Sugar transport in reversibly hemolyzed avian erythrocytes.

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37 degrees C, 88% of the ghosts regained their permeability barrier to L-glucose. In these ghosts, the carrier-mediated rate of entry of 3-O-methylglucose was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.     

Anaerobic stimulation of sugar transport in avian erythrocytes

The kinetic parameters of the sugar transport in avian erythrocytes were evaluated under aerobic and anaerobic conditions. In anaerobic cells, transport measurements with $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose resulted in a set of similar dissociation-like constants. Thus the Michaelis constants of $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose entry and exit, $\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>si</mtext>}}$, were 8 and 7 mM, respectively. The equilibrium exchange constant, $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$, and the counterflow constant, $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$, were 9 and 11 mM, respectively. The activity constant for $\text{3-O-}\text{methylglucose}$ transport, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, defined as $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$, was 4 ml/h per g. This set of kinetic constants was compatible with a symmetrical mobile-carrier model. In contrast, the Michaelis constant for glucose entry, $\text{K}{}_{\mathrm{<mtext>go</mtext>}}$, was 2 mM and less than the counterflow constant, $\text{R}{}_{\mathrm{<mtext>g</mtext>}}$ (8 mM). This result could be accounted for by slower movement of the glucose-carrier complex than the free carrier. The activity constant for glucose transport, $\text{F}{}_{\mathrm{<mtext>g</mtext>}}$, was 5 ml/h perg.Under aerobic conditions, two of the dissociation-like constants ($\text{K}{}_{\mathrm{<mtext>si</mtext>}}$ and $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$) for $\text{3-O-}\text{methylglucose}$ transport were significantly larger than those obtained in anaerobic cells, but the remaining two ($\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$) remained unchanged. The values, for $\text{K}{}_{\mathrm{<mtext>so</mtext>}}\text{, K}{}_{\mathrm{<mtext>si</mtext>}}\text{, B}{}_{\mathrm{<mtext>s</mtext><mtext>\; and</mtext>}}\text{R}{}_{\mathrm{<mtext>s</mtext>}}$ were 8, 26, 20 and 8 mM, respectively. The activity constant, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, decreased to 2 ml/h per g. These changes in kinetic constants were consistent with the hypothesis that anoxia accelerated sugar transport by releasing free carrier that was previously sequestered on the inside of the cell membrane.     

Alteration of hexose transport in avian erythrocytes by vinblastine and colchicine

Vinblastine and colchicine, compounds which effect the state of aggregation of microtubules, were investigated to determine if changes in the rate of sugar transport were produced by these compounds. Vinblastine accelerated 3-O-methylglucose entry into avian erythrocytes. At a concentration of 1.5 mm, transport was accelerated two-fold. The effect of vinblastine was not attributed to cell energy depletion or to increased entry by simple diffusion. Stimulation of transport did not require preincubation of the cells with vinblastine, and the effect was reversible. Colchicine (2 mm) inhibited 3-O-methylglucose entry in aerobic or anoxic intact red cells and in red cell ghosts. A change in the state of aggregation or activity of the microtubular system could represent a model for regulation of a membrane carrier. The present results would lend support to this model.     

NADH-ferrihemoglobin reductase in avian erythrocytes

The assay for NADH-ferrihemoglobin reductase (NADH-FR) was optimized for avian blood samples. In this assay the pH optimum for Japanese quail red cell NADH-FR was 5.5, which was close to the enzyme's pI. Enzyme kinetic parameters were determined for quail, chicken and turkey NADH-FR. Preparation of erythrocyte ghost-cells and subsequent fractionation showed that the enzyme was present in the plasma membrane as well as in the nuclear membrane, while Triton X-100 treatment gave a release of enzyme activity from the membrane. In the cytosolar fraction of avian red cells no NADH-FR could be detected.     

Amino ketone synthesis in avian erythrocytes

1. The relative rates of synthesis of aminolaevulate and aminoacetone by particles prepared from avian erythrocytes were measured under various conditions. 2. The production of both amino ketones by fresh particles was about three times greater in anaemia caused by phenylhydrazine and acetylphenylhydrazine than in anaemia caused by removal of 20-30ml. of blood. 3. The synthesis of aminolaevulate by freeze-dried particles decreased more than that of aminoacetone in the absence of added pyridoxal phosphate, in the presence of cyanide and of tris buffer, and after preincubation of the erythrocyte particles. Other differences in the rates of synthesis of the two amino ketones were observed after (a) incubation of particles at different temperatures and (b) storage of homogenized freeze-dried particles at different pH values. 4. It is suggested that these differences in the production of the two amino ketones are due to the presence of two amino ketone synthetases or to two or more isoenzymes of aminolaevulate synthetase. 5. The metabolic significance of aminoacetone in erythrocytes is discussed.     

RNA polymerase in normal avian erythrocytes

Significant RNA polymerase activity was demonstrated in isolated nuclei from mature avian erythrocytes. This activity was shown to have characteristics common to mammalian systems, including sensitivity to α-amanatin. A crude fraction of RNA polymerase was solubilized from these nuclei and characterized to provide further support for the existence of the enzyme in these cells.