Enzymes sulfate-precipitated

In vitro studies were conducted with enzymes extracted from peanut (7), pea (. ), and onion (9). The enzymes were fractionated by ammonium sulfate precipitation, dialyzed, and stored frozen until used. The enzymes were assayed for various activities as described In the Results and Discussion. 

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-... 

In view of the high stability of the enzyme most samples have been prepared by the procedure described by Kunitz (16) and modified by McDonald (17) to remove all traces of proteolytic activity. During this procedure the minced bovine pancreas is exposed to 0.25 N sulfuric acid, ammonium sulfate precipitation, 10 min at 95°-100° and pH 3, and, finally, reprecipitation. The product can be crystallized it was also shown later to contain a number of components all with ribonuclease activity. A practical summary of all details is given by Kunitz and McDonald (18). 

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.

Once the crude cellulase solution was obtained, it was concentrated and at the same time separated from most of the salts (left from the fermentation) in the enzyme solution. The steps which accomplished this were (1) addition of ammonium sulfate (75% saturation) to precipitate protein (2) recovery of the protein as a pellet by centrifugation at 12,000 rpm for 15 min (3) redissolution of the protein in 0.1M sodium phos-phate-0.2mM EDTA buffer (pH 6.8) (4) desalting on a 1.5 X 45 cm Sephadex G-25 column and (5) lyophilization to obtain concentrated enzyme followed by a final ammonium sulfate precipitation to obtain precipitated enzyme. 

ATP-extraction followed by backextraction showed a considerable improvement over purification with protamine sulfate precipitation. A multistage ATP extraction followed by multistage backextraction should further improve the enzyme recovery. This would be in keeping with the observations of Lee et al.66 and of Sebastiao et al.69... 

ALS was isolated from barley seedlings as a 0-33% Ammonium Sulfate precipitate and examined for inhibition by TP. It is apparent from Figure 5 that the enzyme is very sensitive to the compound. The 1(50) value (concentration required for 50% inhibition) was calculated to be 0.047 uM. This value is within the range reported for CS tested against ALS from different species (19). Imidazolinones are less potent with 1(50) values in the range 2-12 uM (26). ALS isolated from several species and their 1(50) values for TP is shown in Table I. 

The overall recovery of the enzyme was >60 % with a 235-fold purification. The final preparation, however, was not homogenous. Barley has two isoforms of ALS which can be separated on a phenyl agarose column immediately after the ammonium sulfate precipitation. One of the forms does not bind to the column and was too unstable to attempt purification. The details of purification in Table III pertain to the fraction with affinity for phenyl agarose. 

The detailed study of enzyme mechanisms requires the use of purified if not homogeneous enzymes. This experiment presents three procedures commonly used in protein purification ammonium sulfate precipitation, heat denaturation, and ion-exchange chromatography. Although the purification procedure outlined in this experiment is useful in the isolation of glutamate-oxaloacetate transaminase (GOT), the same techniques can be modified to aid in the purification of many other proteins of interest. 

Figure 5.7 The use of HPLC to monitor enzyme purification. These profiles were obtained by gel filtration chromatography during the purification of the enzyme sAMP synthetase. The column was a TSK-250 (BioSil, 7.5 mm X 30 cm), and the mobile phase was 0.1 M potassium phosphate (pH 6.0). The column was monitored at 280 nm. Profiles obtained after (A) 30 to 50% ammonium sulfate precipitation, (B) affinity chromatography, (C) ion-exchange chromatography on DE-52, and (D) HPLC ion-exchange chromatography on AX-300.

Retinal oxidase was assayed in a medium containing 0.1 M phosphate buffer (pH 7.7) and 10 piL of 25 mM retinal dispersed in acetone containing 5% Triton X-100. The reaction was initiated by adding 20 to 40 fih of reconstituted ammonium sulfate precipitate or cytosol. The final volume was 500 /xL. The reaction was continued at 37°C for 30 minutes and was stopped by freezing at -70°C, whereupon 50 juL was injected directly onto the column. Enzyme activity was linear with protein concentration up to 2.4 mg/mL, and with time up to 30 minutes. 

Figure 9.151 Determination of strictosidine synthetase activity by HPLC. Codeine (a), tryptamine (b), and strictosidine (c) were separated on a 4.0 (i.d.) X 250 mm LiChrosorb RP-8 Select B column at a flow rate of 1.0 mL/min. Incubation was for 30 minutes at 30°C with enzyme from Catharanthus roseus after ammonium sulfate precipitation (35-50% saturation) and gel filtration on Sephadex G-25, in the presence of 100 mM fi-D-gluconolactone. Injection volume was 8 pL and the UV detector was set at 0.02 AUFS. (From Pennings et al., 1989.)...

The highest specific activity of dehydrocyclopeptine epoxidase was measured after acetone treatment of hyphal cells and conidiospores. More than one-third of the measurable activity sedimented with the cell wall-plasma membrane fraction, from which it could be solubilized by a 1% solution of deoxycholate. The soluble enzyme fraction was purified by ammonium sulfate precipitation and gel chromatography, and its molecular weight was estimated to be near 500,000. 

In early experiments, glucoamylase was purified by ammonium sulfate precipitation, followed by an acid treatment to eliminate traces of alp/ia-amylase, but gel filtration and ion-exchange chromatography have now been successfully applied to purification of the A. niger, R. delemar, and gamma-amylases. Crystallization of the enzymes from A. niger and R. delemar has also been reported. ... 

For many years salting-out by high concentrations of ammoniiun sulfate has been one of the classical methods of protein separation. There is very little literature on the theoretical basis of the method, particularly as applied to the isolation of enzymes, where it has mainly been used quite empirically. The underlying assumption in most cases seems to have been that the different proteins are precipitated at different fixed ammonium sulfate concentrations, provided the pH and temperature are fixed. For example one may commonly read in instructions for the piuification of an enzyme that the enzyme is precipitated at 65% saturation with ammonium sulfate or that the fraction precipitating between 0.62 and 0.68 saturation should be taken. It is, however, a fairly common experience that when one repeats a published method the enzyme fails to precipitate within the limits given. Furthermore, where the purification of a protein involves more than one salt-fractionation stage, the limits are usually found to be different for the different stages. 

Add 0.1 ml of lysine solution and leave the mixture at 4° for 2 hr. Dialyze against several changes of PBS at 4°. If desired remove free enzyme by precipitation with saturated ammonium sulfate as described in steps 8-10. 

A crude cell-free extract of skeletal muscle contained 32 mg orotein/ml. Ten microliters of the extract catalyzed a reaction-aJL rate of. mole/min ) under standard optimum assay conditions, (gifty millilitei ot qhe extract were fractionated by ammonium sulfate precipitation. The fraction precipitating between 20% and 40% saturation was redissplved in 10 ml. This solution was found to contain 50 mg protein/ml. Ten microliters of this purified fraction catalyzed the reaction ata rate of 0 65 mole/min. Calculate (a) the percent recovery of the enzyme in the purified fraction, and (b) the degree of purification obtained by the fractionation (the purification factor). 

Fifty milliliters of the cell-free extract described above was fractionated by ammonium sulfate precipitation. The fraction precipitating between 30 and 50% saturation was redissolved in a total volume of 10 ml and dialyzed. The solution after dialysis occupied 12 ml and contained 30 mg protein/ml. Twenty microliters of the purified fraction catalyzed the phosphorylase reaction at a rate of 5.9 nmoles/min under the standard assay conditions. Calculate (a) the recovery of the enzyme and (b) the degree of purification obtained in the ammonium sulfate step. 

The intact enzyme can be kept for up to 4 months without major loss of activity, if stored as an ammonium sulfate precipitate in the presence of lactate, PMSF, and EDTA at 4°C under an atmosphere of nitrogen. The best preparations of intact enzyme yield around 1 jumol kg" of dried yeast (20). 

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. 

In this laboratory, attempts (G6, G8) have been made to purify and crystallize human placental alkaline phosphatase enzyme by a number of procedures involving homogenization with 0.05 M Tris buffer (pH 8.6), extraction with butanol, ammonium sulfate precipitation, exposure to heat, ammonium sulfate fractionation, dialysis, repeated ethanol fractionation, gel filtration with Sephadex G-200 (Fig. 18), continuous curtain electrophoresis on paper (Beckman Model CP), multiple TEAE-cellulose anion exchange chromatography, and equilibrium dialysis. Variant A (electrophoretically fast-moving) of human placental alkaline... 

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