Using simple batchwise adsorption and elution, large-scale purification of recombinant Z. mobilis glucose 6-phosphate dehydrogenase and glucokinase has been achieved. In each case adsorption on the dye Procion Yellow HE-3G was followed by elution, precipitation with ammonium sulphate, and gel filtration, although the latter two steps effected only marginal additional purification. These two enzymes can be used in quantitative diagnostic kits for glucose and for creatine kinase.

A simple, economical and rapid purification procedure for muscle lactate dehydrogenase is described. It makes use of batch adsorption on Procion Red H-3B coupled to agarose through an amine linkage. The enzyme is eluted with NAD(+) and sulphite, and for many purposes no more processing is necessary. Only 70 ml settled volume of adsorbent is required to process extract containing 1 g of lactate dehydrogenase, which is recovered with up to 90% yield, at over 98% purity. The method is readily scalable up or down, and makes an excellent class demonstration of affinity techniques.

Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined.

Gold-labeled antibodies were used to examine the subcellular locations of 11 glycolytic and fermentative enzymes in Zymomonas mobilis. Glucose-fructose oxidoreductase was clearly localized in the periplasmic region. Phosphogluconate lactonase and alcohol dehydrogenase I were concentrated in the cytoplasm near the plasma membrane. The eight remaining enzymes were more evenly distributed within the cytoplasmic matrix. Selected enzyme pairs were labeled on opposite sides of the same thin section to examine the frequency of colocalization. Results from these experiments provide evidence that glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and alcohol dehydrogenase I form an enzyme complex.

The gene encoding glucose-fructose oxidoreductase (gfo) from Zymomonas mobilis was cloned in Escherichia coli and sequenced. An open reading frame of 439 amino acids encoded a protein of 49 kDa. A leader sequence of 52 amino acids preceded the N-terminal sequence of the enzyme, indicating cleavage of the precursor protein at an Ala-Ala site to give rise to an active form of the enzyme of 43 kDa. Processing of the glucose-fructose oxidoreductase leader sequence, although not complete, was demonstrated in an in vitro translation system. The two Z. mobilis promoters of the gfo gene show considerable homology to other highly expressed Z. mobilis genes (pdc, adhB, gap, and pgk) as well as to the E. coli consensus sequence. Although translation of the gfo gene was demonstrated in vitro in an E. coli S30 coupled transcription-translation system, a functional stable protein was not produced in the E. coli clone. However, the gfo gene cloned into a shuttle vector was shown to overexpress glucose-fructose oxidoreductase to levels of up to 6% of the soluble protein in Z. mobilis.

The 13 major enzymes which compose the glycolytic and fermentative pathways in Zymomonas mobilis are particularly abundant and represent one-half of the soluble protein in exponential-phase cells. One- and two-dimensional polyacrylamide gel electrophoresis maps were developed for 12 of these enzymes. Assignments were made by comigration with purified proteins, comparison with overexpressed genes in recombinant strains, and Western blots (immunoblots). Although most glycolytic enzymes appeared resistant to turnover and accumulated in stationary-phase cells, the protein levels of pyruvate kinase, alcohol dehydrogenase I, and glucokinase declined. Alcohol dehydrogenase II was identified as a major stress protein and was induced both by exposure to ethanol and by elevated temperature (45-degrees-C). This enzyme, encoded by the adhB gene, is expressed from tandem promoters which share partial sequence identity with the Escherichia coli consensus sequence for heat shock proteins.

Pyruvate decarboxylase (EC 4.1.1.1) from Zymomonas mobilis purified to homogeneity by using dye-ligand and ion-exchange chromatography. Antibodies produced against the enzyme and the amino-terminal sequence obtained for the pure enzyme were used to select and confirm the identity of a genomic clone encoding the enzyme selected from a genomic library of Z. mobilis DNA cloned into pUC9. The genomic fragment encoding the enzyme expressed high levels of pyruvate decarboxylase in Escherichia coli. Possible RNA polymerase and ribosome-binding sites have been identified in the 5'-untranslated region of the pyruvate decarboxylase gene.

Pyruvate decarboxylase (EC 4.1.1.1), the penultimate enzyme in the alcoholic fermentation pathway of Zymomonas mobilis, converts pyruvate to acetaldehyde and carbon dioxide. The complete nucleotide sequence of the structural gene encoding pyruvate decarboxylase from Zymomonas mobilis has been determined. The coding region is 1704 nucleotides long and encodes a polypeptide of 567 amino acids with a calculated subunit mass of 60,790 daltons. The amino acid sequence was confirmed by comparison with the amino acid sequence of a selection of tryptic fragments of the enzyme. The amino acid composition obtained from the nucleotide sequence is in good agreement with that obtained experimentally.

The enzymes responsible for sorbitol formation in Zymomonas mobilis were investigated. A previously undescribed enzyme catalyzes the intermolecular oxidation-reduction of glucose and fructose to form gluconolactone and sorbitol. This enzyme has been purified; it had a subunit size of 40,000 daltons and is probably tetrameric at low pH. It contained tightly bound NADP as the hydrogen carrier and did not require any added cofactor for activity. In addition, a gluconolactonase has been isolated, although not completely purified. Together these two enzymes were capable of completely converting a 54% (wt/vol) equimolar mixture of glucose and fructose to sorbitol and sodium gluconate at the optimum pH of close to 6.2. The oxidoreductase had low affinities for its substrates, but natural environmental conditions would expose it to high concentrations of sugars. The amount of the enzyme in Z. mobilis cells was sufficient to account for the rate of sorbitol formation in vivo. However, the enzyme was present in the highest amounts when the cells were grown on glucose alone, and it was repressed by the presence of fructose; this was not the case with the gluconolactonase.

The enzyme gluconate kinase EC 2.7.1.12 has been found at high levels in glucose-grown Zymomonas mobilis cells. A simple procedure, based on differential dye-ligand chromatography, has been used to isolate the enzyme, purifying it some 600-fold. The purified enzyme is a monomer of molecular weight 18,000 Da, which is much smaller than other gluconate kinases reported. It has a relatively low affinity for ATP. (Km = 1.5 mM), but high for gluconate (Km = 0.33 mM), and has little activity with any other potential substrates.

1. An isolation procedure for preparing gram quantities of a previously undescribed protein from muscle is presented. 2. The protein amounts to over 2% of the sarcoplasmic proteins of pig longissimus dorsi muscle, but does not correspond to any glycolytic enzyme, nor to any of several other enzymes that have been tested for. 3. The protein is isoelectric between pH8.5 and 9.0, has mol.wt. 34500+/-500 and has E(1%) (281mmu) 20.7. 4. The identities of nearly all the major protein bands obtained in electrophoresis on starch gel of pig longissimus dorsi muscle sarcoplasm have been established.

ULTRASONIC VIBRATIONS AND MICROSOMES 201 4. The activity of ultrasonically treated micro- somes could not be restored by polyuridylic acid.5. The application of ultrasonic vibrations on microsomes caused a several-fold increase in phosphodiesterase activity as measured by the breakdown of [8H]polyuridylic acid.As a break- down product, an increased amount of 5'-UMP was obtained.There was no significant effect on the breakdown by ultrasonically treated ribonucleo- protein particles.6. Electron microscopy showed that on treat- ment by ultrasonic vibrations the microsomal fraction retained membranes and particles.No apparent change in the structure of isolated ribonucleoprotein particles was observed.The membrane-bound enzyme glucose 6-phosphatase was not destroyed by treatment with ultrasonic vibrations for 1 min.