues corresponding to Mr = 134,000 and some complex type N-linked oligosaccharide chains. A fraction (15-20%) of the gp155 is recovered in nonionic detergent-extracted ghosts along with 25-30% of band 3 and other cytoskeletal proteins and is completely released into solution by extraction with 1 M KCl. Immunoprecipitation with anti-gp155 and anti-ankyrin antibodies of detergent-solubilized membranes separated on a gel permeation chromatography column showed that a part of the gp155 is tightly linked to band 3 with a molar ratio of 1:2 to 1:3. This gp155-band 3 complex in turn is associated to ankyrin through the binding of band 3 to ankyrin. These data indicate that, in native erythrocyte membranes, as well as in detergent solution, gp155 could play a physiological role in controlling cellular integrity and elasticity by forming the gp155-band 3-ankyrin complex. Partial amino acid sequences of the tryptic peptides are also determined.     

Calcitonin-induced phosphorylation of rat liver cytosolic proteins

Calcitonin (CT) stimulated phosphorylation of two liver cytosolic proteins whose molecular weights are 67,000 and 93,000. Stimulation of 67,000-Mr protein phosphorylation began shortly after subcutaneous injection of CT, reaching a maximum at 5 min and decreasing to below the control level at 30 min. The reaction was independent of cyclic AMP or Ca2+, and was not influenced by a calmodulin antagonist, W7. Stimulation of 93,000-Mr protein phosphorylation became evident by 30 min. This reaction was also stimulated by administration of vasopressin or epinephrine, which is known to cause increased phosphorylation of glycogen phosphorylase having the same molecular weight. The phosphorylation of 93,000-Mr protein, stimulated by CT, was dependent on Ca2+ but not on cyclic AMP, and appeared to be inhibited by W7. In addition, CT did not influence the phosphorylation of 61,000-Mr protein, a major protein phosphorylated in a cyclic AMP-dependent manner. These results suggest that CT may exert its effect on liver cells through protein phosphorylation, most probably in a cyclic AMP-independent manner.     

Chemiluminescence in L-tyrosine-H2O2-horseradish peroxidase system: possible formation of tyrosine cation radical

Tyrosine-H2O2-horseradish peroxidase system at pH 7.4 emitted light in visible region. Phenolic compounds other than tyrosine were also emissive, whereas methoxy phenylalanine and phenyl compounds were not, in H2O2-peroxidase systems. Chemiluminescence spectrum of tyrosine of tyrosine-H2O2-horseradish peroxidase system showed two prominent peaks at 478 nm and 500 nm (Luminescence 1) and additional two or three peaks near 550 and 610 nm (Luminescence 2). Luminescence 1 is quite similar to the phosphorescence originated from an excited tyrosine in triplet state, while Luminescence 2 is quite similar to the phosphorescence originated from an indole in triplet state. Possible formation of tyrosine cation radical (a precursor of the excited tyrosine) and indole cation radical in the enzyme protein (a precursor of the excited tryptophan residue) were discussed.     

Acute inhalation of cigarette smoke augments hypoxic chemosensitivity in humans

Hypoxic and hypercapnic ventilatory responses were measured after two levels of acute inhalation of cigarette smoke, minimum-level nicotine smoke (smoke 1) and nicotine-containing smoke (smoke 2), in 10 normal men. Chemosensitivity to hypoxia and hypercapnia was assessed both in terms of slope factors for ventilation-alveolar PO2 curve (A) and ventilation-alveolar PCO2 line (S) and of absolute levels of minute ventilation (VE) at hypoxia or hypercapnia. Ventilatory response to hypoxia and absolute level of VE at hypoxia significantly increased from 23.5 +/- 22.6 (SD) to 38.6 +/- 31.3 l . min-1 . Torr and from 10.6 +/- 2.5 to 12.6 +/- 3.5 l . min-1, respectively, during inhalation of cigarette smoke 2 (P less than 0.05). Inhalation of cigarette smoke 2 tended to increase the ventilatory response to hypercapnia, and the absolute level of VE at hypercapnia rose from 1.42 +/- 0.75 to 1.65 +/- 0.58 l . min-1 . Torr-1 and from 23.7 +/- 4.9 to 25.5 +/- 5.9 l . min-1, respectively, but these changes did not attain significant levels. Cigarette smoke 2 inhalation induced an increase in heart rate from 64.7 +/- 5.7 to 66.4 +/- 6.3 beats . min-1 (P less than 0.05) during room air breathing, whereas resting ventilation and specific airway conductance did not change significantly. On the other hand, acute inhalation of cigarette smoke 1 changed none of these variables. These results indicate that hypoxic chemosensitivity is augmented after cigarette smoke and that nicotine is presumed to act on peripheral chemoreceptors.     

Serum and tissue coenzyme Q9 in rats with thyroid dysfunctions

Serum and tissue CoQ9 levels were determined in hypothyroid, euthyroid and hyperthyroid rats. A significant negative correlation was demonstrated between serum FT4 or T3 and CoQ9 in rats with various states of thyroid functions. Liver CoQ9 was significantly increased in rats rendered mildly hyperthyroid. There was a significant positive correlation between serum FT4 or T3 and liver CoQ9. While liver CoQ9 did not significantly change in severely hyperthyroid animals, liver mitochondrial CoQ9 showed a significant positive correlation with serum T3. Kidney and heart CoQ9 levels did not significantly change in hyperthyroid rats, but those in hypothyroid rats showed a tendency to increase. It was suggested that the synthesis of CoQ9 was increased in the liver in hyperthyroidism.     

Increase of Na+ gradient-dependent L-glutamate and L-aspartate transport in high K+ dog erythrocytes associated with high activity of (Na+, K+)-ATPase

As reported previously, some dogs possess red cells characterized by low Na+, high K+ concentrations, and high activity of (Na+, K+)-ATPase, although normal dog red cells contain low K+, high Na+, and lack (Na+, K+)-ATPase. Furthermore, these red cells show increased activities of L-glutamate and L-aspartate transport, resulting in high accumulations of such amino acids in their cells. The present study demonstrated: (i) Na+ gradient-dependent L-glutamate and L-aspartate transport in the high K+ and low K+ red cells were dominated by a saturable component obeying Michaelis-Menten kinetics. Although no difference of the Km values was observed between the high K+ and low K+ cells, the Vmax values for both amino acids' transport in the high K+ cells were about three times those of low ones. (ii) L- and D-aspartate, but not D-glutamate, competitively inhibited L-glutamate transport in both types of the cells. (iii) Ouabain decreased the uptake of the amino acids in the high K+ dog red cells, whereas it was not effective on those in the low K+ cells. (iv) The ATP-treated high K+ cells [(K+]i not equal to [K+]o, [Na+]i greater than [Na+]o) showed a marked decrease of both amino acids' uptake rate, which was almost the same as that of the low K+ cells. (v) Valinomycin stimulated the amino acids' transport in both of the high K+ and the ATP-treated low K+ cells [( K+]i greater than [K+]o, [Na+]o), suggesting that the transport system of L-glutamate and L-aspartate in both types of the cells might be electrogenic. These results indicate that the increased transport activity in the high K+ dog red cells was a secondary consequence of the Na+ concentration gradient created by (Na+, K+)-ATPase.