Masato Ohe

Ligand-dependent polymerization of tetrameric hemoglobin from the blood clam Anadara broughtonii

Hemoglobin (Hb II) of the blood clam Anadara broughtonii has a alpha 2 beta 2 sub-unit structure in athe oxy form with a sedimentation constant of 4.8 S. When deoxygenated, Hb II polymerizes with a major component, S20,w = 11.5 (above 150 microM in heme). Deoxy polymerization was not observed in a highly diluted protein below 20 microM (in heme). Gel filtration of Hb II in the deoxygenated state indicated that the major component has an apparent molecular weight of 195 000, which corresponds to a dodecamer. However, the sedimentation pattern and the elution profile of gel filtration showed the polymerization to be somewhat asymmetric. These results suggest that deoxy Hb II may polymerize with different polymerization states. We examined oxygen equilibria of Hb II in a range of 3--180 microM (in heme). Influences of the polymerization on its oxygen affinity and cooperativity were found to be very small. We have also found that the deoxy polymerization was completely prevented when all the sulfhydryl groups of the hemoglobin molecule were modified with p-chloromercuribenzoate.

Ligand-dependent allosteric transformation of hemoglobins from the blood clam, Anadara Broughtonii

1. Oligomeric Hb I and II of Anadara broughtonii, which are unusual with respect to having no Bohr effect, were shown to have a R-T transformation on ligand-binding on the basis of the following experimental results. (a) Iodoacetamide reacted preferentially with the oxyiforms of the hemoglobins. (b) CD spectra at the far-ultraviolet regions significantly changed on ligand-binding. (c) 1-Anilinonaphthalene-8-sulfonate bound to the hemoglobins with a preference for deoxyforms. From these results and previous findings [1], it is concluded that the absence of the Bohr effect in these hemoglobins is due to the lack of the Bohr proton ionizing groups in the molecules. 2. Hb I and II treated with p-chloromercuribenzoate, designated as PMB-I and PMB-II, showed greatly increased oxygen affinity and decreased cooperativity. CD spectra at the far-ultraviolet of the PMB-Hb in the oxygen liganded state gave similar patterns to those of native oxygenated Hb. However no changes in the spectra were observed on deoxygenation. These findings suggest that the PMB-I and PMB-II retain their native oxy conformation even in the deoxy states. The PMB-modification might prevent the initial ligand-induced conformational change within the protomers.

Putative occurrence of lysine decarboxylase isoforms in soybean (Glycine max) seedlings.

The activity of lysine decarboxylase was studied in 3-day-old soybean (Glycine max (L.) Meer cv. Sakai) seedlings also in relation to light conditions. Lysine decarboxylase activity was mainly localized in the roots and to a lesser extent in the hypocotyls and was detectable in both the soluble and particulate fractions. The enzyme activity levels were similar during germination under light and dark conditions. With respect to lysine concentration, the initial decarboxylation rate of the soluble fraction showed a saturating curve. Conversely, the initial decarboxylation rate of the particulate fraction showed a sigmoidal curve. These results could suggest that at least two isoforms of lysine decarboxylase are present in different organs of soybean seedlings. In the root soluble fraction, the suicide inhibitor α-difluoromethyl-lysine suppressed the activity of lysine decarboxylase and of ornithine decarboxylase to the same extent, but had no effect on arginine decarboxylase activity.

Apoptosis in the Epididymal Epithelium of Adult Male Golden Hamster Exposed to Diethylstilbestrol

Apoptosis in the testis and prostate exposed to disrupters of endocrine function, including diethylstilbestrol (DES), during neonatal or postnatal periods has repeatedly been demonstrated, but not in the mature epididymis. We investigated the effects of DES, a potent and synthetic estrogen, on apoptosis in the adult. Adult male golden hamsters received an SC injection of DES and were then sacrificed to collect epididymides after 1, 4, or 7 days of treatment. A significant decrease in epididymal weight and an increase in apoptotic cells were shown on the first day after DES injection. Flow cytometry showed that DES treatment (1 mg/kg) for 1, 4, or 7 days induced significant apoptosis both in the caput and the cauda epididymides. Greater numbers of apoptotic cells were detected in the caput than in the cauda at a fixed time after DES treatment. Serum levels of testosterone decreased markedly within 24 hr after DES administration, reaching undetectable levels of 0.1 ng/ml at 4 days and thereafter. These results indicate that DES administration can increase epididymal apoptosis with a decrease in serum testosterone levels. Because DES used to be injected into domestic animals, adult males also have a chance to take this substance through food. Our study indicates that exposure to DES in adults is as toxic as that in the perinatal period.

Cadaverine turnover in soybean seedlings using 15N-labelled lysine and cadaverine.

The synthesis and translocation of the diamine cadaverine during soybean (Glycine max L. Meer cv. Sakai) germination were studied using 15N-labelled lysine (the cadaverine precursor) and 15N-labelled cadaverine, both under light/dark (12 h/12 h) and total dark germinating conditions. 15N-cadaverine and non-labelled polyamines were simultaneously detected using ionspray ionization-mass spectrometry. Both 15N-cadaverine and 15N-lysine were taken up by soybean. 15N-lysine was transported to the shoot and root and converted into 15N-cadaverine, whereas relatively little 15N-cadaverine was formed from 15N-lysine in the cotyledon. The acropetal translocation of 15N-cadaverine from the cotyledon to the shoot seemed to predominate over basipetal transport to the root. Although no other 15N-derivatised polyamines were found, supplying exogenous 15N-lysine seemed to indirectly affect the metabolism of 14N putrescine, spermidine and spermine, while no significant effect was detected after supplying 15N-cadaverine.