Hisaharu Kohzuki

Oxygen affinities (p50) of myoglobins from four vertebrate species (Canis familiaris, rattus norvegicus, mus musculus and Gallus domesticus) as determined by a kinetic and an equilibrium method

Rate constants of the reaction with oxygen of myoglobins from four vertebrate species (Canis familiaris, Rattus norvegicus, Mus musculus and Gallus domesticus) and the isolated alpha A and beta A chains of human adult hemoglobin (HbA) were determined by the stopped-flow-spectrophotomeric method. Half-saturation oxygen pressure (P50) of the proteins calculated from the rate constants, assuming a simple bimolecular reaction model, agreed very well with those directly determined by oxygen equilibria. The proteins used were freshly prepared, and fully characterized by electrophoretic and ultracentrifugal analyses. Sulphydryl groups in the Hb chains were ascertained to be completely regenerated.

In vitro erythropoietin assay based on erythroid colony formation in fetal mouse liver cell culture

Summary. Critical studies were made on erythroid colony formation from cultured fetal mouse liver cells in an attempt to develop a simple and sensitive erythropoietin (Epo) assay procedure. The maximum colony formation was observed 24 h after plating of the cells when an evident dose–response relation was found for Epo added. The colony forming ability decreased steadily as the gestational age of the fetus advanced and was gradually lost by postnatal days 10–11. By morphological and cytochemical criteria almost all the colonies were found to be erythroid. 59Fe‐labelling experiments revealed a fairly good correlation between the colony number and 59Fe incorporation into both cells and haem. Dose–response curves for plasma were parallel to the Epo standard curve. Based on these findings we developed a procedure which could measure as little as 0·4 mU of Epo without requiring 59Fe. Using this method, plasma Epo titres were determined in 16 normal and 69 anaemic subjects.

HB hope, β136(H14)Gly→Asp, in a Diabetic Japanese Female and its Functional Characterization

A beta-variant hemoglobin, first misjudged as a marked elevation of Hb A1, was found in a 68-year-old Japanese female with diabetes mellitus. This hemoglobin was isolated by Bio-Rex 70 chromatography combined with chromatofocusing, and was found to be Hb Hope, beta 136(H14)Gly----Asp, by classical and high performance liquid chromatographic peptide mapping techniques. Intrinsic oxygen affinity of this hemoglobin was approximately one-third as compared with that of Hb A0. This property was still observed in the constituent beta subunits isolated. Effects of such allosteric effectors as H+ (at a fixed concentration of Cl-), anion (Cl-), 2,3-diphosphoglycerate and carbon dioxide were more or less depressed. Among others, a marked reduction in the carbamate effect should be noted in a structural interpretation of the functional modifications. Subunit cooperativity, on the contrary, was not different from that in Hb A0 (n = 2.8-2.9). Explanation of these altered functions were attempted on the basis of the altered structure. The reduced stability of Hb Hope is also described.

Wide Variation of Myoglobin Contents in Gizzard Smooth Muscles of Various Avian Species

We determined myoglobin contents of gizzards (muscular stomach) and breast muscles in 34 avian species by a modification of Reynafarjes spectrophotometric procedure. The birds were apparently differentiated into two groups in respect of the gizzard, one with a high myoglobin content (7.74 +/- 1.81 mg/g muscle) and the other with a low (1.54 +/- 0.41 mg/g). In the former group of 15 species all but one were herbivorous, and all but one were carnivorous or else omnivorous in the latter group of 19 species. The myoglobin level was considered to closely correlate with mechanical performance and therefore oxygen demands of the gizzards. It might also be relevant to a circulatory situation during the tonic contractions of this organs.

Energy expenditure by Ba2+contracture in rat ventricular slices derives from cross-bridge cycling

To clarify the energy-expenditure mechanism during Ba(2+) contracture of mechanically unloaded rat left ventricular (LV) slices, we measured myocardial O(2) consumption (VO(2)) of quiescent slices in Ca(2+)-free Tyrode solution and VO(2) during Ba(2+) contracture by substituting Ca(2+) with Ba(2+). We then investigated the effects of cyclopiazonic acid (CPA) and 2,3-butanedione monoxime (BDM) on the Ba(2+) contracture VO(2). The Ca(2+)-free VO(2) corresponds to that of basal metabolism (2.32 +/- 0.53 ml O(2). min(-1). 100 g LV(-1)). Ba(2+) increased the VO(2) in a dose-dependent manner (from 0.3 to 3.0 mmol/l) from 110 to 150% of basal metabolic VO(2). Blockade of the sarcoplasmic reticulum (SR) Ca(2+) pump by CPA (10 micromol/l) did not at all decrease the Ba(2+)-activated VO(2). BDM (5 mmol/l), which specifically inhibits cross-bridge cycling, reduced the Ba(2+)activated VO(2) almost to basal metabolic VO(2). These energetic results revealed that the Ba(2+)-activated VO(2) was used for the cross-bridge cycling but not for the Ca(2+) handling by the SR Ca(2+) pump.

High affinity of blood for oxygen reduces oxygen uptake in contracting canine gracilis muscle

To clarify the influence of blood flow with high‐oxygen (O2)‐affinity blood on oxygen consumption (VO2) in submaximally exercising skeletal muscle, we perfused the isolated dog gracilis (n = 8) contracting under 1 Hz stimulation alternatively with normal and high‐O2‐affinity blood, with a constant arterial O2 content (Ca,O2) and varying perfusion rates. The average P50 (oxygen partial pressure (PO2) for half‐saturation at pH 7.40, PCO2 of 40 mmHg at 37 degrees C) of the high‐O2‐affinity blood prepared by carbamylation was 15.5 mmHg, and that of the normal blood 33.7 mmHg. With normal blood perfusion, the average VO2 above 6 ml min‐1 (100 g)‐1 of O2 delivery (Ca,O2 x flow) was 4.38 ml min‐1 (100 g)‐1 (range 4.17–4.68 ml min‐1 (100 g)‐1, and VO2 at the O2 delivery range of 6–5 and 4‐2.5 ml min‐1 (100 g)‐1 decreased to 3.96 and 2.43 ml min‐1 (100 g)‐1, respectively. The PO2 of venous effluent (Pv,O2) at the O2 delivery of 6 ml min‐1 (100 g)‐1 was 33 mmHg. With low‐P50 blood perfusion, VO2 was significantly less than with normal blood, both below the O2 delivery level of 6 ml min‐1 (100 g)‐1 and above it, even in the fairly high O2 delivery range of 8.5–12 ml min‐1 (100 g)‐1 (P < 0.05). Thus, high blood flow did not compensate for the reduced VO2 caused by high‐O2‐affinity blood. At values of Pv,O2 less than 33 mmHg, VO2 with low‐P50 blood was not significantly different from that with normal blood (P > 0.05). The reduced VO2 in submaximally exercising skeletal muscle might be due to a slower O2 dissociation from the high‐O2‐affinity red cells and to a limited O2 diffusion resulting from the lower Pv,O2 value (which reflects mean end‐capillary PO2).

Preparation of native α and β subunits from canine hemoglobin

Preparation of native alpha and beta subunits from non-primate hemoglobins has never been successful using the procedure of Bucci, E. and Fronticelli, C. ((1965) J. Biol. Chem. 240, PC551-552). We describe a method for isolating the constituent subunits from canine hemoglobin. Human-canine hybrid hemoglobins (alpha 2Can beta 2A and alpha 2A beta 2Can) were prepared by acid hybridization techniques and DEAE-Sephadex chromatography. The hybrids were then reacted with an excess of p-chloromercuribenzoate under slightly acid conditions and the resultant alpha Can and beta Can subunits were isolated by either anion- or cation-exchange chromatography. Identifications of the subunits were made by gel electrophoresis and amino acid analysis. After removal of the bound mercurials with beta-mercaptoethanol, both subunits exhibited two reactive sulfhydryl groups per chain and were found to be fully in the native form, as judged by spectroscopy. By ultracentrifugal analysis, the alpha subunit was shown to be in a dimer-monomer equilibrium while the beta subunit was largely in a tetrameric form.

Characterization and Quantitation of Glycosylated Hemoglobins in Canine Blood

Canine hemolysates exhibited two minor and one major hemoglobins when chromatographed on Bio Rex-70 column with 0.05 M phosphate-0.01 M KCN (pH 6.5) containing 0.015 M NaCl. The first minor fraction was electrophoretically heterogeneous, constituting at least two hemoglobins. The second was completely homogeneous with somewhat faster anodal mobility than the major one and the difference was shown to be localized solely in the beta subunits. Thiobarbituric acid reaction was strongly positive for this hemoglobin, indicating the presence of glycosyl ketoamine linkage. We describe a simple and reliable procedure for quantitation of the canine glycosylated hemoglobins with a home-made Bio Rex-70 mini-column.

Mixed disulphide formation in myoglobin of mouse (Mus musculus)

The myoglobin, isolated from murine skeletal muscles by chromatofocusing, showed the three components, one major and two minor, with different electrophoretic mobilities. The major component with the isoelectric point (pI) of 7.55 had one reactive SH/mole, while the others with pI of 7.32 and 7.16 showed none, which could be rendered fully reactive by treating the proteins with beta-mercaptoethanol. The three components were the same in their molecular weight (18 kDa), amino-acid composition with one Cys residue and oxygenation properties. By a sensitive high-performance liquid chromatography method, the occurrence of cysteine or glutathione mixed disulphide was verified in the two minor components. We conclude from these results and incubation experiments with low-molecular-weight thiols that the two minors were derived from the major by a mixed disulphide formation with either cysteine or glutathione of the cysteinyl SH at the 66th sequence.

Postnatal transition of γ-globin gene expression in normal Japanese population

Temporal course of postnatal changes in the γ isoform composition of human fetal haemoglobin (Hb F) was studied in 259 cord, 272 infantile and 216 adult Japanese blood samples. Reversed phase high‐performance liquid chromatography was used for determination of the three isoforms of the γ chain (AγT, Aγ1 and cγ), and the adult samples, usually with less than 1% of Hb F, were enriched for Hb F by an alkali denaturation‐salting out procedure. The results show that the cγ gene expression, kept at a higher and strictly‐controlled level in the neonatal period, undergoes a gradual change during 1 year, beginning 3–4 months after birth, to the adult stage which is characterized by a generally lower and loosely‐controlled expression of the gene. Further change seems to occur after 1 year, settling to the final adult level. The AγT gene frequency is estimated as 0‐1 39 in the present Japanese adult population, and is essentially identical to the value for the newborns in the same population (0‐141).