Manganese-binding proteins of the oxygen-evolving complex

The extrinsic 33-kDa protein (P33) was cross-linked covalently to the binding site on P33-depleted PSII preparations which is responsible for reconstitution of photosynthetic water oxidation after PSII preparations have been washed with 1 M CaCl2. Conditions were found in which more than half of the cross-linked protein complexes formed in the PSII preparations retained the ability to catalyze the oxidation of water. The complex is composed of the P33 cross-linked to the D1 and D2 proteins and a 34-kDa protein, which is present in lower abundance than the other three proteins. After solubilization of the membranes with SDS and purification by preparative SDS-PAGE, the complex retains bound manganese and can catalyze the conversion of H2O2 to O2. Calcium and chloride increased the catalase activity of the purified cross-linked complex while lanthanum or hydroxylamine abolished the activity. By use of the specific activity of the H2O2-dependent reaction to follow the extent of purification of the cross-linked complex, the most highly purified complex was determined to contain 0.34 microgram of manganese/180 micrograms of protein. The mole ratio of Mn/protein was calculated to range from 3.6 to 4.5 depending on the assumed stoichiometry of the protein subunits. The results presented here provide direct evidence that one or more of the three proteins that have cross-linked to the P33 are responsible for binding the manganese of the oxygen-evolving complex.     

Protein PSII-G. An additional component of photosystem II identified through its plastid gene in maize

An unidentified open reading frame, 248 or 255 amino acids in length, on the maize chloroplast DNA fragment Bam5 was sequenced. It encodes a protein which contains a high proportion of hydrophilic amino acids, of which 22% are hydroxylated, interrupted by hydrophobic domains. A synthetic peptide corresponding to a hydrophilic sequence was used to generate antibodies. Western blots of photosystem I and II complexes prepared from maize and spinach thylakoids indicate that the psbG gene product is a membrane-associated protein of the photosystem II complex that migrates as a 24-kDa species on polyacrylamide gel electrophoresis.     

Effect of chronic cocaine administration and cocaine withdrawal on coronary flow rate and heart rate responses to epinephrine and cocaine in isolated perfused rat hearts

The acute dose-dependent effects of epinephrine and cocaine on heart rate and coronary flow rate (CFR) were examined in isolated, perfused (Langendorff) rat hearts from animals: i) pretreated with daily cocaine injections (20 mg/kg/day) for 8 weeks; ii) after 2-day withdrawal from 8-week cocaine pretreatment; iii) vehicle-treated controls. Chronic cocaine (CC) hearts were significantly less sensitive to the chronotropic effects of epinephrine than control (C) or withdrawal (CW) hearts. CW hearts exhibited significantly higher heart rates in response to epinephrine than C and CC hearts. Epinephrine alone (2.5 x 10(-7) M) decreased CFR 11% (C), 9%(CC), 14%(CW) from respective baseline levels. Cocaine alone had no significant effect on CFR in C hearts but produced slight dose-dependent decrements in CFR in CC and particularly CW hearts at higher doses. Cocaine plus epinephrine markedly decreased CFR in all groups, particularly in CW hearts. The results indicate that chronic daily cocaine administration produces a functional tolerance of the heart to the chronotropic actions of epinephrine but a 2-day withdrawal from chronic cocaine results in a rebound supersensitivity to adrenergic stimulation and cocaine's sympathomimetic effects. In addition, cocaine produces only minor decrements in coronary flow in the rat heart, while cocaine acts synergisticallly with epinephrine to produce a marked decrease in CFR.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

1,5-diamino-2-pentyne is both a substrate and inactivator of plant copper amine oxidases.

1,5-diamino-2-pentyne (DAPY) was found to be a weak substrate of grass pea (Lathyrus sativus, GPAO) and sainfoin (Onobrychis viciifolia, OVAO) amine oxidases. Prolonged incubations, however, resulted in irreversible inhibition of both enzymes. For GPAO and OVAO, rates of inactivation of 0.1-0.3 min(-1) were determined, the apparent KI values (half-maximal inactivation) were of the order of 10(-5) m. DAPY was found to be a mechanism-based inhibitor of the enzymes because the substrate cadaverine significantly prevented irreversible inhibition. The N1-methyl and N5-methyl analogs of DAPY were tested with GPAO and were weaker inactivators (especially the N5-methyl) than DAPY. Prolonged incubations of GPAO or OVAO with DAPY resulted in the appearance of a yellow-brown chromophore (lambda(max) = 310-325 nm depending on the working buffer). Excitation at 310 nm was associated with emitted fluorescence with a maximum at 445 nm, suggestive of extended conjugation. After dialysis, the color intensity was substantially decreased, indicating the formation of a low molecular mass secondary product of turnover. The compound provided positive reactions with ninhydrin, 2-aminobenzaldehyde and Kovacs' reagents, suggesting the presence of an amino group and a nitrogen-containing heterocyclic structure. The secondary product was separated chromatographically and was found not to irreversibly inhibit GPAO. MS indicated an exact molecular mass (177.14 Da) and molecular formula (C10H15N3). Electrospray ionization- and MALDI-MS/MS analyses yielded fragment mass patterns consistent with the structure of a dihydropyridine derivative of DAPY. Finally, N-(2,3-dihydropyridinyl)-1,5-diamino-2-pentyne was identified by means of 1H- and 13C-NMR experiments. This structure suggests a lysine modification chemistry that could be responsible for the observed inactivation.     

REVIEW ARTICLE: Cyanogenesis in cassava (Manihot esculenta Crantz)

Cassava is the most agronomically important of the cyanogeniccrops. Linamarin, the predominant cyanogenic glycoside in cassava,can accumulate to concentrations as high as 500 mg kg^–1fresh weight in roots and to higher levels in leaves. Recently,the pathway of linamarin synthesis and the cellular site oflinamarin storage have been determined. In addition, the cyanogenicenzymes, linamarase and hydroxynitrile lyase, have been characterizedand their genes cloned. These results, as well as studies onthe organ- and tissue-specific localization of linamarase andhydroxy-nitrile lyase, allow us to propose models for the regulationof cyanogenesis in cassava. There remain, however, many unansweredquestions regarding the tissue-specific synthesis, transport,and accumulation of cyanogenic glycosides. The resolution ofthe sequestions will facilitate the development of food processing,biochemical and transgenic plant approaches to reducing thecyanogen content of cassava foods. Key words: Cyanide, cyanogenic glycosides, linamarin, cyanogens