Ethanol exhibits alpha receptor blocking-like properties in anesthetized rats

The ability of ethanol to reduce alpha-adrenergic receptor-mediated pressor responsiveness in vivo was investigated in chloralose-anesthetized male Sprague-Dawley rats. Catheters were inserted in the jugular vein and the femoral artery of rats for the injection of drugs and the measurement of blood pressure, respectively. Dose-response curves for phenylephrine and norepinephrine were constructed by plotting the change in mean arterial pressure following a bolus dose of the agent against the dose of the pressor agent used. Following construction of an initial dose-response curve, animals were challenged with either a 1 g/kg dose of ethanol or an equivalent volume of saline (iv) and the dose-response curves were repeated. Using a similar protocol, pressor responsiveness was evaluated in animals pretreated with either yohimbine (1 mg/kg) or prazosin (3.9 micrograms/kg), a dose sufficient to produce partial blockade of alpha receptor-mediated pressor responsiveness, and then treated with ethanol. Ethanol produced a partial blockade of alpha receptors when the animals were challenged with either phenylephrine or norepinephrine. This blockade produced by ethanol was shown to be similar to that produced by the receptor blocking agents used in this study. To rule out any nonspecific effects of ethanol in reducing vascular reactivity, some animals were challenged with angiotensin II both before and after treatment with ethanol, yohimbine, or prazosin and after both drugs were administered together. Ethanol, as well as the alpha 1- and alpha 2-adrenergic blocking agents tested failed to have any significant effect on angiotensin II-pressor responsiveness, ruling out any nonspecific effect of ethanol on the vasculature. It is concluded, therefore, that ethanol has alpha receptor blocking-like activity in vivo.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Cloning of DNA involved in sporulation of Streptomyces griseus

Twenty-two bald mutants of Streptomyces griseus were isolated and classified into four phenotypic groups, two of which showed conditional sporulation. A 3-kilobase fragment of DNA was cloned in a high-copy-number vector and detected by its ability to restore sporulation to one class of conditionally bald mutants. Analysis of subclones demonstrated that the sporulation property was contained within a 2.5-kilobase fragment. Hybridization studies and restriction analysis indicated that this DNA fragment was present in several Streptomyces species and was distinct from DNA that has been shown to complement afsA mutants of S. bikiniensis and bldA mutants of S. coelicolor.     

Directed mutagenesis indicates that the donor to P+680 in photosystem II is tyrosine-161 of the D1 polypeptide

Photosystem II contains two redox-active tyrosines. One of these, YZ, reduces the reaction center chlorophyll, P680, and transfers the oxidizing equivalent to the oxygen-evolving complex. The second, YD, has a long-lived free radical state of unknown function. We recently established that YD is Tyr-160 of the D2 polypeptide by site-directed mutagenesis of a psbD gene in the unicellular cyanobacterium Synechocystis 6803 [Debus, R. J., Barry, B. A., Babcock, G. T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430]. YZ is most likely the symmetry-related Tyr-161 of the D1 polypeptide. To test this hypothesis, we have changed Tyr-161 to phenylalanine by site-directed mutagenesis of a psbA gene in Synechocystis. The resulting mutant assembles PSII, as judged by its ability to produce the stable Y+D radical, but is unable to grow photosynthetically and exhibits altered fluorescence properties. The nature of the fluorescence change indicates that forward electron transfer to P+680 is disrupted in the mutant. These results provide strong support for our identification of Tyr-161 in the D1 polypeptide with YZ.     

Similarity of EPR Signal IIf rise and P-680+ decay kinetics in Tris-washed chloroplast Photosystem II preparations as a function of pH

The rise time, of Signal II_f and the decay time of P-680⁺ have been measured kinetically as a function of pH by using EPR. The Photosystem II-enriched preparations which were used as samples were derived from spinach chloroplasts, and they evolved oxygen before Tris washing. The onset kinetics of Signal II_f are in agreement, within experimental error, with the fast component of the decay of an EPR signal attributable to P-680⁺. The signal II_f rise kinetics also show good agreement with published values of the pH dependence of the decay of P-680⁺ measured optically (Conjeaud, H. and Mathis, P. (1980) Biochim. Biophys. Acta 590, 353–359). These results are consistent with a model where the species Z (or D₁) responsible for Signal II_f is the immediate electron donor to P-680⁺ in tris-washed Photosystem II fragments.     

Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum

Brefeldin A (BFA) has been reported to block protein transport from the ER and cause disassembly of the Golgi complex. We have examined the effects of BFA on the transport and processing of the vesicular stomatitis virus G protein, a model integral membrane protein. Delivery of G protein to the cell surface was reversibly blocked by 6 micrograms/ml BFA. Pulse-label experiments revealed that in the presence of BFA, G protein became completely resistant to endoglycosidase H digestion. Addition of sialic acid, a trans-Golgi event, was not observed. Despite processing by cis- and medial Golgi enzymes, G protein was localized by indirect immunofluorescence to a reticular distribution characteristic of the ER. By preventing transport of G protein from the ER with the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or by use of the temperature-sensitive mutant ts045, which is restricted to the ER at 40 degrees C, we showed that processing of G protein occurred in the ER and was not due to retention of newly synthesized Golgi enzymes. Rather, redistribution of preexisting cis and medial Golgi enzymes to the ER occurred as soon as 2.5 min after addition of BFA, and was complete by 10-15 min. Delivery of Golgi enzymes to the ER was energy dependent and occurred only at temperatures greater than or equal to 20 degrees C. BFA also induced retrograde transport of G protein from the medial Golgi to the ER. Golgi enzymes were completely recovered from the ER 10 min after removal of BFA. These findings demonstrate that BFA induces retrograde transport of both resident and itinerant Golgi proteins to the ER in a fully reversible manner.     

Optical and resonance Raman spectroscopy of the heme groups of the quinol-oxidizing cytochrome aa3 of Bacillus subtilis.

The cytochrome aa3-type terminal quinol oxidase of Bacillus subtilis catalyzes the four-electron reduction of dioxygen to water. It resembles the aa3-type cytochrome-c oxidase in using heme A as its active-site chromophores but lacks the CuA center and the cytochrome-c oxidizing activity of the mitochondrial enzyme. We have used optical and resonance Raman spectroscopies to study the B. subtilis oxidase in detail. The alpha-band absorption maximum of the reduced minus oxidized enzyme is shifted by 5-7 nm to the blue relative to most other aa3-type oxidases, and accordingly, we designate the Bacillus enzyme as cytochrome aa3-600. The shifted optical spectrum cannot be ascribed to an alteration in the strength of the hydrogen bond between the formyl group of the low-spin heme and its environment, as the Raman line assigned to this mode in aa3-600 has the same frequency and degree of resonance enhancement as the low-spin heme a formyl mode in most other aa3-type oxidases. Raman modes arise at 194 and 214 cm-1 in aa3-600, whereas a single band at about 214 cm-1 is assigned to the iron-histidine stretch for the other aa3-type oxidases. Possible explanations for the occurrence of these two modes are discussed. Comparison of formyl and vinyl modes and heme skeletal vibrational modes in different oxidation states of aa3-600 and of beef heart cytochrome-c oxidase shows a strong similarity, which suggests conservation of essential features of the heme environments in these oxidases.