X-ray diffraction and flash photolysis studies of M intermediate of lattice-contracted purple membrane

The effects of cross-linking and lattice contraction of purple membrane (PM) on the photodynamics of bacteriorhodopsin (bR) and on the tertiary structure were studied by flash photolysis and X-ray diffraction. To get a contracted lattice form of PM, native PM, and/or PM cross-linked by glutaraldehyde were treated with deoxycholate or Triton X-100. Part of the Triton-treated cross-linked PM was further incubated with Bio-Beads SM-2 to remove Triton X-100. In the modified PM, several long-lived components of the M intermediate appeared, the features of which were related to the environment of bR. Also, X-ray diffraction studies using synchrotron radiation were performed on the modified PM under intense light irradiation (lambda greater than 500 nm) in which 40-80% of bR was photoconverted to the M state. In the Triton-treated cross-linked PM dispersed in 0.25% Triton X-100, the unit cell of membrane crystalline lattice was enlarged from 58.8 to 59.8 A and the crystalline order decreased with irradiation. The analysis of X-ray diffraction patterns suggests that light-induced conformational changes of bR correlated with the Triton content of the environment and an increase of substitution disorder was caused by these changes, but the average location of bR was unchanged. However, the other modified PM showed no significant changes of diffraction, upon light irradiation.     

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase: effect of substrates on the sensitivity of the enzyme to trypsin and identification of tryptic cleavage sites

The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner mitochondrial membrane. The enzyme (1043 residues) is composed of an N-terminal hydrophilic segment (approximately 400 residues long) which binds NAD(H), a C-terminal hydrophilic segment (approximately 200 residues long) which binds NADP(H), and a central hydrophobic segment (approximately 400 residues long) which appears to form about 14 membrane-intercalating clusters of approximately 20 residues each. Substrate modulation of transhydrogenase conformation appears to be intimately associated with its mechanism of proton translocation. Using trypsin as a probe of enzyme conformation change, we have shown that NADPH (and to a much lesser extent NADP) binding alters transhydrogenase conformation, resulting in increased susceptibility of several bonds to tryptic hydrolysis. NADH and NAD had little or no effect, and the NADPH concentration for half-maximal enhancement of trypsin sensitivity of transhydrogenase activity (35 microM) was close to the Km of the enzyme for NADPH. The NADPH-promoted trypsin cleavage sites were located 200-400 residues distant from the NADP(H) binding domain near the C-terminus. For example, NADPH binding greatly increased the trypsin sensitivity of the K410-T411 bond, which is separated from the NADP(H) binding domain by the 400-residue-long membrane-intercalating segment. It also enhanced the tryptic cleavage of the R602-L603 bond, which is located within the central hydrophobic segment. These results, which suggest a protein conformation change as a result of NADPH binding, have been discussed in relation to the mechanism of proton translocation by the transhydrogenase.     

Potentiation by BL191 of differentiation of neuroblastoma cells induced by dibutyryl cAMP and prostaglandin E1

BL191, a newly developed phosphodiesterase inhibitor, markedly potentiated a differentiation of neuroblastoma cell clones (Neuro2a, NS-20Y, and N1E115) induced by dibutyryl cyclic adensoine 3′:5′-monophosphate(dibutyryl cAMP) and prostaglandin E₁ (PGE₁). BL191 (1 mM) inhibited DNA synthesis more strongly when used together with PGE₁ (0.5 μg/ml) and dibutyryl cAMP (0.5 mM) than papaverine (1.6 μg/ml) alone did. The inhibition rates of DNA synthesis were 72.5% for N1E-115, 75.3% for Neuro2a, and 82.5% for NS-20Y. After the treatment with BL191. PGE₁, and dibutyryl cAMP for 48 h all of three cell lines became enlarged and flattened, and extended long processes. The specific activities of choline acetyl transferase (EC 2.3.1.9) of NS-20Y and dopamine β-hydroxylase (EC 1.14.17.1) of N1E-115 increased about 3-fold as compared to the controls. The tumorigenicities of Neuro2a and N1E-115 cells were decreased, but not of NS-20Y. These data suggest the heterogenous responsiveness in neuroblastoma cells to drug treatment.     

Effects of Phosphate in Medium on Protein Secretion in a Protein-Producing Bacterium, Bacillus brevis 47

Bacillus brevis 47 secreted up to 1 mg of protein per ml in a chemically defined medium, depending on phosphate concentration. The composition of exoproteins was altered quantitatively by the concentration of external phosphate. Morphologically, B. brevis 47 showed a distinct three-layered cell wall structure and shed the outer two layers during growth.     

Species specificity of radioreceptor assay and radioimmunoassay for rat FSH

Species specificity of the radioreceptor assay (RRA) for rat FSH, in which pregnant mare serum gonadotropin (PMSG)-treated immature rat ovary was employed as the receptor, was compared with that of NIAMDD rat FSH radioimmunoassay (RIA). In the RIA system, pituitary preparations from mammals only showed significant crossreaction. Their inhibition curves, however, were not always parallel to the standard curve. On the other hand, in the RRA system, the pituitary preparations from mammals, avians, lizard and amphibians competitively inhibited the binding of radioactive rat FSH to the ovarian receptor. Only the pituitary preparation from dog salmon failed to show any crossreaction in the RRA system. These results indicated that this RRA system would be useful for the measurement of FSH or gonadotropins of the pituitaries from mammals to amphibians.     

A case of histiocytosis X associated with panhypopituitarism and hyperimmunoglobulinemia G and E.

A 43 year old man with diabetes insipidus who showed panhypopituitarism and marked hypergammaglobulinemia due to histiocytosis X is reported. His low basal plasma adrenocorticotropin (ACTH) and growth hormone (GH) failed to respond to insulin-induced hypoglycemia. His basal serum thyroid hormone level was below normal and normal basal plasma thyrotropin (TSH) showed a delayed response with normal peak value to TSH-releasing hormone (TRH). Normal basal plasma pituitary gonadotropin also showed a delayed response with normal peak value to luteinizing hormone-releasing hormone (LH-RH). Suppression of plasma prolactin (PRL) by levodopa (l-dopa) was impaired and elevation of basal plasma PRL was noted at the second admission. These results, combined with diabetes insipidus, suggested that the panhypopituitarism in these patients was hypothalamic in origin. The polyclonal hypergammaglobulinemia was characterized by elevated serum IgG and IgE levels which returned to normal after corticosteroid treatment with concomitant clinical improvement. Elevated serum IgE levels, tissue and peripheral eosinophilia, and the effectiveness of corticosteroid therapy support the hypothesis that some allergic mechanism may be involved in the pathogenesis of this disease.     

An increase of pituitary 3', 5' cyclic adenosine monophosphate produced by estradiol benzoate in vitro: possible implication of this increase in the secretion of luteinizing hormone.

In an attempt to study the site and mechanism of action of estrogen in producing positive feedback control, porcine anterior pituitary slices were incubated in vitro in the presence of estradiol benzoate (EB). EB elevated pituitary cyclic AMP concentration within 5 min and augmented pituitary release of luteinizing hormone (LH). The magnitude of increase of cyclic AMP and LH release was related to the doses of EB used. Also, luteinizing hormone releasing hormone (LH-RH) elevated pituitary cyclic AMP concentration and stimulated pituitary release of LH. The magnitude of increase of cyclic AMP and LH release was inversely related to the doses of LH-RH used. EB and LH-RH were additive in increasing cyclic AMP. Progesterone and clomiphene citrate interfered with an increase of pituitary cyclic AMP produced by EB, but did not significantly affect the basal level of pituitary cyclic AMP. Testosterone propionate, human chorionic gonadotropin and hexestrol were without effect on either basal or stimulated level of pituitary cyclic AMP. Since cyclic AMP and dibutyryl cyclic AMP (DBC) stimulated LH release, it is suggested that EB directly stimulates the release of LH by augmenting cyclic AMP synthesis in the anterior pituitary.     

Pumpkin (Cucurbita sp.) seed globulin I. Purification, characterization, and subunit structure

A heat stable globulin present in the cotyledons of pumpkinseeds was prepared as crystals which were soluble in a dilutesaline solution below pH 4.5 or in a solution with a high ionicstrength at neutral pHs. The protein was nearly homogeneousby ultracentrifuge analysis, and had a molecular weight of about112,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisseparated the globulin into two subunits, and ß,corresponding to molecular weights of about 63,000 and 56,000daltons, respectively. By reduction of disulfide bonds, thetwo subunits were each separated into two polypeptide chainswith molecular weights of around 36,000 and 22,000 daltons,judged by gel electrophoresis. The amino acid composition ofwhole globulin indicated high contents of arginine, glutamicacid and aspartic acid. The total number of half-cystine residuewas nine and only one residue was shown to be free. The subunitstructure of the globulin is discussed. The protein has beenshown to have oxaloacetate decarboxylase activity, and thisfact was confirmed. However, the activity decreased markedlyat pH 4.5 in a fairly short period. It did not require Mn⁺⁺,and the K_m for oxaloacetate was determined to be 4.1 mM. (Received April 9, 1976; )     

Effects of x-ray irradiation on the subsequent gonadotropin secretion in normal and neonatally estrogenized female rats.

Female rats were irradiated with 190R of X-rays at 10 days of age and sacrificed 4, 7 or 12 months later. Their ovaries were histologically examined and serum levels and pituitary contents of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined by radioimmunoassay. Both serum levels and pituitary contents of LH and FSH rose significantly 4 and 7 months after irradiation, although the ovaries were markedly reduced in weight. On the contrary, 12 months after irradiation, the ovaries increased in weight and consisted mostly of polyhedral, hyperplastic interstitial cell masses, and both LH and FSH in the serum and pituitary were reduced to normal levels. These characteristic changes in the ovarian weight and histological appearance could not be observed in the similarly irradiated animals which were received daily injections of estrone for the first 30 days of postnatal life, i.e., daily injections of 50 mug for the first 10 days, 100 mug for the middle 10 days and 200 mug for the last 10 days. Serum LH levels of the estrogenized irradiated rats at 7 or 12 months of age did not elevate although those of FSH were significantly higher than the non-irradiated intact levels. From these results, a rise in the blood levels of LH and the FSH may be attributed to the increase in weight and the histological changes in the ovaries of the irradiated female rats, and the elevation of only FSh level may not result in the abnormal growth of the irradiated ovaries.