Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37°C, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37°C. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50 percent higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 æM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 æM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.

The calcium-dependent protease of Loxosceles gaucho venom acts preferentially upon red cell band 3 transmembrane protein

Eighty micrograms red blood cell (RBC) ghosts from patients who had previously exhibited the cutaneous form of loxoscelism (presenting localized dermonecrosis) and the viscerocutaneous form of loxoscelism (presenting dermonecrosis, hemoglobinuria, hematuria, and jaundice) and from controls were incubated with 2.5 æg crude Loxosceles gaucho venom in 5 mM phosphate buffer, pH 7.4, at 37ºC. Among all membrane proteins, quantitative proteolysis of the important integral transmembrane protein 3 increased with venom dose and with incubation time from 30 to 120 min, as demonstrated by gel densitometry. Similar quantitative data were obtained for RBC ghosts from patients and from control subjects, a fact that argues against the possibility of genetic factors favoring the hemolytic viscerocutaneous form. These data suggest that the clinical forms may be different types of the same disease, with the viscerocutaneous form being the result of large amounts of intravascularly injected venom and the superficial form being the result of in situ venom action. Since protein 3 is a housekeeping integral membrane protein, whose genetic deficiency leads to hemolytic anemia, it is reasonable to relate it to the hemolysis which occurs in the viscerocutaneous form of loxoscelism. The venom protease responsible for the process was not inhibited after 120-min incubation by 0.2 mM paramethylsulfonyl fluoride or by 0.2 mM N-ethylmaleimide but was inhibited by 25 mM ethylenediaminetetraacetic acid (a calcium-chelating agent) in 5 mM phosphate buffer at pH 7.4, which suggests that the enzyme is a calcium-dependent metalloprotease

Increased fetal hemoglobin levels in patients infected with human immunodeficiency virus (HIV1/2)

Fetal hemoglobin was measured in HIV1/2 patients under treatment with combined therapy (zidovudine and a protease inhibitor). A total of 143 patients and 103 normal individuals were investigated by the quantitative method of Betke and the semi-quantitative acid elution method of Kleihauer. In the normal person, hemoglobin F makes up less than 1 percent and an increase higher than 1.5 percent was observed in 21.4 percent of HIV patients by the method of Betke and in 24.8 percent of HIV-infected patients by the method of Kleihauer. The quantitative biochemical method of Betke showed that the populations were significantly different (two-tailed Mann-Whitney test). The reason for this hemoglobin F increase might be ascribed to the effect of zidovudine or to direct viral action on gamma chain expression. The finding of a higher F cell frequency indicated by the method of Kleihauer rather suggests that there is an increased F cell clone proliferation rather than an increase in hemoglobin F level in every cell

The erythrocyte cytoskeleton protein 4.2 is not demonstrable in several mammalian species

Erythrocyte membrane proteins from 44 representative mammals were studied. Protein 4.2 was not detected in guinea pigs (Cavia porcellus) (N = 14), Southern Brazilian swamp large rats (Myocastor coypus) (N = 2), cutias (Dasyprocta sp) (N = 4), and horses (Equus caballus) (N = 13). These animals also presented high ankyrin concentrations except for the horse which did not exhibit a sharp band, although minor components located between proteins 2 and 3 could account for the ankyrin family. The rodents studied did present band 6, which was not detectable in other common rodents such as white rats (Rattus norvegicus) (N = 9) and mice (Mus musculus) (N = 12). Since the absence of protein 4.2 does not disrupt the cytoskeleton membrane, we suggest that it is not an essential protein. Its absence may be compensated physiologically by the higher ankyrin concentration observed.

Erythrocyte glucose-6-phosphate dehydrogenase activity assay and affinity for its substrate under "physiological" conditions

Glucose-6-phosphate dehydrogenase (G6PD) activity and the affinity for its substrate glucose-6-phosphate were investigated under conditions similar to the physiological environment in terms of ionic strength (I: 0.188), cation concentration, pH 7.34, and temperature (37oC). A 12.4, 10.4 and 21.4 percent decrease was observed in G6PD B, G6PD A+ and G6PD A- activities, respectively. A Km increase of 95.1, 94.4 and 95.4 percent was observed in G6PD B, G6PD A+ and G6PD A-, respectively, leading to a marked decrease in affinity. In conclusion, the observation of the reduced activity and affinity for its natural substrate reflects the actual pentose pathway rate. It also suggests a much lower NADPH generation, which is crucial mostly in G6PD-deficient individuals, whose NADPH availability is poor

Correlation of discocyte frequency and ATP concentration in preserved blood: a morphological indicator of red cell viability

Red blood cells (RBC) are viable if kept in an adequate preservative solution, although gradual changes in morphology and metabolism may occur. There is a gradual decrease in adenosine-5'-triphosphate (ATP) cocentration, pH, glucose consumption, and enzyme activity during preservation. The normal discocyte shapes are initially replaced by echimocytes and stomatocytes and, at final stages, by spherocytes, the last before eplenic sequestration. Post-transfusional survival has been correlated with the ATP concentration. RBC preserved in ADSOL, a solution containing adenine, dextrose, sodium chloride, and mannitol, are viable for transfusion for up to 6 weeks. Erythrocytes from 10 blood units taken from healthy adult donors were preserved for 12 weeks in ADSOL at 4 graus Celsius. We now report a significant correlation (r2 = 0.98) between the percentage of discocytes (89 to 7 percent) and ATP (100 to 10 percent) concentration in ADSOL-preserved RBC. The results suggest that the percent of discocyte shapes used as an indicator OF ATP concentration may be a useful indicator for quality control of RBC viability in centers which have limited assay facilites.

Erythrocyte vitamin B2 and B6 dependent enzymes in adult and newborn Thoroughbred horses

Com o propósito de determinar o estado nutricional de cavalos Puro-Sangue Inglês (PSI) em relaçäo à riboflavina (vitamina B2) e à piridoxina (vitamina B6), 10 animais adultos e 30 recém-nascidos foram investigados. Foi observado um bom estado nutricional quanto à riboflavina, notando-se moderada deficiência de piridoxina nos animais adultos, mas näo nos recém-nascidos. Estes fatos sugerem que os animais adultos devam receber suplementaçäo com piridoxina

Gd(-) Carapicuiba, a rare glucose-6 phosphate dehydrogenase variant associated with moderate enzyme deficiency and chronic hemolysis

A 27 year old Brazilian male of both Portuguese and Spanish origin presenting nonspherocytic chronic hemolytic anemia was found to have a rare glucose-6-phosphate dehydrogenase variant herein named Gd(-) Carapicuiba. The red blood cell enzyme variant is characterized by a moderate enzyme deficiency (47%), high Km for its substrates G6P and NADP, decreased activity against deamino-NADP, increased Ki for NADPH and decreased heat stability. The clinical signs of the patient are probably related to these properties of the enzyme variant

Viabilidade de sangue conservado em recipientes de varias procedencias. / Viability of blood stored in recipients of different sources

Os autores estudaram os sangues contidos em recipientes de plastico e de vidro de cinco procedencias diferentes, utilizando para isto as determinacoes de 2-3-difosfoglicerato (2-3-DPG), adenosina trifosfato eritrocitaria (ATP), hemoglobina (Hb), latico desidrogenase (LDH), sodio e potassio plasmaticos, e pressao parcial sanguinea do oxigenio e do dioxido de carbono e pH sanguineo. Quanto ao 2-3-DPG e ATP, hemoglobina e LDH plasmatica e pH, houve paralelismo, mas houve leves diferencas nos demais parametros entre as diversas procedencias

Efeito da anfotericina B sobre a viabilidade eritrocitaria.Perspectiva do seu uso na prevencao da doenca de Chagas induzida pela transfusao de sangue. / Effect of amphothericin B on the erythrocyte viability. Perspective of its use in the prevention of Chagas' disease induced by blood transfusion

O efeito do antibiotico anfotericina B sobre o sangue armazenado em recipientes plasticos foi estudado. Foram determinados os parametros adenosina-trifosfato, 2-3-difosfoglicerato, pressao parcial de oxigenio, pressao parcial de dioxido de carbono, pH, hemoglobina e latico-desidrogenase plasmaticas e sodio e potassio plasmaticos. Leves diferencas foram notadas em relacao aos controles, mas houve significativo aumento do potassio e diminuicao do sodio nos recipientes com anfotericina B

Distribuicao do sistema ABO e Rh destacando-se a pesquisa do antigeno Du, en Santo Andre, S.P. / Distribution of ABO and Rh blood-group systems, with special reference to the study of the Du antigen, in Santo Andre, S.P

Foi realizada a determinacao do grupo sanguineo ABO e Rh (D) en 1.198 doadores de sangue da cidade de Santo Andre distribuidos em grupos etnicos, destacando-se a pesquisa do antigeno Du em individuos identificados inicialmente como sendo Rh negativo (dd). Os resultados encontrados na pesquisa em relacao aos sistemas ABO e Rh, sao semelhantes a outros trabalhos encontrados na bibliografia nacional. Ressaltamos a importancia da pesquisa do antigeno Du, pois foram por nos encontrados nove individuos portadores do antigeno Du, em 143 individuos identificados inicialmente como sendo do grupo sanguineo Rh negativo, quando na realidade se tratavam de individuos pertencentes ao grupo sanguineo Rh positivo (D)

Nova variante da glicose-6-fosfato desidrogenase eritrocitaria: Gd Sao Paulo. / New variant of erythrocyte glucose-6-phosphate dehydrogenase: Gd Sao Paulo

Nova variante da glicose-6-fosfato desidrogenase eritrocitaria (E.C.1.1.1.49) e descrita em menino negroide do sexo masculino, em que os parametros atividade contra substratos, constante de Michaellis Menten, termoestabilidade, curva de pH foram determinados.Trata-se de variante pertencente a classe tres da classificacao da Organizacao Mundial da Saude, com moderada deficiencia, a qual se denominou Gd Sao Paulo

Aplasia medular e grupos sanguineos ABO e RHo (D). / Bone marrow aplasia and ABO blood groups and RHo (D)

Em 55 pacientes portadores de anemia aplastica, nao foi encontrada incidencia significativa quanto a distribuicao do sistema ABO,mas certificou-se na amostra estudada que houve menor prevalencia de individuos Rh negativos (Rho), o que revelaria menor susceptibilidade de contrair a doenca