Purification and Some Properties of Soluble Cytochromes, Cytochrome c-551 and Cytochrome c-550, from a Facultative Methylotroph, Protaminobacter ruber strain NR-1

Two soluble cytochromes of the C-type, cytochrome c-551 andcytochrome c-550, were purified from the bacteriochlorophyll-containingcells of a facultative methylotroph, Protaminobacter ruber StrainNR-1, by ion-exchange chromatography and gel-filtration. Cytochrome c-551 had absorption maxima at 551, 522 and 416 nmin the reduced form, and at 525, 410 and 273 nm in the oxidizedform. This cytochrome was a slightly basic protein with an isoelectricpoint of 8.4. It had a mid-point redox potential of 272 mV atpH 7.0. The molecular weight of this protein was 13,500 and13,700 by sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE) and gel-filtration, respectively. Cytochrome c-550 had absorption maxima at 550, 522 and 415 nmin the reduced form, and at 527, 409 and 278 nm in the oxidizedform. This cytochrome was acidic, having an isoelectric pointof 4.3. It had a mid-point redox potential of 227 mV at pH 7.0.Its molecular weight was 19,500 and 22,000 by SDS-PAGE and gel-filtration,respectively. (Received August 4, 1984; Accepted October 22, 1984)     

Spinach nitrate reductase: purification, molecular weight, and subunit composition

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.     

Modulation of postinhibitory increase in plasma prolactin by domperidone in patients with prolactin secreting adenoma

To evaluate the PRL secretory mechanism in patients with PRL-secreting adenoma (PRL-oma), plasma PRL responses to dopamine (DA) were studied in these cases and in normal subjects. Plasma PRL values showed clear decreases during the infusion of DA (5 micrograms/kg/min for 90 min) in both 6 normal and 7 PRL-oma subjects (%decrease: 43.8 +/- 3.9% vs. 53.9 +/- 5.6%; NS) and postinhibitory increases after the termination. However, the postinhibitory increase occurred more promptly and markedly in PRL-oma patients than in normal subjects, i.e. the postinhibitory increase exceeded the basal level 45 min after the termination of DA infusion in PRL-oma patients, whereas the increase in normal subjects did not exceed the basal level even 90 min after the infusion. When domperidone was injected at the termination of DA infusion, the postinhibitory increases were significantly enhanced in either PRL-oma or normal subjects. The maximal increments in plasma PRL in the combination test of DA plus domperidone were significantly larger in PRL-oma patients, but were almost the same in normal controls, compared to the single domperidone test. In contrast, TRH did not modify the postinhibitory rises in 9 PRL-oma patients. These results indicate that the secretory properties and the sensitivities of lactotrophs to decreasing action of DA might be different between PRL-oma patients and normal controls. Further, the postinhibitory rebound phenomenon in PRL-oma patients is possibly determined by an overshoot of PRL storage concomitantly with a decreasing DA action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of mibolerone to androgen receptor of benign hypertrophic human prostate. Comparison with R1881

The binding nature of mibolerone in cytosols and nuclear extracts from hypertrophic human prostate was examined in comparison with that of R 1881. The binding of mibolerone in the cytosol and nuclear extract was single and of high affinity when evaluated by the method of Scatchard (1949). Binding of mibolerone with testosterone-binding globulin was not detected. The sedimentation coefficients of the binder for mibolerone in the cytosol and nuclear extract were 10.6 S and 3.6 S, respectively. When triamcinolone acetonide was induced in the binding medium, inhibition of mibolerone binding in the cytosol by testosterone and dihydrotestosterone was potentiated and this may imply that the binding observed in the presence of triamcinolone acetonide was responsible for the binding of the androgen receptor. In the nuclear extract, the binding was attributable mainly to the androgen receptor irrespective of the presence or absence of triamcinolone acetonide. These properties of the binding observed in the hypertrophic human prostate were almost same as those of the binding with R 1881. Although maximum binding sites measured using mibolerone were correlated with those using R 1881 in the cytosols as well as in the nuclear extracts, the values obtained with mibolerone were slightly greater than those with R 1881. Thus, mibolerone seems to be a suitable ligand for measuring the androgen receptor, but when compared with R 1881 no special merits in using mibolerone were detected.     

Long-term survival of functional hepatocytes from adult rat in the presence of phenobarbital in primary culture

In the presence of phenobarbital (PB) at 3 mM, hepatocytes isolated from adult rats by a collagenase-perfusion technique survived well on plastic dishes for at least 49 days after initiation of primary culture. PB at concentrations less than 3 mM was ineffective for the maintenance of hepatocytes, and the maintenance of them was attained only in the continuous presence of 3 mM PB. The hepatocytes surviving in the presence of 3 mM PB were morphologically indistinguishable from the hepatocytes after 1-day attachment period, except for the presence of prominent nucleoli in the former. Although both the albumin secretion and tyrosine aminotransferase (TAT) activities of the cells decreased gradually up to day 7 with time in culture, both were thereafter maintained at relatively high levels at least up to day 35 of primary culture. The addition of 10 microM dexamethasone caused a 3-5-fold induction in TAT activity, and the cells were capable of responding to the hormone in this manner at least up to day 28 of primary culture. Furthermore, the cells also had glucose-6-phosphatase activity, even though the level of this enzyme activity was relatively low as compared with that of TAT activity. Survival of hepatocytes in the presence of 3 mM PB was further enhanced by simultaneous addition of dexamethasone (10 microM) and insulin (10 micrograms/ml). The sensitivity of hepatocytes to 3'-methyl-4-dimethylaminoazobenzene (0.24 mM) was remarkably reduced by treatment with PB at 3 mM. PB treatment decreased efficiently the falling rate of total cytochrome P-450 content, but did not induce P-450PB, which is the specific form of cytochrome P-450 induced by PB, in primary cultured hepatocytes. On the other hand, 3-methylcholanthrene (MC, 10 microM) caused an increase of both contents of total cytochrome P-450 and P-450MC, which is the specific form of cytochrome P-450 induced by MC, in primary cultured hepatocytes. However, MC was ineffective for the maintenance of hepatocytes in primary culture. The possible biological actions of PB on primary cultured hepatocytes are discussed on the basis of the experimental data obtained.     

Nucleotide sequence of the gene determining plasmid-mediated citrate utilization.

The citrate utilization determinant from transposon Tn3411 has been cloned and sequenced, and its polypeptide products have been characterized in minicell experiments. The nucleotide sequence was determined for a 2,047-base-pair BglII restriction endonuclease fragment that includes the citrate determinant. This region contains an open reading frame that would encode a 431-amino-acid very hydrophobic polypeptide and which is preceded by a reasonable ribosomal binding site. However, the single polypeptide found in minicell experiments had an apparent molecular weight of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Isolation and characterization of a monoclonal antibody against pig kidney sodium- and potassium-activated ATPase

A hybridoma cell line producing mouse monoclonal antibody against pig kidney Na,K-ATPase was established. The antibody, named 38 (mAb38, IgG1), was purified from mouse ascites fluid by chromatography on a protein A-Sepharose column. Antigens immobilized on microplate wells with p-benzoquinone were used for titer assays. mAb38 cross-reacted with both dodecyloctaethyleneglycol monoether (C12E8)-solubilized enzyme and membranous sodium dodecyl sulfate (SDS)-treated enzyme from kidney with high affinity (50% binding = 0.6 nM). However, the antibody bound to neither alpha- nor beta-subunit separated by preparative SDS-polyacrylamide gel electrophoresis (PAGE). The stoichiometry of antibody binding to the purified enzyme was estimated to be about 0.86 mol of IgG per mol of alpha beta-protomer. Na,K-ATPase proteins were recovered from a column of mAb38-coupled Affi-Gel by elution with pH 3 buffer when C12E8-solubilized kidney enzyme or detergent extracts of brain microsomes were applied to it, confirming that the mAb is directed to Na,K-ATPase. mAb38 at saturation level concentrations had no effect on kidney Na,K-ATPase activity or on ouabain-sensitive Rb uptake in erythrocytes. In an immunofluorescence study, the antibody bound to intact erythrocytes much more strongly than control IgG1 (mAb50c), but the extent of the antibody binding to inside-out vesicles under hypotonic conditions was lower than that of the control. Most of the antibody binding activity remained when the kidney enzyme was treated with sialidase. These results suggest that this mAb38 was raised against an intact conformation of a cell-surface-exposed site of Na,K-ATPase.     

In vitro establishment of human fibroblasts of lysosomal diseases,GM1-gangliosidosis and Sandhoff disease,by transformation with origin-minus SV40 DNA

The permanent human cell lines preserving defects of lysosomal enzymes, G_M1-1019-SV and SA-1077-SV, were established from the respective fibroblasts from patients with G_Ml-gangliosidosis and Sandhoff disease by transfection with replication origin-minus simian virus 40 DNA. These ceils grow rapidly without entering senescence during more than 120 population doublings. The activity of -galactosidase in G_M1-1019-SV and of B-N-acetylhexosaminidase in SA-1077-SV was respectively 40- and 180-fold lower than that of normal fibroblasts.     

Arterial perfusion of frog tongue for intracellular recording of taste cell receptor potential

The frog tongue was perfused through its artery with a Ringer solution using a peristaltic pump, and a method was developed to record stable intracellular receptor potentials of taste cells. Perfusing at 0.05 ml/min with a Ringer solution containing 5% dextran did not cause tongue edema, but perfusing at the same rate with Ringer without dextran caused edema. After perfusion at 0.05 ml/min with 100 mM K Ringer, the membrane potential of taste cells gradually decreased and reached a constant level in about 30 min, indicating that the intercellular fluid of the tongue could be replaced within this time period. While the artery of the frog tongue was perfused at 0.05 ml/min with Ringer containing 5% dextran, intracellular receptor potentials of taste cells elicited by four basic taste stimuli (1 M NaCl, 10 mM quinine-HCl (Q-HCl), 1 mM acetic acid and 1 M galactose) were similar to those obtained from the control taste cells under normal blood flow.