Topological mapping of the active sites of cytochromes P4502B1 and P4502B2 by in situ rearrangement of aryl-iron complexes.

Cytochrome P4502B1 reacts with phenylhydrazine or phenyldiazene to give an iron-phenyl complex that oxidatively rearranges in situ to the two N-phenylprotoporphyrin IX regioisomers with the phenyl group on pyrrole rings A (NA) and D (ND) [Swanson, B. A., Dutton, D. R., Lunetta, J. M., Yang, C. S., & Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 19258-19264]. The conclusion that the active site of cytochrome P4502B1 is open above pyrrole rings A and D but not B and C is extended here by studies with larger arylhydrazines. The N-arylprotoporphyrin IX standards required for product identification were obtained by reaction of the arylhydrazines with equine myoglobin. Cytochrome P4502B1 aryl-iron complex formation followed by oxidative shift of the aryl group produces the following N-aryl-protoporphyrin IX NA:ND regioisomer ratios: phenylhydrazine (39:61), 3,5-dimethylphenylhydrazine (29:71), 4-tert-butylhydrazine (25:75), 2-naphthylhydrazine (less than 2:greater than 98), and 4-(phenyl)phenylhydrazine (87:13). Electron-withdrawing substituents (as in 3,5-dichlorophenyl) prevent the aryl group shift. The increase in the proportion of the ND regioisomer with increasing bulk of the aryl group suggests that the region over pyrrole ring A is more sterically encumbered than that over pyrrole ring D. The regiospecificity is reversed, however, with 4-(phenyl)phenylhydrazine, which primarily gives the NA regioisomer. This reversal suggests that the active site has a sloping roof that is higher over pyrrole ring A than pyrrole ring D and that provides a larger steric barrier to the shift of tall aryl moieties than the barrier over pyrrole ring A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoinactivation of photosystem II by cumulative exposure to short light pulses during the induction period of photosynthesis

Photoinactivation of Photosystem (PS) II in vivo was investigated by cumulative exposure of pea, rice and spinach leaves to light pulses of variable duration from 2 to 100 s, separated by dark intervals of 30 min. During each light pulse, photosynthetic induction occurred to an extent depending on the time of illumination, but steady-state photosynthesis had not been achieved. During photosynthetic induction, it is clearly demonstrated that reciprocity of irradiance and duration of illumination did not hold: hence the same cumulative photon exposure (mol m^–2) does not necessarily give the same extent of photoinactivation of PS II. This contrasts with the situation of steady-state photosynthesis where the photoinactivation of PS II exhibited reciprocity of irradiance and duration of illumination (Park et al. (1995) Planta 196: 401–411). We suggest that, for reciprocity to hold between irradiance and duration of illumination, there must be a balance between photochemical (qP) and non-photochemical (NPQ) quenching at all irradiances. The index of susceptibility to light stress, which represents an intrinsic ability of PS II to balance photochemical and non-photochemical quenching, is defined by the quotient (1-qP)/NPQ. Although constant in steady-state photosynthesis under a wide range of irradiance (Park et al. (1995). Plant Cell Physiol 36: 1163–1169), this index of susceptibility for spinach leaves declined extremely rapidly during photosynthetic induction at a given irradiance, and, at a given cumulative photon exposure, was dependent on irradiance. During photosynthetic induction, only limited photoprotective strategies are developed: while the transthylakoid pH gradient conferred some degree of photoprotection, neither D1 protein turnover nor the xanthophyll cycle was operative. Thus, PS II is more easily photoinactivated during photosynthetic induction, a phenomenon that may have relevance for understorey leaves experiencing infrequent, short sunflecks.Abbreviations D1 protein psbA gene product - DTT dithiothreitol - F_v, F_m, F_o variable, maximum, and initial (corresponding to open traps) chlorophyll fluorescence yield, respectively - NPQ non-photochemical quenching - PS Photosystem - Q_A primary quinone acceptor of PS II - qP photochemical quenching coefficient     

Specificity of binding of beta-glucoside activators of ryegrass (1-->3)-beta-glucan synthase and the synthesis of some potential photoaffinity activators.

Structure-activity relationships among glycoside activators of ryegrass (Lolium multiflorum) (1-->3)-beta-glucan synthase were investigated using a number of natural and synthetic glycosides, including some carrying photoaffinity functions. There is an absolute requirement for a beta-D-glycosyl moiety in the activator, both S- and N-glucosides are active, and the position of the glucosidic linkage in beta-glucose disaccharides has a significant effect on the affinity of binding. However, the binding requirement does not extend beyond a single beta-D-glucosyl residue, and beta-D-oligoglucosides are less effective than disaccharides. The nature of the aglycon has a major influence on the binding affinity. Hydrophobic aglycons lower the concentration required for half-maximal stimulation of the enzyme obtained from an Eadie-Hofstee plot of kinetic data (Ka) for activation, but charge aglycons increase Ka. Relative to methyl-beta-D-glucoside and cellobiose (Ka 1.1 mM), the most potent compounds tested were N-[4-(benzoyl)benzoyl]-beta-D-glucosylamine and 2'-[4-azidosalicylamino]ethyl-1-thio-beta-D-glucoside with K(a)s of approximately 30 microM. The latter also was tested for its potential to specifically label the beta-glucoside-binding site on the synthase, but under the conditions used the binding was found to be nonspecific.     

Distribution of the catabolic transposon Tn5271 in a groundwater bioremediation system.

The distribution of Tn5271-related DNA sequences in samples of groundwater and a groundwater bioremediation system at the Hyde Park (Niagara Falls, N.Y.) chemical landfill site was investigated. PCR amplification of target sequences within the cha genes of Tn5271 revealed similar sequences in the groundwater community and in samples from the sequencing batch reactors treating that groundwater. Cell dilution combined with PCR amplification indicated that cha sequences were carried in about 1 of 10 culturable bacteria from the treatment system. Characterization of isolates involved in chlorobenzoate and toluene biodegradation in the treatment system indicated that two phenotypic clusters, Alcaligenes faecalis type 2 and CDC group IVC-2, contained all of the Tn5271 probe-positive isolates from the community. These two groups differed phenotypically from recipient groups isolated following horizontal transfer of pBRC60 (Tn5271) in pristine freshwater microcosms. A genetic rearrangement in Tn5271 attributable to the intramolecular transposition of the flanking element IS1071R was detected in an isolate from the treatment system. Comparison of the structure of the intramolecular transposition derivative from groundwater isolate OCC13(pBRC13) with a laboratory-derived intramolecular transposition derivative of pBRC60 revealed similarities. The rearrangement was shown to increase the stability of the plasmid under starvation conditions.     

Diversity and conservation of blackwater fishes in Peninsular Malaysia,particularly in the North Selangor peat swamp forest

One of the most extreme freshwater habitats in Peninsular Malaysia is the peat swamp forest, with dark-coloured and highly acidic waters. Surprisingly, little is known about blackwater fishes in Peninsular Malaysia. Until 1968, only 26 fish species were known from blackwaters throughout Peninsular Malaysia, of which only one can be regarded as stenotopic. A recent intensive survey of part of the North Selangor peat swamp forest yielded 47 species, of which 14 are probably stenotopic taxa. These include four undescribed species and several new records for western Peninsular Malaysia. These discoveries are significant in that they include the family Chaudhuriidae which until 1985, was not reported from Sundaic Southeast Asia, and the rare genus Encheloclarias which had not been encountered for over 50 years. The rapid rate of destruction of the peat swamp forest owing to development, forestry and agricultural activities must be halted or slowed significantly to enable the proper zoological surveys and studies to be conducted. Conservation plans and environmental impact assessments based on inadequate sampling and knowledge of species present is acutely dangerous. There are no longer substantial undisturbed blackwater peat swamp forests left in most of Peninsular Malaysia. Conservation of the remaining blackwater biotopes is critically important if extinction of many species, here regarded as economically valuable renewable resources, is to be prevented.     

The systematics and ecology of snakeheads (Pisces: Channidae) in Peninsular Malaysia and Singapore

Seven species of snakeheads (Channidae) are known from Peninsular Malaysia and Singapore, viz. Channa bankanensis, C. gachua, C. lucius, C. marulioides, C. melasoma, C. micropeltes and C. striata. Up-to-date distribution maps of each species are presented, including new records. Their systematics is reviewed and partially revised. The taxonomic status of C. marulioides and C. melanoptera is clarified. Specimens from Peninsular Malaysia identified as C. melanoptera sensu Weber & de Beaufort, 1922, proved to be the adult form of C. marulioides s.str. The real C. melanoptera appears to be restricted to Borneo and possibly Sumatra. The life history of a blackwater species C. bankanensis is also documented, with regards to the morphological and colour-pattern changes associated with growth. An updated key to the seven species based on morphometric measurements and meristic counts is presented.     

Phosphorylation and Palmitoylation of the Human D2L Dopamine Receptor in Sf9 Cells

Abstract: We have expressed and biochemically characterized the human D2_long (D2_L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2_L receptors expressed in mammalian cell lines. Dopamine binding to D2_L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2_L receptor-specific antibody identified two major protein species at ∼44 kDa and at ∼93 kDa in immunoblots, suggesting the presence of D2_L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin-solubilized preparation of cells expressing D2_L receptor prelabeled with ³²P_i or [³H]-palmitate. These results constitute the first direct evidence for D2_L receptor phosphorylation and palmitoylation.