Purification of a flavoprotein having NADPH-cytochrome c reductase and transhydrogenase activities from Nitrobacter winogradskyi and its molecular and enzymatic properties

A flavoenzyme which showed NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase EC 1.6.2.4) and transhydrogenase (NADPH-NAD⁺ oxidoreductase, EC 1.6.1.1) activities was purified to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The reductase was a flavoprotein which contained one FAD per molecule but no FMN. The oxidized form of the enzyme showed absorption maxima at 272, 375 and 459 nm with a shoulder at 490 nm, its molecular weight was estimated to be 36,000 by SDS polyacrylamide gel electrophoresis, and the enzyme seemed to exist as a dimer in aqueous solution. The enzyme catalyzed reduction of cytochrome c, DCIP and benzylviologen by NADPH, oxidation of NADPH with menadione and duroquinone, and showed transhydrogenase activity. NADH was less effective than NADPH as the electron donor in the reactions catalyzed by the enzyme. The NADPH-reduction catalyzed by the enzyme of N. winogradskyi cytochrome c-550 and horse cytochrome c was stimulated by spinach ferredoxin. The enzyme reduced NADP⁺ with reduced spinach ferredoxin and benzylviologen radical.Abbreviations DCIP dichlorophenolindophenol - Tris trishydroxy-methylaminomethane - Mops 3-(N-morpholino) propanesulfonic acid - SDS sodium dodecylsufate     

Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA.

The expression of human basic fibroblast growth factor (bFGF) cDNA in mouse BALB/c 3T3 clone A31 cells induced morphological transformation. These transformed cells grew well and reached more than a sixfold-higher saturation density than parental A31 cells even in serum-free medium. They were able to form colonies in soft agar. The phenotypic alteration in the transformed cells was reversed by the addition of anti-human bFGF antibodies to the medium. These results suggest that the cellular transformation mediated by bFGF is caused by autocrine stimulation with secreted bFGF molecules.     

Inhibition of rat brain adenylate cyclase activity by benzodiazepine through the effects on Gi and catalytic proteins

The effect of benzodiazepines on adenylate cyclase system was examined in rat brain. Micromolar concentrations of diazepam inhibited the enzyme activity in synaptic membranes in dose- and time-dependent manners. The inhibitory effect of diazepam was more evident on the enzyme activity in the presence of guanylyl-5'-imidodiphosphate (GppNHp) or NaF-AlCl3 than on that in the basal state. In the pertussis toxin-treated membranes, the effect of diazepam in the presence of GppNHp or NaF-AlCl3 was markedly suppressed. In addition, other benzodiazepines, such as medazepam, flurazepam, flunitrazepam, and clonazepam, had similar effects to those of diazepam, whereas Ro15-1788, an antagonist of a high affinity receptor in the central nervous system, had no effect on adenylate cyclase activity and did not antagonize the effect of diazepam. These findings indicate that benzodiazepines inhibit rat brain adenylate cyclase activity through the effects on both a low affinity benzodiazepine receptor coupled with the inhibitory GTP-binding regulatory protein (Gi) and catalytic protein.     

The standardization of the candidate national standard anti-D blood-typing serum with the Groupamatic MG50 system

The utility of the Groupamatic MG50 system for quantitative expression of agglutination in terms of continuous response was studied. By use of the characteristics of the design of this system, in which the change in voltage reflects the degree of agglutination, a linear regression relationship between the log ratio of light flux obtained by transformation of the voltage and the log dilution factor of the serum was demonstrated. The availability of the parallel line assay method to the standardization of the blood grouping antisera was also described.     

Effect of metabolic acidosis on phosphate transport by the renal brush-border membrane

Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (P_i) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH₄Cl (acidosis). At killing, blood pH and plasma bicarbonate were $\text{7.36 ± 0.01}$ and $\text{21.8 ± 0.8}\text{mequiv./l}$, respectively, in control and $\text{7.12 ± 0.03}$ ($\text{P < 0.01}$) and $\text{11.1 ± 1.2}$ ($\text{P < 0.01}$) in acidotic rats. Serum P_i was similar in both groups, while 24 h urine P_i excretion was higher in the acidotic group ($\text{P < 0.01}$). Peak sodium-dependent uptake of P_i, measured after 1.5 min of incubation, was higher in controls than acidotic rats ($\text{4442 ± 464}$ vs. $\text{2412 ± 259}\text{pmol/mg}$ protein, $\text{P < 0.01}$), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for P_i and glucose uptake were similar in the two groups. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for P_i uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits P_i uptake by the brush border of the proximal tubule by decreasing the availability of P_i carriers of the renal brush-border membrane.     

Cadaverine Involved in Urate Degrading Activity (Uricase Activity) in Soybean Radicles

Cadaverine, a 5-carbon diamine, was identified as the cofactorof uricase activity previously found in soybean seedlings. Thesubstance purified from freeze dried hypocotyls was subjectedto liquid chromatography, mass spectrometry, ¹H- and ¹³C-nuclearmagnetic resonance spectrometry for identification. The concentrationsof cadaverine in 3-day-old radicles and hypocotyls were 2.37mM and 5.09 mM, respectively. Other polyamine concentrationswere low. Biogenic polyamines (cadaverine, putrescine, spermidineand spermine) functioned as cofactors, whereas conjugated polyamines(tyramine and histamine) and amino acids had no effect. Theaddition of catalase to the assay system counteracted the effectof cadaverine. Peroxide at appropriate concentrations actedlike cadaverine with an identical K_m value, suggesting thaturate degrading activity can be ascribed to the diamine oxidase-peroxidasesystem. (Received October 19, 1982; Accepted December 23, 1982)     

Expression of human immunoglobulin E epsilon chain cDNA in E. coli.

Using the cDNA of human epsilon chain, three expression plasmids that code directly the constant portion of the epsilon chain (C epsilon 1-C epsilon 4, C epsilon 2-C epsilon 4 and C epsilon 3-C epsilon 4 domains) were constructed. These epsilon chain peptides were synthesized in E. coli under the control of the trp promoter-operator. The bacterially produced peptides have the antigenicity of human epsilon chain and gave the molecular weights equal to the values calculated from the amino acid sequence of the constructed plasmids.