Donald R. Harkness

Studies on human placental alkaline phosphatase. II. Kinetic properties and studies on the apoenzyme.

Abstract Human placental alkaline phosphatase is a non-specific phosphatase capable of hydrolyzing compounds containing phosphomonoesters and pyrophosphates and of catalyzing both phosphorylation and transphosphorylation reactions. The heat of activation for hydrolysis of 5′-AMP was calculated to be 10,375 cal/mole. A turnover number of 40,500 moles/mole enzyme/min with the substrate p -nitrophenyl phosphate is reported. The enzyme is competitively inhibited by inorganic phosphate, arsenate and trimetaphosphate. Inactive apoenzyme, prepared by dialysis against EDTA at pH 5.0, is optimally reactivated by Zn ++ or Co ++ . The Zn ++ -reactivated enzyme is stable, whereas the Co ++ -apoenzyme is not. It is concluded that the placental phosphatase is a Zn ++ -metalloenzyme.

Studies on human placental alkaline phosphatase: 1. Purification and crystallization☆

Abstract Placental alkaline phosphatase has been purified by butanol extraction, methanol precipitation, and column chromatography on DEAE-cellulose and crystallized from a solution 42% saturated with ammonium sulfate. The colorless crystals are needleshaped and measure approximately 1 × 20 mμ. The crystalline enzyme appeared homogeneous by ultracentrifugation and immunoelectrophoresis. The enzyme has a spectrum of a pure protein with a 280:260 ratio of 1.93. The average molecular weight from several determinations was 125,000. Enrichment in zinc during purification was demonstrated. The amino acid analysis is reported.

Studies on avian erythrocyte metabolism. Effect of organic phosphates on oxygen affinity of embryonic and adult-type hemoglobins of the chick embryo.

The effects of 2,3 diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), and inositol hexaphosphate (IHP) on the oxygen affinity of whole “stripped” hemoglobin (WSH), hemoglobin H (Hb-H), hemoglobin A (Hb-A) and hemoglobin D (Hb-D) isolated from 18-day chick embryo blood have been determined. The effect of the three organic phosphates upon the oxygen dissociation curves is similar and the following order of decreasing oxygen affinity of the organic phosphates was observed for each hemoglobin: 2,3-DPG < ATP < IHP. 2,3-DPG appears to have a slightly greater effect upon the P50 of Hb-H than upon that of either of the two adult-type hemoglobins. However, this effect seems insufficient to suggest a preferential interaction of 2,3-DPG with Hb-H which would account for either the large amounts of 2,3-DPG in the erythrocytes of embryos or the higher oxygen affinity of the whole blood. The effects of the organic phosphates upon the Hill constant of the purified hemoglobins are variable. It is concluded that since the distribution of hemoglobins H, A, and D in the erythrocytes during the developmental period from 18-day embryos to 6-day chicks remains fairly constant, the previously described progressive decrease in oxygen affinity of the whole blood during this period results from changes in the total amount and distribution of the intraerythrocytic organic phosphates.2

A comparative study on the phosphoglyceric acid cycle in mammalian erythrocytes.

Abstract 1. 1. The activities of the enzymes and intracellular concentrations of substrates and cofactors associated with phosphoglyceric acid metabolism in erythrocytes of nine mammals are reported. 2. 2. In every case 2,3-DPG phosphatase activity was lower than that of the other enzymes measured: glyceraldehyde phosphate dehydrogenase, diphosphoglycerate mutase, phosphoglycerate kinase and phosphoglycerate mutase. 3. 3. The red blood cells of man, rabbit, dolphin and pig contain high concentrations of 2,3-DPG and ATP whereas bovine, caprine and feline erythrocytes contain little of either compound. Equine erythrocytes contain large amounts of 2,3-DPG but little ATP. 4. 4. No 2,3-DPG and only very small amounts of diphosphoglyceric acid mutase was detected in the erythrocytes of the goat.

Studies on avian erythrocyte metabolism--II. Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in chick embryos and chicks.

Abstract 1. The changes in organic phosphates of chicken erythrocytes (RBC) have been determined in relation to the changes in oxygen affinity of whole blood during growth of the embryo and chick. 2. 2,3-diphosphoglyceric acid is the major organic phosphate (42–47%) in the RBC from 15-days of incubation until hatch. The amount of 2,3-DPG during this period is ∼4–6 μmoles/cm3 RBC, decreases abruptly at hatching, and disappears from the RBC within 8 days after hatching. 3. Adenosine triphosphate (ATP) is the major organic phosphate (45–50%) during the first 5 days post-hatching. 4. The P50 of the whole blood during the last week of incubation and the first 5 days post-hatching correlates best with the amount of ATP in the cells. 5. The effect of inositol pentaphosphate on increasing P50 of the whole blood is more gradual and appears to become of major influence in the chick after 4–5 days post-hatching.

Studies on avian erythrocyte metabolism--I. Procedure for separation and quantitation of the major phosphorylated metabolic intermediates by anion exchange chromatography.

Abstract 1. A method for fractionation of the phosphorylated metabolic intermediates in extracts of chicken red blood cells by anion exchange chromatography using a linear ammonium formate gradient, pH 3·45, is described. 2. The method will resolve myoinositol pentaphosphate (IPP) and/or 2,3-diphosphoglyceric acid (2,3-DPG) as well as the other major phosphorylated intermediates of red cells. 3. The amounts of AMP, Pi, ADP, ATP, and IPP in terms of μmoles of Pi per cm 3 of red blood cells (RBC) of the adult chicken were found to be 0·3, 1·2, 1·0, 3·1, and 17·3, respectively.

The 2,3-diphosphoglyceric acid phosphatase activity of phosphoglyceric acid mutase purified from human erythrocytes☆

Abstract The partial purification of phosphoglyceric acid mutase from human erythrocytes is described. This enzyme has an absolute requirement for 2,3-diphosphoglyceric acid under the conditions used for the assay. 2,3-Diphosphoglyceric acid phosphatase activity copurified with the mutase, and evidence that both of these activities are carried out by the same enzyme is reported. The phosphatase activity is markedly enhanced by inorganic pyrophosphate, 20- to 50-fold by 20 m m concentrations. In experiments utilizing 32P-labeled pyrophosphate, direct involvement in the reaction by pyrophosphate was excluded. Intracellular compounds in physiological concentrations failed to augment significantly the phosphatase activity of phosphoglyceric acid mutase. A second 2,3-diphosphoglyceric acid phosphatase which is stimulated by sodium bisulfite and unassociated with mutase activity was separated from the pyrophosphate-stimulated enzyme. The relative significance of these two enzymes in the metabolism of 2,3-diphosphoglyceric acid in the erythrocyte is discussed.

Inositol pentaphosphate in erythrocytes of a freshwater fish, Piraracú Arapaima gigas.

Abstract Inositol pentaphosphate (IPP), a characteristic component of avian erythrocytes (RBC), has been found for the first time in teleost. IPP is present in the erythrocytes of a freshwater air-breathing fish, Piraracu, at a level of 1.8 μmoles per ml RBC, representing 39.0 per cent of the total cell phosphate. Guanosine triphosphate (GTP) represents 22.0 per cent of the cell phosphate.

Some studies on blood of the Florida manatee, Trichechus manatus latirostris.

Abstract 1. 1. Hematological data on the Florida manatee, Trichechus manatus latirostris have been collected. 2. 2. The red blood cells (129.5μ3) of the manatee are larger than those of the dolphin or man and contain 6.84 and 0.27μmoles/cm3 of 2,3-diphosphoglyceric acid and adenosine triphosphate, respectively. 3. 3. Serum electrolytes, blood chemistries and serum enzyme activities are very similar to those of man except for unusually low levels of glutathione reductase activity (15 μmoles/min per 100cm3 RBC). 4. 4. The whole blood P50 values for the manatee and baby manatee are 16.5 and 20.6mm Hg, respectively. 5. 5. The diploid chromosome number of this species is 48.

Erythrocyte metabolism in the bottle-nosed dolphin, Tursiops truncatus

Abstract 1. 1. Erythrocyte metabolism in the bottle-nosed dolphin and in man are compared and found to be similar in most regards. 2. 2. Erythrocytes of dolphins and man contain similar concentrations of 2,3-diphosphoglyceric acid, ATP and ADP. Dolphin cells contain five times as much DPN and a third as much TPN. 3. 3. Dolphin erythrocytes glycolyze less actively and a greater percentage of the glucose is utilized via the hexose monophosphate shunt. 4. 4. Sodium and potassium concentrations are slightly higher in the dolphin erythrocyte; sodium efflux is less than in the human erythrocyte. 5. 5. No significant differences in the activities of the enzymes of glycolysis, the hexose monophosphate shunt and the phosphoglyceric acid cycle were noted except that dolphin erythrocytes contain several times as much glucose-6-phosphate dehydrogenase. 6. 6. Dolphin hemoglobin migrates on electrophoresis as a single band approximately as human adult hemoglobin and is more alkali stable than human hemoglobin.