Isolation and characterisation of a full-length genomic clone encoding a plastidic glucose 6-phosphate dehydrogenase from Nicotiana tabacum.

We describe here the isolation and characterisation of the first full-length genomic clone encoding a plant glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) from Nicotiana tabacum L. cv Samsun. The gene was expressed in all tissues, including roots, leaves, stems and flowers. Comparison of the gene with other known plant G6PDH cDNAs grouped this sequence with plastidic isoforms. The protein, minus a putative plastidic transit sequence, was overexpressed in Escherichia coli as a glutathione S-transferase fusion protein. The resulting protein was shown to be immunologically related to the potato plastidic G6PDH. This suggests that the sequence described here codes for a plastidic isoform. Plastidic G6PDH mRNA was induced in both roots and leaves in response to KNO3, and the induction in roots was approximately 4 times the response seen in leaves. Sequence analysis of the 5'-untranslated region of the genomic clone indicated the presence of several NIT2 elements, which may contribute to the control of the expression of this gene. Plastidic G6PDH mRNA levels did not appear to respond to light.

Evidence for channeling of intermediates in the oxidative pentose phosphate pathway by soybean and pea nodule extracts, yeast extracts, and purified yeast enzymes.

Evidence is presented that intermediates of the oxidative pentose phosphate pathway (OPPP) are channeled from one pathway enzyme to the next. CO2 produced from [1-14C]glucose in the presence of unlabelled pathway intermediates contained much more radioactivity than predicted by a model in which pathway-produced intermediates are in equilibrium with identical molecules in the bulk phase. This was the case whether glucose 6-phosphate (Glc6P), 6-phosphogluconolactone, or 6-phosphogluconate was added. Assumptions involved in calculating the amount of 14CO2 predicted for free mixing of 14C-labelled and unlabelled intermediates are discussed, together with the following results. (a) 14CO2 production by pea nodules in the presence of 3 mM 6-phosphogluconate was higher than in its absence. (b) Apparent channeling of intermediates was much higher for purified yeast enzymes than for yeast extract. (c) 6-Phosphogluconate and 6-phosphogluconolactone were channeled between yeast Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase despite the absence of 6-phosphogluconolactonase in the purified yeast enzyme mixture. (d) When purified yeast hexokinase was physically separated from Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase by a dialysis membrane, there was no apparent channeling. (e) Poly(ethylene glycol), high salt and detergents had little effect on apparent channeling of OPPP intermediates, which is consistent with a stable complex of enzymes. On the other hand, density gradient centrifugation experiments suggested a more transient interaction between the enzymes. Taken together, the results support channeling of OPPP pathway intermediates.

Effects of low and high relative molecular protein mass on four methods for total protein determination in urine.

Four commonly used methods for the determination of total protein in urine were compared. These were two biuret methods using different precipitants, a Ponceau S method and a Coomassie Brilliant Blue method. The protein content of the urines was also evaluated by sodium dodecylsulphate polyacrylamide gel electrophoresis. The biuret method with ethanolic phosphotungstic acid as precipitant correlated best with the Coomassie Brilliant Blue method (r = 0.944; p less than 0.001) but less well with the Ponceau S (r = 0.895; p less than 0.001) or biuret-trichloroacetic acid (r = 0.874; p less than 0.001) methods. For urines with normal electrophoretic protein patterns, the imprecise biuret-trichloroacetic acid method (cv = 18.5%) gave the greatest number of false high results (23 in 36 urines) as assessed by electrophoresis. False low results were common in low relative molecular mass (Mr) proteinuria, especially with the biuret-tricholoroacetic acid and Ponceau S methods. High Mr proteinuria rarely caused false low results. Discrepancies between methods appear to have resulted from incomplete precipitation of low Mr protein by trichloroacetic acid.