The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5

The role of cytochrome $\text{b}$₅ in the $\text{p}$-nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome $\text{b}$₅. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome $\text{b}$₅ reductase, and Triton X-100 in addition to cytochrome $\text{b}$₅. The omission of cytochrome $\text{b}$₅ from the complete system entirely abolished the activity. These results clearly show that cytochrome $\text{b}$₅ is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome $\text{b}$₅ in such a way that the second electron is transferred from cytochrome $\text{b}$₅ and thus exhibits the demethylase activity.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins,pigment System II particles and chlorophyll a in diethylether solution by the curve-fitting method

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25°C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol. 49, 421–429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm.The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0–2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6–4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively.Absorption spectra at 25°C and at ?196°C of the water-soluble chlorophyll proteins were compared by the curve-fitting method. The component bands at ?196°C were blue-shifted by 0.8–4.1 nm and narrower in half widths as compared to those at 25°C.     

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase.

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.     

Cytokinin activity of O6-substituted guanine and hypoxanthine derivatives

O⁶-Substituted guanine and hypoxanthine derivatives were prepared and tested for their cytokinin activity by the tobacco callus, radish cotyledons and lettuce seed bioassay systems. The results indicated that some derivatives of both types possess cytokinin activity.     

The location of ribosomal cistrons (rDNA) in chromosomes of the rat

In situ hybridization of ³H-labelled ribosomal RNA to the chromosomes of rat bone marrow cells revealed that clusters of ribosomal cistrons (rDNA) are located in the secondary constrictions of chromosomes No. 3 and 12 and near the centromere of chromosome No. 11, both associated with the late DNA-replicating regions. They were not found in Nos. 1, 2, 13, 19, 20, and the Y chromosome.     

Detection of Clostridium botulinum Toxin by Local Paralysis Elicited with Intramuscular Challenge

Clostridium botulinum toxin can be identified by a characteristic, acute local paralysis that follows its injection into the gastrocnemius ("calf" muscle) of mice. The local botulism can be elicited with slightly less than one-tenth the toxin amount that is needed to kill mice by the intraperitoneal (i.p.) challenge route. The practical sensitivities of the intramuscular (i.m.) versus i.p. tests are about equal, however, because maximum sample volume injectable i.m. is 0.1 ml as compared to the 0.5-ml range that can be given i.p. i.m. injection of 10 or more mouse i.p. mean lethal doses causes paralysis in about 1 h, and an i.m. injection of about 0.5 i.p. mean lethal doses causes paralysis in 3 to 4 h. Toxin neutralization by homologous type of antitoxin only can be demonstrated with an incubated mixture of toxin and antitoxin. Although not as convenient as the i.p. method for routine use to detect botulinum toxin, the i.m. method has characteristics which could make it a useful supplement to the presently accepted i.p. procedure.     

Inhibition and uncoupling of the ADP-regulated electron transport in isolated chloroplasts

The effects of energy transfer inhibitors, electron transport inhibitors and uncouplers on the ADP-regulated and ADP-independent activity of ferricyanide reduction in isolated spinach chloroplasts were studied. Phlorizin and sulfate did not affect the ADP-independent ferricyanide reduction. In the ADP-regulated reduction, these reagents did not affect the ADP inhibition process but inhibited the activity restoration process due to phosphorylation. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and linolenic acid depressed both ADP-regulated and ADP-independent activity of ferricyanide reduction. Gramicidin S and 2-amino-1-butanol depressed ADP-regulated activity and stimulated ADP-independent activity. The decrease in the ADP-regulated ferricyanide-reducing activity (restoration) due to (incomplete) uncoupling paralleled the decrease in phosphorylation activity (P/Δe=1).     

A crystallographic investigation on NADPH-adrenodoxin oxidoreductase

Single crystals of NADPH-adrenodoxin oxidoreductase were grown in 50 mM potassium phosphate (pH 7.4) containing 5% glycerol and ammonium sulfate. The crystals are monoclinic, belong to space group P21 and have dimensions of a= 83.4 A, b = 62.6 A, c = 59.3 A, alpha = gamma = 90 degrees, and beta = 107.1 degrees. There is one molecule per asymmetric unit.