Purification and Characterization of the Carboxypeptidase Isoinhibitors from Potatoes

Abstract

Three zones of carboxypeptidase inhibitory activity were observed when heat-stable extracts of potato tubers (cv. Russet Burbank) were chromatographed on carboxymethyl celulose. The isoinhibitors found in these zones were denoted I, II, and III based upon their order of elution from this column. The predominant form (II) had previously been suggested to be a mixture of two polypeptides (IIa and IIb) MATERIALS AND METHODSMaterials. Bovine CPB was prepared from trypsin-activated acetone powder extract (1) and the polypeptide chymotrypsin inhibitor was prepared from Russet Burbank potato tubers as described earlier (15). Bovine CPA, horse heart Cyt c, lima bean trypsin inhibitor, hippuryl-L-arginine, and hippuryl-L-phenylalanine were purchased from Sigma. Sephadex G-25 SF, G-50 SF, and G-75 were obtained from Pharmacia, and CM32 and DE52 were from Whatman. Pyridine was distilled from ninhydrin prior to its use in chromatographic systems (16) and all other chemicals were used without further purification.Purification of Isoinhibitors. The polypeptide fraction was obtained by chromatography of crude inhibitor I, the heat-stable fraction of extracts of Russet Burbank potatoes on Sephadex G-75 as described previously (21). After lyophilization, polypeptide material derived from about 14 kg of potatoes was dissolved in 5 mM sodium citrate (pH 4.3) and chromatographed on a column (2.5 x 40 cm) of CM-cellulose. Following elution of the breakthrough peak, the column was developed with a linear gradient (0-0.5 M) of NaCl in equilibration buffer. The flow rate was 150 ml/h. Fractions of 12 ml were collected and monitored for A at 280 nm and inhibitory activity against CPA (see below). Three zones of inhibitory activity were observed and the appropriate fractions were pooled, lyophilized, desalted by chromatography on a column (2.5 x 80 cm) of Sephadex G-25 SF, and relyophilized.CPI-I, which eluted in the breakthrough of the CM-cellulose column, was purified to apparent homogeneity by chromatography on a column (1.5 x 30 cm) of DEAE-cellulose. A 400-ml linear gradient (0-0.2 M) of NaCl in equilibration buffer (10 mm Tris-HCl [pH 7.5]) was employed to develop the column.CPI-II, the form of the CPI which had been characterized previously (25), was separated into two closely related species (14) by equilibrium chromatography on a column (2.5 x 40 cm) of CM-cellulose using 10 mm sodium citrate (pH 4.0) as eluant. Fractions of 10 ml were collected and pooled based on A at 280 nm. Partially purified CPI-IIa and CPI-IIb were then rechromatographed in this system. CPI-III, noted previously (14), was nearly pure after desalting on Sephadex G-25 as described above.Electrophoretic Procedures. Polyacrylamide gel electrophoresis was performed as described by Panyim and Chalfley (23). Gels (15% acrylamide/0.1% bisacrylamide containing 6 M urea) were used at pH 3.2. Samples were applied in 40%o sucrose and run at 3 mamp/gel. The gels were stained for 2 h in 1% Amido black in 7.5% acetic acid and then destained by diffusion using ...

Distribution of Carboxypeptidase Isoinhibitors in the Potato Plant

“…Thus, immobilized carboxypeptidase ( Fig. 1) was an effective means of eliminating unwanted protein, and the three isoinhibitor families described previously (7) accounted for most, if not all, of the CPI activity in the potato plant. The relative amounts of each of the isoinhibitor families present in each tissue were estimated by densitometry of the stained polyacrylamide gels (Fig.…”

“…In a companion manuscript the purification and characterization of three isoinhibitor forms of the CPI are discussed (7). The distribution of CPIs in the potato plant and the relative amounts of each form are presented herein.…”

“…Although inhibitors of the serine proteases are widely distributed among plant species, the potato is the only plant which has been shown to have an inhibitor of the pancreatic carboxypeptidases (14,15). The purification and amino acid sequence analysis of three families of carboxypeptidase isoinhibitors have been presented (7).…”

Specificity of the Carboxypeptidase Inhibitor from Potatoes

“…Carboxypeptidase isoinhibitor II (CPI4) was purified from Russet Burbank potatoes as described by Ryan et al (28,15). CPI was coupled to CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals) as described previously (I).…”

“…Alanine was detected after paper chromatography as described for the lemon peel enzyme. Tomato fruit (Lycopersicon esculentum) was homogenized at 4 C for I min and the homogenate was centrifuged at 9,000 rpm for 30 min at 4 C. The clear extract was adjusted to 0.5 M NaCl, 50 mm Tris-HCl (pH 7.5) and chromatographed on a column of CPDase A-Sepharose 4B to remove the endogenous CPDase inhibitor (15,16). The breakthrough fraction was adjusted to 70% saturation with solid ammonium sulfate and then centrifuged at 9,000 rpm for 30 min.…”