Purification and Characterization of ?-Amylase Inhibitors in Wheat (Triticum Aestivum Var. Anza)

C. Postal 6001, 84051-970 -Londrina, PR -Brazil 2Dept. Alimentos e Nutricdo Experimental Faculdade de Ci6ncias Famc8uticas Universidade de Sdo Paul0 C. Postal 66083. 05389-970 -SaO Paulo, SP -Brazil ABSTRACTA total of six a-amylase inhibitory proteins (isoinhibitors) were extracted from triticale (Triticum x Secale) seeds and two of rhem were pur$ed to homogeneity. The isoinhibitors were extracted by 70% ethanol and produced, by Sephade-r G l 0 0 chromatography, two peaks that exhibited a-amylase inhibitory activity. Further purijkation ofthe most active peak by DEAE-cellulose chromatography resulted in six activefractions. Two of them designated as TAl-5 and TAl-6, considered to be homogeneous by both acidic and alkaline electrophoresis, were partially characterized. Ihe isoelectric points were 4.80 and 4.70, and the molecular weights 39,200 and 29,200, respectively. Under dissociating conditions the molecular weights were 13,500 and 13,000, suggesting that the isoinhibitors are composed of three and two subunits, respectively. Both isoinhibitors were stable at different pHs, relatively stable at 98C, and resistant to proteolysis by trypsin, chymotrypsin and pepsin. The optimum interaction pH for both isoinhibitors with human salivary amylase was 7.9. They exhibited specijkiry to human salivary and porcine pancreatic a-amylases, but had no inhibitory activiry on Bacillus subtillis, Aspergillus oryzae and endogenous tnticale a-amylases.

Section: Specificity Of the Inhibitorssupporting

confidence: 79%

“…Several properties of the two purified isoinhibitors are similar between them and similar to those of wheat and rye inhibitors (Silano 1978;Granum and Whitaker 1977;Granum 1978). Preincubation for 40 min at 37C was needed for maximum inhibition.…”

Section: Physical and Chemical Propertiesmentioning

confidence: 72%

Purification and Partial Characterization of Two Proteinaceous ?-Amylase Inhibitors From Triticale

Ida

Finardi-Filho

Lajolo

1994

“…The a-amylase inhibitors in wheat have received the most attention Sandstedt 1943, 1946;Shainkin and Birk 1970;Silano et al 1973;Petrucci et ul. 1974;O'Donnell and McGeeney 1976; Granum and Whitaker 1977). Although extracts from diploid wheats do not contain detectable inhibitor activity, all tetraploid and hexaploid wheats as well as Aegilops species contain several inhibitor components having different electrophoretic mobilities (Bedetti et al 1974).…”

Section: Introductionmentioning

confidence: 99%

PURIFICATION AND PARTIAL CHARACTERIZATION OF TWO ?-AMYLASE INHIBITORS FROM BLACK BEAN (Phaseolus vulgaris)

Frels¹,

Rupnow²

1984

Tho a-amylase inhibitors from black bean (Phaseolus vulgaris) were purified to homogeneity using ammonium sulfate fractionation, DEAE -Sephadex chromatography, phenyl-Sepharose hydrophobic interaction chromatography and gel filtration with Sephadex G-100. The inhibitors were designated I-1 and I-2 based on their order of elution from the phenyl-Sepharose column. Both inhibitors are mannose containing glycoproteins, composed of subunits; active against porcine pancreatic, human salivary, and insect a-amylases and inactive against bacterial, mold, and plant a-amylases. The inhibitors I-1 and I-2 have molecular weights of 49,000 and 47,000 and isoelectric points 4.93 cznd 4.86, respectively. Both inhibitors have similar amino acid compositions and are rich in aspartic acid, serine, glutamic acid, valine, and threonine and are low in sulfur containing a,mino acids. I-2 is more resistant to heat denaturation than I-1.

“…Some other reports of the presence of amylase inhibitors in wheat include those by Militzer et al (1946a); Shainkin and Birk (1965); Saunders et al (1971); Strumeyer (1972); Silano et al (1975); Petrucci et al (1974); Bedette et at. (1974); O'Donnell and McGeeney (1976); Granum andWhitaker (1977 andWarchalewski (1977a,b). a-Amylase inhibitors have been reported in beans by Bowman (1943Bowman ( , 1945; Jaffk and Vega-Lette (1968); Hernandez and Jaffk (1968); Marshall and Lauda (1975); Powers and Whitaker (1975); in rye Strumeyer (1972); in mango Matto and Modi (1970); in taroroot Rao et al (1967) and in acorn Stankovic and Markovic (1963).…”

Section: Introductionmentioning

confidence: 99%

Purification and Characterization of an ?-Amylase Inhibitor From Maise (Zea Maize)

Blanco‐Labra

Iturbe-Chiñas

1981

α‐Amylase inhibitor is presented in maize seeds. It is a protein as indicated by precipitation with ammonium sulfate and trichloroacetic acid, denaturation by heat, digestion with proteases and by dye‐staining. It was purified to homogeneity by ammonium sulfate precipitation and Sephadex G‐75 gel filtration. It had an apparent molecular weight of 29,600 and did not contain any carbohydrate. Its properties differed from those of previously reported α‐amylase inhibitors, since it was active against α‐amylase of maize, produced during germination as well as against Bacillus subtilis α‐amylase. It was also active against α‐amylase from the insects Tribolium castaneum, Sitophilus zeamais and Rhyzopertha dominica, but it was inactive against α‐amylase from human saliva, hog pancreas, Aspergillus oryzae, wheat, rye, barley, triticale, and sorghum. It was stable for 5 min at 96°C at pH 7. Maximal inhibition required at least 10 min of preincubation with the enzyme at pH 6.8 and 257deg;C. Polyacrylamide gel electrophoresis gave three protein bands, but only one was obtained in S.D.S. and mercaptoethanol.