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 ...

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ABSTRACrThe total amount of carboxypeptdase inhibitor was estmated in ex (14,15) and roundworms (9). Studies on the inhibitor from potato tubers have included purification and general characterization (15), amino acid sequence determination (8) and disulfide bond pairng (11), chemical modifications (6), and estimation of levels in a variety of potato cultivars (16). In addition, the use of immobilized CPI3 to purify target enzymes has been explored (1).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. MATERIALS AND METHODSPlant MateriaL Four Solanum tuberosum L. cv. Russet Burbank tuber seed pieces were planted in artificial media 1:1:1, v,v) fertilizer added, pH adjusted to 6, and grown under greenhouse temperatures of 24 C/18 C night. Metal halide lighting, supplying a minimum of 320 lux, was used for supplementary lighting and also to extend the photoperiod to 16 h. Single stem plants were established by pinching lateral shoot growth and secondary shoots arising from the tuber. Harvest took