Application of characteristic reaction paths: Rate-limiting capability of phosphofructokinase in yeast fermentation.

The intracellular rate-limiting capability of phosphofructokinase in baker's yeast (Saccharomyces cerevisiae) fermentation is investigated using the reaction path analysis discussed by Liao and Lightfoot in a previous article. It is found that the yeast cells under our experimental conditions indeed exhibit the characteristic behavior, and the characteristic reaction path on the G6P-ATP phase plot is determined for cells fed with different sugars, cells of different strains, and cells in the transition period following rehydration. Results suggest that phosphofructokinase does not limit the CO(2) production rate of the cells under investigation. However, it is not present in a great excess either: ca. 80% of phosphofructokinase activity is utilized by the glycolytic pathway of the cell under investigation.

Differential transcription in vivo and in vitro of two adjacent maize chloroplast genes: The large subunit of ribulosebisphosphate carboxylase and the 2.2-kilobase gene.

The transcription of cloned maize plastid DNA sequences in vitro by maize plastid DNA-dependent RNA polymerase has been studied to expose the roles of the enzyme, polypeptide cofactors, and DNA sequences in the regulation of gene expression. The 4.35-kilobase pair BamHI fragment 9 carries the maize plastid gene for the large subunit of ribulosebisphosphate carboxylase and part of the gene for a 2.2-kilobase RNA. These two genes are separated by approximately 330 base pairs and are transcribed divergently. Transcripts of the gene for the large subunit of ribulosebisphosphate carboxylase are abundant in bundle sheath cells of maize leaves and we show here that transcripts of the 2.2-kilobase RNA gene are present in both mesophyll cells and the adjacent bundle sheath cells. In vitro, in the presence of the S factor, maize chloroplast DNA-dependent RNA polymerase produces a transcript of the gene for the large subunit of ribulose-bisphosphate carboxylase with a 5' terminus like that of the corresponding mRNA isolated from plastids, transcribes chloroplast DNA sequences of Bam fragment 9 in a chimeric plasmid in preference to the vehicle RSF 1030 and, in a ratio of 3:1, preferentially transcribes the gene for the large subunit of ribulosebisphosphate carboxylase over the 2.2-kilobase RNA gene from supercoiled chimeric plasmid DNA.

Overlapping divergent genes in the maize chloroplast chromosome and in vitro transcription of the gene for tRNA.

In the presence of the S polypeptide, maize chloroplast DNA-dependent RNA polymerase preferentially transcribes sequences within the 2200-nucleotide-pair-long maize chloroplast chromosome fragment Eco [unk] from a supercoiled chimeric plasmid cloned in Escherichia coli [Jolly, S. O. & Bogorad, L. (1980) Proc. Natl. Acad. Sci. USA 77, 822-826]. Eco [unk] contains one gene for tRNA(His) and one for a 1.6-kilobase RNA that includes an open reading frame. These two genes overlap by at least a few nucleotides and are transcribed divergently from complementary DNA strands. This indicates possible transcriptional regulation of chloroplast DNA at the nucleotide level. The 5' end of tRNA(His) (G-U-G) isolated from maize chloroplasts is indistinguishable from that of the transcript produced from Eco [unk] in vitro by maize chloroplast DNA-dependent RNA polymerase. This purified system initiates RNA synthesis faithfully and exhibits preference for some chloroplast genes. Maize chloroplast DNA for tRNA(His) lacks the sequence C-C-A at its 3' terminus; it is presumably added post-transcriptionally. Maize tRNA(His) has both prokaryotic and eukaryotic features.

Preferential transcription of cloned maize chloroplast DNA sequences by maize chloroplast RNA polymerase.

Zea mays chloroplast DNA-dependent RNA polymerase in vitro preferentially transcribes maize chloroplast DNA sequences incorporated in cloned chimeric bacterial plasmids. Preferential transcription is dependent on the presence of a 27.5-kilodalton polypeptide, the S factor, which has been purified from maize chloroplasts, and also on the template's being in the supercoiled form.

NADH-Nitrate Reductase Inhibitor from Soybean Leaves.

A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Merr. Var. Amsoy) leaves that had been in the dark for 54 hours. The presence of the inhibitor was first suggested by the absence of nitrate reductase activity in the homogenate until the inhibitor was removed by diethylaminoethyl (DEAE)-cellulose chromatography. The inhibitor inactivated the enzyme in homogenates of leaves harvested in the light. Nitrate reductases in single whole cells isolated through a sucrose gradient were equally active from leaves grown in light or darkness, but were inhibited by addition of the active inhibitor.The NADH-nitrate reductase inhibitor was purified 2,500-fold to an electrophoretic homogeneous protein by a procedure involving DEAE- cellulose chromatography, Sephadex G-100 filtration, and ammonium sulfate precipitation followed by dialysis. The assay was based on nitrate reductase inhibition. A rapid partial isolation procedure was also developed to separate nitrate reductase from the inhibitor by DEAE-cellulose chromatography and elution with KNO(3). The inhibitor was a heat-labile protein of about 31,000 molecular weight with two identical subunits. After electrophoresis on polyacrylamide gel two adjacent bands of protein were present; an active form and an inactive form that developed on standing. The active factor inhibited leaf NADH-nitrate reductase but not NADPH-nitrate reductase, the bacterial nitrate reductase or other enzymes tested. The site of inhibition was probably at the reduced flavin adenine dinucleotide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The inhibitor did not appear to be a protease. Some form of association of the active inhibitor with nitrate reductase was indicated by a change of inhibitor mobility through Sephadex G-75 in the presence of the enzyme. The inhibition of nitrate reductase was noncompetitive with nitrate but caused a decrease in V(max).The isolated inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity returned. Equal amounts of inhibitor were present in leaves harvested from light or darkness, except that the inhibitor was at first inactive when rapidly isolated from leaves in light. Photoinactivation of yellow impure inhibitor required no additional components, but inactivation of the purified colorless inhibitor required the addition of flavin.Preliminary evidence and a procedure are given for partial isolation of a component by DEAE-cellulose chromatography that stimulated nitrate reductase. The data suggest that light-dark changes in nitrate reductase activity are regulated by specific protein inhibitors and stimulators.