Tyrosinase-catalyzed binding of 3,4-dihydroxyphenylalanine with proteins through the sulfhydryl group

The cytotoxicity of catechols has been ascribed to covalent binding of the omicron-quinone oxidation products to proteins through sulfhydryl groups. The nature of the covalent binding was studied with dopaquinone formed on tyrosinase oxidation of 3,4-dihydroxyphenylalanine (DOPA). After acid hydrolysis of the reaction products, cysteinyldopas liberated (protein-bound cysteinyldopas) were determined by HPLC with electrochemical detection. When 0.1 mM DOPA was oxidized in the presence of 0.2 mM bovine serum albumin, alcohol dehydrogenase or isocitrate dehydrogenase, protein-bound cysteinyldopas were formed in yields of 5.4, 44, or 33%, respectively. The covalent binding was almost completely inhibited by 1 mM cysteine or 1 mM ascorbic acid, but 10 mM lysine had no effect. These results unambiguously demonstrate that dopaquinone can bind with proteins mostly through sulfhydryl groups.     

Fluorescence resonance energy transfer between the nucleotide binding site and Cys-10 in G-actin and F-actin

Intramonomer fluorescence resonance energy transfer between the donor epsilon-ATP bound to the nucleotide site and the acceptor N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) or 4-dimethylaminophenyl-azophenyl-4'-maleimide bound to Cys-10 in G-actin was measured. The donor-acceptor distance was calculated to be about 40 A. The intermonomer energy transfer in F-actin occurring between epsilon-ADP and DABMI was also measured. The radial coordinate of Cys-10 was calculated to be 25 A based on the helical symmetry of F-actin and the recently calculated radial coordinate of the nucleotide binding site in F-actin i.e. 25 A (Miki, M., Hambly, B. and dos Remedios, C.G. (1986) Biochim. Biophys. Acta 871, 137-141). (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Corresponding distances separating the donor nucleotide in one monomer from acceptors on Cys-10 in the first and second nearest neighbours in F-actin are 39-40 A and 41-43 A.     

Cytochrome c peroxidase activity of bovine heart cytochrome oxidase incorporated in liposomes and generation of membrane potential

Cytochrome oxidase vesicles catalyzed the peroxidatic oxidation of ferrocytochrome c. The maximal peroxidase activity in the absence of an uncoupling agent was 9.8 mol ferrocytochrome c oxidized/(s X mol heme a), indicating a 5-fold activation compared with the soluble enzyme system. The peroxidase activity was further enhanced 1.2 to 2.1 times upon addition of an uncoupler, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The stoichiometry of the reduction of hydrogen peroxide by ferrocytochrome c was established to be 1 : 2, indicating water formation. Potassium cyanide (0.14 mM) completely inhibited the peroxidase activity. The inhibition by 1 mM CO was 40-77% depending on the energized state of cytochrome oxidase vesicles, but in contrast, 85% inhibition was observed with the soluble enzyme. In the energized state the enzyme showed a slightly lower affinity for CO than in the deenergized state. Coupled with the peroxidase activity, a membrane potential of 72 mV was registered transiently; this may be physiologically significant in relation to the energy transduction mechanism.     

High temperature induction of a stringent response in the dnaK(Ts) and dnaJ(Ts) mutants of Escherichia coli

The cellular concentrations of ppGpp in the dnaK(Ts) and dnaJ(Ts) mutants of Escherichia coli were examined, since the thermosensitive RNA synthesis of these mutants is relaxed by an additional mutation in the relA gene. The results showed that ppGpp accumulated extensively in the dnaK(Ts) and dnaJ(Ts) mutants after a temperature shift up, reaching levels of 5 mM and 0.5 mM, respectively. This unusual accumulation of ppGpp was suppressed by the relA1 mutation, implying that it results from induction of a stringent response in these mutants at a nonpermissive temperature.     

Calcitonin-induced phosphorylation of rat liver cytosolic proteins

Calcitonin (CT) stimulated phosphorylation of two liver cytosolic proteins whose molecular weights are 67,000 and 93,000. Stimulation of 67,000-Mr protein phosphorylation began shortly after subcutaneous injection of CT, reaching a maximum at 5 min and decreasing to below the control level at 30 min. The reaction was independent of cyclic AMP or Ca2+, and was not influenced by a calmodulin antagonist, W7. Stimulation of 93,000-Mr protein phosphorylation became evident by 30 min. This reaction was also stimulated by administration of vasopressin or epinephrine, which is known to cause increased phosphorylation of glycogen phosphorylase having the same molecular weight. The phosphorylation of 93,000-Mr protein, stimulated by CT, was dependent on Ca2+ but not on cyclic AMP, and appeared to be inhibited by W7. In addition, CT did not influence the phosphorylation of 61,000-Mr protein, a major protein phosphorylated in a cyclic AMP-dependent manner. These results suggest that CT may exert its effect on liver cells through protein phosphorylation, most probably in a cyclic AMP-independent manner.     

Proton translocation by cytochrome oxidase vesicles catalyzing the peroxidatic oxidation of ferrocytochrome c

Coupled with the peroxidatic oxidation of ferrocytochrome c under anaerobic conditions, proteoliposomes reconstituted with a purified preparation of bovine heart cytochrome oxidase ejected protons into the external medium with an apparent H+/e- ratio of 0.9. At the same time, protons in the intravesicular space were consumed. Dicyclohexylcarbodiimide significantly inhibited the proton translocation. Cyanide (0.14 mM) completely inhibited both the peroxidase and proton translocating activities. On the contrary, in the presence of 1 mM CO the proton ejection was abolished almost completely, but 50% of the peroxidase activity persisted. This result suggests the operation of multiple mechanisms in the peroxidase reaction and that the CO-sensitive one is coupled to the proton translocation.