Robert K. Scopes

Measurement of protein by spectrophotometry at 205 nm

Abstract A method is described for the measurement of protein concentration by using the peptide bond absorption at 205 nm. ϵ205 is estimated, allowing for the absorption due to Trp and Tyr residues, by measuring the absorbance at 280 nm as well as at 205 nm. The estimated ϵ205 is compared with the actual ϵ205 for a number of proteins, the mean error being less than 2%. This is about three times better than using an average ϵ2051 mg/ml of 31 and approaches the range of experimental error inherent in any method of protein estimation.

Purification and analysis of an extremely halophilic β-galactosidase from Haloferax alicantei

As a first step in the development of a reporter system for gene expression in halophilic archaea, a beta-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180 +/- 20 kDa in size, consisting of two monomers (each 78 +/- 3 kDa). It cleaves several different beta-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has beta-D-fucosidase activity. No beta-glucosidase, beta-arabinosidase or beta-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.

[79] Purification of all glycolytic enzymes from one muscle extract

Publisher Summary Muscle tissue cytoplasm is a very convenient source of the glycolytic enzymes. This chapter describes a scheme to purify all the glycolytic enzymes (except hexokinase), creatine kinase, adenylate kinase, and phosphorylase b kinase from the one extract of rabbit muscle. The chapter also presents enzyme assays, and the extraction procedures for glycolytic enzymes. The chapter proposes to develop a fractionation procedure that allows purification of 17 enzymes associated with the conversion of glycogen to lactate in muscle tissue. Although rabbit muscle is the source, many enzymes from muscle of pig, chicken, and some other species are purified by similar methods. The whole procedure can theoretically be completed in about three days, if limitless apparatus and personnel are available. However, storage of the redissolved ammonium sulfate precipitates for a few days at 0°C, or longer at –30°C allows the whole process to be comfortably extended over a couple of weeks. The enzymes are stored as crystalline suspension in ammonium sulfate at 5°C or frozen concentrated solutions without salt; they mostly retain their activity for many months or years.

Studies on cell-free metabolism: Ethanol production by a yeast glycolytic system reconstituted from purified enzymes

Abstract A reconstituted glycolytic system has been established from individually purified enzymes to simulate the conversion of glucose to ethanol plus CO 2 by yeast. Sustained and extensive conversion occurred provided that input of glucose matched the rate of ATP degradation appropriately. ATPase activity could be replaced by arsenate, which uncoupled ATP synthesis from glycolysis. The mode of uncoupling was investigated, and it was concluded that the artificial intermediate, 1-arseno-3-phosphoglycerate, has a half-life of no more than a few milliseconds. Arsenate at 4 mM concentration could simulate the equivalent of 10 μmol ml −1 min −1 of ATPase activity. The reconstituted enzyme system was capable of totally degrading 1 M (18% w/v) glucose in 8 h giving 9% (w/v) ethanol. The levels of metabolites during metabolism were measured to detect rate-limiting steps. The successful operation of the reconstituted enzyme system demonstrates that it is possible to carry out complex chemical transformations with multiple enzyme systems in vitro.

Activity and stability of glycolytic enzymes in the presence of ethanol

SummaryAn investigation of the effects of ethanol on both the stabilities and activities of glycolytic enzymes of yeast and Zymomonas mobilis is presented. It is concluded that enzyme denaturation is unlikely to play a direct part in ethanol tolerance, but inhibition by ethanol may be responsible for slowing some of the glycolytic reactions.

An iron-activated alcohol dehydrogenase

An alcohol dehydrogenase isolated from Zymomonas mobilis was found to be activated by ferrous ions but not by zinc, after inactivation with metal‐complexing agents. Cobaltous ions also re‐activated to a lesser extent. It is suggested that in this species the alcohol dehydrogenase naturally contains iron. Kinetic studies on the iron‐treated enzyme indicate an ‘alcohol activation’ phenomenon, which may have physiological relevance in overcoming product inhibition during fermentation.

Studies on cell-free metabolism: Ethanol production by extracts of Zymomonas mobilis

Using a cell-free extract of Zymomonas mobilis, it has been possible to achieve rapid and sustained ethanol production from added glucose. In one example 18% glucose was totally converted to 9% (w/v) ethanol. The controls on the glycolytic enzymes have been investigated by measuring metabolite levels during the experiment. No substantial accumulations of intermediates occurred when ATP production by the glycolytic metabolism was correctly balanced by ATPase activity. But as alcohol levels increased, some inhibitions of glucose 6-phosphate and pyruvate removal became apparent.

The structure of glucose-fructose oxidoreductase from Zymomonas mobilis: an osmoprotective periplasmic enzyme containing non-dissociable NADP.

BACKGROUND The organism Zymomonas mobilis occurs naturally in sugar-rich environments. To protect the bacterium against osmotic shock, the periplasmic enzyme glucose-fructose oxidoreductase (GFOR) produces the compatible, solute sorbitol by reduction of fructose, coupled with the oxidation of glucose to gluconolactone. Hence, Z mobilis can tolerate high concentrations of sugars and this property may be useful in the development of an efficient microbial process for ethanol production. Each enzyme subunit contains tightly associated NADP which is not released during the catalytic cycle. RESULTS The structure of GFOR was determined by X-ray crystallography at 2.7 A resolution. Each subunit of the tetrameric enzyme comprises two domains, a classical dinucleotide-binding domain, and a C-terminal domain based on a predominantly antiparallel nine-stranded beta sheet. In the tetramer, the subunits associate to form two extended 18-stranded beta sheets, which pack against each other in a face to face fashion, creating an extensive interface at the core of the tetramer. An N-terminal arm from each subunit wraps around the dinucleotide-binding domain of an adjacent subunit, covering the adenine ring of NADP. CONCLUSIONS In GFOR, the NADP is found associated with a classical dinucleotide-binding domain in a conventional fashion. The NADP is effectively buried in the protein-subunit interior as a result of interactions with the N-terminal arm from an adjacent subunit in the tetramer, and with a short helix from the C-terminal domain of the protein. This accounts for NADPs inability to dissociate. The N-terminal arm may also contribute to stabilization of the tetramer. The enzyme has an unexpected structural similarity with the cytoplasmic enzyme glucose-6-phosphate dehydrogenase (G6PD). We hypothesize that both enzymes have diverged from a common ancestor. The mechanism of catalysis is still unclear, but we have identified a conserved structural motif (Glu-Lys-Pro) in the active site of GFOR and G6PD that may be important for catalysis.

Separation by Precipitation

In the early days of protein chemistry, the only practical way of separating different types of proteins was by causing part of a mixture to precipitate through alteration of some property of the solvent. Precipitates could be filtered off and redissolved in the original solvent. These procedures remain a vitally important method of protein purification, except that filtration has mostly been replaced by centrifugation. Protein precipitates are aggregates of protein molecules large enough to be visible and to be centrifuged at reasonably low g forces. In some cases, the aggregation continues and the precipitate flocculates, but usually the motions and collisions of the particles in suspension keep their size small. This results in clogging of filter papers and necessitates centrifugation at considerably more than 1 g. The various methods of obtaining a precipitate are described in the separate sections below.

Isolation and characterization of the gene encoding gluconolactonase from Zymomonas mobilis.

The gene encoding the enzyme gluconolactonase (D-glucono-delta-lactone lactonohydrolase, EC 3.1.1.17) has been isolated from a recombinant library of genomic Zymomonas mobilis DNA, by detection of enzyme activity in recombinant clones. The gene encoded a protein of 320 amino acids, which is processed to the mature enzyme of 285 amino acids (31079 Da) by cleavage at an Ala-Ala bond, as determined from N-terminal sequencing of the purified enzyme. A minor sequence commencing at amino acid 6 is suggestive of an alternative start of translation at the ATG codon of amino acid 5; in this case the expressed enzyme would remain cytoplasmic, whereas it is presumed that the main portion is directed to the membrane of periplasm by the leader sequence.