Ammonium sulfate-precipitated enzyme preparation from

Figure 1. Elution patterns of the ammonium sulfate-precipitated enzyme preparation from a DEAE-Sephadex A-50 column. (0) CMC-saccharifying activity (5-min incubation) of eluates diluted 60-fold, (O) Avicel-saccharifying activity (1-hr incubation), ( ) protein concentration measured in terms of the absorbance at 280 nm column 5.0 X 50 cm flow rate 20 mL/8 min one fraction 20 mL.

The multiple activities in T. thermophila share some of the characteristics of both the squid-type OPA anhydrase and classical Mazur-type OPA anhydrase found in hog kidney. In crude preparations, the OPA anhydrase activity has the characteristics of the hog kidney OPA anhydrase in that it hydrolyzes soman faster than DFP, is stimulated by Mn2+, and is inhibited by mipafox. Further purification has revealed that the hydrolysis of soman and the stimulation of this hydrolysis by Mn2+ is principally due to the Tt DFPase-4. The Tt DFPase-1, Tt DFPase-2, and Tt DFPase-3 hydrolyze soman and DFP at approximately the same rates and demonstrate only moderate stimulation of soman hydrolysis by Mn2+ and yet are inhibited by mipafox. The Tetrahymena OPA anhydrases fall within a narrow range from 96,000 Da to 67,000 Da. However, this range of molecular weights is larger than that typically ascribed to the Mazur-type enzymes. The Tetrahymena OPA anhydrases can be purified by ammonium sulfate precipitation, like the squid-type OPA anhydrase. 

The enzyme, which in this case is glucose isomerase, was obtained from Novo Laboratories and is derived from Bacillus coagulans NRL-5666. The soluble enzyme was isolated from a dried cell preparation, as supplied by Novo Laboratories, by suspension in buffer and centrifugation to remove cellular material and other insoluble impurities. The resulting solution was fractionated by ammonium sulfate precipitation retaining the 55-70% fractions followed by dialysis to obtain a purified enzyme suitable for immobilization onto the microporous support. 

The reaction mixture (final volume 0.34 ml) contained (in /umol and including compounds added with the enzyme) potassium phosphate, pH 6.3, 102 Tris, pH 7.6, 5 sucrose, 13 GSH, 16 2-mercaptoethanol, 0.7 Na EDTA, 0.5. Methyl donors were added at the specified final concentrations. The various stereoisomeric forms of S-adenosylmethionine were prepared as described by de la Haba el al. (1959) using adenosylmethionine cyclotransferase (E.C. 2.5.1.4) to resolve (/(S)-S-adenosyl-L-methionine to (f )-5-adenosyl-L-methionine. The reaction was started by addition of enzyme dissolved in Tris buffer, pH 7.6, 25 mM, containing Na EDTA, 1 mM and 2-mercaptoethanol, 7 mM, and partially purified from an extract of cabbage leaves by successive ammonium sulfate precipitation (45-60% saturation), gel filtration (Sephadex G-25) and DEAE-cellulose column chromatography. After incubation at 26°C for 1 h, each reaction mixture was diluted with 0.76 ml cold water, and a 1.0 ml aliquot was immediately added to a column of Dowex-50(NHJ) 1 x 3 cm. Unreacted [ C]methionine was removed by washing the column with 25 ml water. The 5-[" C]methylmethionine sulfonium salt was then eluted with 8 ml 0.6 N NH OH. Radioactivity was determined by scintillation spectrophotometry (Mudd et al., 1965). A blank of 540 cpm has been subtracted from each value. The low activity with (R)-5-adenosyl-L-methionine may have been due to the presence of a small residual amount of (5)-5-adenosyl-L-methionine (de la Haba eta/., 1959). 

This conception works out as shown in Scheme 10 CTP 23 formed by the above described sequence is directly consumed by -acetyl neuraminic acid 26 under the catalytic influence of cytidine-5 -monophosphosialate synthase (E.C. 2.7.7.43). This enzyme is isolated from calf brain by ammonium sulfate precipitation (2 5) and subsequent affinity chromatography. The stationary phase consists of CNBr-activated Sepharose 4B reacted with p-[3-(2-amino ethylthio)propyl]-iV-acetyl neuraminic acid 27, which is synthesized by radiating a mixture of the allyl glycoside and cysteamine to achieve radical C-S bond formation (24), The behavior of methyl p-N-acetyl-neuraminic acid as an inhibitor is in accordance with Zbiral s findings (25), where the methyl a-glycoside has been shown to compete with the native substrate for the enzyme, and thus 27 is recommended to be an ideally suited ligand (Scheme 9). A typical analytical run is shown in Scheme 9. Due to elution of the protein fraction by a salt gradient, the transfer to a preparative scale is rather difEcult denaturation occurs and thus a drop in activity down to 6% is observed. 

Antibodies were raised in rabbits against the individual polypeptides components of the ftc. complex that were purified by preparative SDS-polyacrylamide gel electrophoresis p]. IgG was purified from the antisera by ammonium sulfate precipitation, and immunoblotting was performed as described by Towbin et al.[5] using peroxidase-labeled goat anti-rabbit IgG as the second antibody and 4-chloro-l-naphthol for the staining reaction. Assessment of the specificity of the antisera by this procedure indicated that of the four subunits, only the 14 MDa component was non-immunogenic. Antibody titers were measured by enzyme-linked immunosorbant assay. For the iron-sulfur protein, sera obtained 11 weeks after immunization exhibited a detectable reaction at dilutions up to 10. ... 

Ti-Aspartic Oxidase. Aspartase and transaminases account for a major part of the metabolism of L-aspartic acid. n-Aspartic acid is oxidized by an enzyme present in liver and kidney. This is an oxidase that converts aspartate to oxalacetate and ammonia while reducing oxygen to hydrogen peroxide. The oxidase was resolved by ammonium sulfate precipitation and dialysis to a protein that could be reactivated by FAD but not by FMN. The enzyme differs from n-amino acid oxidase in its insensitivity to benzoate. The only other known substrate for the partially purified D-aspartic oxidase is D-glutamate, but since the relative rates of oxidation of the two amino acids vary during the preparation of the enzyme, it is... 

Thiamine pyrophosphate has also recently been shown to exert a coenzyme function in the metabolism of pentose phosphate. - The formation of sedoheptulose phosphate from ribulose-5-phosphate by a highly purified enzyme preparation from spinach, which loses activity upon precipitation of the protein with ammonium sulfate at a low pH, was found to be almost completely reactivated by the addition of thiamine pyrophosphate. ... 

This enzyme catalyzes the reaction of epoxides of both aromatic and aliphatic hydrocarbons (Booth et al., 1960, 1961 Boyland and Williams, 1965 Jeriiia et al, 1968). The enzyme was isolated from rat liver supernatant liquor by ammonium sulfate precipitation followed by absorption of impurities on calcium phosphate gel, The best preparation contained... 

The dried bacteria also contain invertase and this hydrolytic enzyme competes with the sucrose phosphorylase for sucrose. However, it is possible to eliminate most of the invertase from the bacterial preparations by several precipitations with ammonium sulfate. Using a partially purified sucrose phosphorylase preparation and a mixture of... 

Evidence was also obtained for a non-cytochrome nitrosamine demethylase. This material was best obtained from "pH 5 enzyme" supernatant (25) by precipitation in 33 percent ammonium sulfate. This preparation requires NADPH but in contrast to cytochrome P-450 enzymes (26), it is not inhibited by carbon monoxide. 

A large scale preparation of E. coli 045 was subjected to enzyme purification using the assay for 3,5-epimerase. Protamin sulfate precipitation, ammonium sulfate fractionation was followed by DEAE-chroma-tography. The fraction containing enzymatic activity, as measured by tritium exchange, was eluted from the DEAE column early. This fraction was incapable of producing any net synthesis of TDP-6-deoxy-L-... 

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