Haruhisa Yoshikawa

The hydrogen ion concentrations and erythrocyte glycolysis

Recently several reports appeared on the control of glycolysis in living cells, notably tumor cells (Wu, 1964) and cerebral tissues (Lowry et al. 1964). In these studies, the glycolytic intermediates of the cells are assayed enzymatically and the controlling points are analyzed by the “crossover theorem” of Chance et aL(1958). Erythrocytes seemed to be one of the most suitable cells for this kind of analysis, as glycolysis is predominant compared to other metabolic activities. The pattern of glycolytic enzymes are systematically studied by Chapman et al. (1962) and some of the intermediates were assayed chromatographically by Bartlett (1959) and Yoshikawa et aL(l960). Hexokinase has been considered as the rate-limiting step in the glycolytic chain of the cells by several investigators (Rapoport et al.,1961, Chapman et al., 1962, Rose and O’Connell, 1964), as the hexokinase activity is the lowest in the glycolytic enzymes and glycolytic rates of the red cells are found to be parallel to those of hexokinase in several conditions, e.g. aging, storage and in different species of animals. The optimum pH for the glycolysis of the cells lies at pH 8.1, which coincides with those of hexokinase. In this paper, the contents of glycolytic intermediates of human erythrocytes except 1,3diphosphoglycerate are reported, especially in relation to the

A bromine compound isolated from human cerebrospinal fluid

Abstract A new bromine compound with properties characteristic of a ketone was isolated from human cerebrospinal fluid. It was degraded by alkali treatment producting acetic acid, glycoli acid, oxalic acid, HBr and 1-methylheptanol. By this treatment, a part of the compound was converted to di-1-methylheptyl-2,5-dioxocyclohexane-1,4-dicarboxylate. These degradation products were also obtained from synthetic 1-methylheptyl-γ-bromoacetoacetate by alkali treatment. 1-methylheptyl-γ-bromoacetoacetate and realted substances were synthesized and compared with the isolated bromine compound in chemical properties, infrared spectra, spectra of nuclear magnetic resonance, and elemental analysis, etc. These results showed that the isolated bromine compound corresponded well to 1-methylheptyl-γ-bromoacetoacetate (synonym of 2-octyl-γ-bromoacetoacetate).

Iron-chelating enzyme from rat liver.

Summary Iron-chelating enzyme was extracted from rat-liver mitochondria. The measurement of the stoichiometry showed that no side reaction occurred. The reaction product was heme, not hemeprotein. Some other properties, such as pH response, Km for iron and protoporphyrin and effect of supernatant are reported.

Iron-chelating enzyme from duck erythrocytes.

Abstract The iron-chelating enzyme, which combines iron and protoporphyrin, was obtained from duck erythrocytes free from hemoglobin. With this enzyme the net heme synthesis was observed and the measurement of the stoichiometry of the reaction showed the absence of a side reaction. It was proved that the reaction product was protoheme, but the formation of hemoglobin was not found. The enzyme had a broad specificity towards 2 carboxylic acid porphyrins, and it was observed that it formed responding hemes with these. On the other hand, porphyrins with 4 or 8 carboxylic groups showed practically no reactions of this kind. Ferrous ion was the most efficient of the metallic ions tested.

Use of sodium borohydride in determination of oxygen dissociation curves of hemoglobin

Abstract Sodium borohydride was used for determination of the oxygen dissociation curves of hemoglobin. The usefulness of the reagent for the purpose was discussed. Data obtained by the method were in good agreement with those of other workers.

Stimulation of amylase formation by an amine from Bacillus subtilis

An active factor which stimulates amylase formation by Bacillus subtilis has been extracted from B. subtilis cells and purified. The factor resembles alkyldiamines or polyalkylamines in its chromatographic behavior, its reaction with ribonucleic acids and other types of reactions. Authentic samples of amines such as putrescine, cadaverine, spermidine and spermine have been found to stimulate amylase formation at high concentrations. None of them however is identical with the factor. The factor also stimulates the production of other exo-enzymes of B. subtilis, e.g., proteases, ribonuclease, and γ-glutamyltransferase. Moreover it stimulates the turnover of RNA and polyphosphate in the cells to a greater extent than it stimulates enzyme production. In vitro experiments showed that in the presence of the factor, RNA associates to larger molecules than those of the original RNA. The chemical structure of the factor and its biological functions are discussed.

Studies on Phenylalanine Metabolism by Use of Tracer Techniques

安定同位体トレーサー法をアミノ酸代謝研究に導入する目的で, まず重水素標識L-フェニルアラニンを合成した。ベンズアルデヒド〔芳香環-d5〕を原料とし, アセチルグリシン縮合, 接触還元, アミノアシラーゼによるラセミ分割を経て, D-およびL-フェニルアラニン〔芳香環-d5〕をそれぞれ収率約18%で得た。ついで本品をGC-MSで測定するために, このエナミソメチルエステルを調製し, この重水素標識体と非標識体との間のMS.およびGCにおける同位体効果をしらべた。MSにおいては開裂彩式において同位体効果はなく, GCにおいては保持時間が標識体のほうが短いことが明らかとなった。

Xylit als Substrat für Methämoglobinreduktion in roten Blutkörperchen

SummaryThe rate of reduction of methemoglobin in fresh erythrocytes in a glucose medium, ranging in concentration from 50–500 mM glucose, is constant; however, in a xylit medium the reduction is dependent upon the concentration of the xylit. The highest rate appears at a substrate concentration of 200 mM xylit. Upon addition of glucose, erythrocytes, which were stored in an acid-citrate-dextrose medium for more than five weeks, barely reduce methemoglobin, whereas upon addition of xylit they still reduce methemoglobin well.ZusammenfassungDie Geschwindigkeit der Reduktion von Methämoglobin in frischen Erythrocyten ist im Glucosemedium im Konzentrationsbereich von 50–500 mM Glucose konstant, im Xylitmedium jedoch von der Konzentration des Xylit abhängig. Die größte Geschwindigkeit tritt bei einer Substratkonzentration von 200 mM Xylit auf. Erythrocyten, die mehr als 5 Wochen in Citronensäure-Dextrose-Medium aufbewahrt wurden, können bei Zugabe von Glucose kaum, bei Zugabe von Xylit noch gut Methämoglobin reduzieren.

Formation in vitro of haemoglobin and myoglobin from iron, protoporphyrin and globin in the presence of an iron-chelating enzyme

Abstract 1. 1. The formation of haemoglobin has been demonstrated in the presence of iron, protoporphyrin, iron-chelating enzyme and apohaemoglobin from duck erythrocytes. Chromatographic as well as spectrophotometric evidence is presented for this. 2. 2. Apohaemoglobin is effective when added to the incubation mixture either before, or after, the incubation. 3. 3. Spectrophotometric evidence of the formation of myoglobin, has been obtained in the same way using apomyoglobin from the myoglobin of horse heart. 4. 4. The presence of apohaemoglobin or of apomyoglobin inhibits the rate of haem formation. 5. 5. The mechanism of the formation of haemoglobin is discussed.

Utilization of Xylitol in Human Erythrocytes

Mature human erythrocytes synthesize no DNA, RNA, heme or protein. They possess no citric acid cycle or electron transfer system, and obtain the energy mainly from carbohydrate metabolism through the Embden-Meyerhof and Warburg-Dickens pathways.