Photosynthesis of herbaceous plants from nutrient-poor tropical forests

Photosynthetic characteristics, specific leaf mass, chlorophyll and total leaf nitrogen concentrations of four herbaceous plants (Dicranopteris linearis, Hanguana malayana, Pentaphragma ellipticum, Tacca integrifolia) from nutrient-poor tropical forests showed that all these plants were well-adapted to their natural growth environments. No photoinhibition was observed even in the understorey plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of multimeric forms of CD4 through a sugar-based cross-linking strategy

We have developed a three-step cross-linking procedure that is specifically targeted at the carbohydrate on a protein and applied it to CD4 as a model system for studying the role of multivalent interactions in function. In the first step CD4 was oxidized with periodate, creating aldehydes that served as targets for the subsequent chemistry. Next the aldehydes were modified with cystamine, converting the reactive group into a thiol. Finally cross-linking through the thiol moiety was generated with the homobifunctional cross-linker bismaleimidohexane. With this procedure, approximately 60% of the CD4 was converted into higher molecular weight complexes that were soluble and retained function as assessed by glycoprotein gp120 binding activity. CD4 dimers and tetramers by mass were 4 and 15 times as active as CD4 monomer in blocking virus infection with HTLV-IIIB in an in vitro cellular assay. The cross-linking chemistry provides an efficient method for producing homomultimers of a glycoprotein.     

Thermal Kinetics of Glutathione Reductase and their Relation to Thermotolerance in Diverse Cultivars of Maize

The characteristics of glutathione reductase from a number ofmaize cultivars with contrasting thermotolerance have been investigated.The apparent K_m (Michaelis constant) for oxidised glutathione(GSSG) was measured between 10 and 45°C at constant pH.The enzyme from highland cultivars adapted to cool environmentshad a slightly lower apparent K_m for GSSG than that from lowlandtropical cultivars at low assay temperatures. Similarly theenzyme from lowland tropical cultivars had a lower apparentK_m for GSSG at high assay temperatures. However these effectswere small and regression lines plotted through the data werenot significantly different in slope or intercept. There wasa strong correlation (r = 0·939) between apparent K_mand V_max (maximum initial velocity) as assay temperature wasvaried. The interpretation of apparent K_m/temperature relationshipsis discussed with hypothetical examples of the effects of temperatureon enzyme activity/substrate concentration plots. It is demonstratedthat an increase in apparent K_m at higher assay temperaturesneed not necessarily reflect any temperature-dependent impairmentof enzyme function. The apparent K_m for GSSG of glutathionereductase from maize increased over four-fold when the temperaturewas raised from 10 to 45°C, but it is concluded that invivo rates of reaction are likely to be increased rather thandecreased by this same change in temperature. Glutathione reductasewould thus appear to be equally well adapted to function atall these temperatures. This suggests that the potential ofenzyme thermal kinetics to predict thermotolerance may be limited.Copyright1994, 1999 Academic Press Zea mays L., maize, glutathione reductase, thermal kinetics, thermotolerance     

High-throughput screening of bacteriorhodopsin mutants in whole cell pastes

A high-throughput screening method has been developed which enables functional analysis of bacteriorhodpsin in whole cell pastes. Reflectance spectra, from as little as 5 ml of Halobacterium salinarum cells, show close correspondence to that obtained from the purified purple membrane (PM), containing bacteriorhodopsin (BR) as the sole protein component. We demonstrate accurate quantification of BR accumulation by ratiometric analysis of BR (A_max 568) and a membrane-bound cytochrome (A_max 410). In addition, ground-state light- and dark-adapted (LA and DA, respectively) spectral differences were determined with high accuracy and precision. Using cells expressing the BR mutant D85N, we monitored transitions between intermediate-state homologues of the reprotonation phase of the light-activated proton pumping mechanism. We demonstrate that phenotypes of three mutants (D85N/T170C, D85N/D96N, and D85N/R82Q) previously characterized for their effect on photocycle transitions are reproduced in the whole cell samples. D85N/T170C stabilizes accumulation of the N state while D85N/D96N accumulates no N state. D85N/R82Q was found to have perturbed the pK_a of M accumulation. These studies illustrate the correspondence between pH-dependent ground-state transitions accessed by D85N and the transitions accessed by the wild-type protein following photoexcitation. We demonstrate that whole cell reflectance spectroscopy can be used to efficiently characterize the large numbers of mutants generated by engineering strategies that exploit saturation mutagenesis.