Rapid zymogen activation and isolation of serine proteases from an individual mouse pancreas by affinity chromatography: genetical heterogeneity of chymotrypsins of Mus musculus.
A rapid method to prepare homogeneous fractions of the various chymotrypsins and trypsins from a single mouse pancreas (130-150 mg wet weight) is described. The method was applied to investigate intra-species variation on a molecular level using chymotrypsins as biochemical indicators. The conditions for optimal extraction of the zymogens in the homogenized pancreas have been studied. DNA had to be removed from the homogenate to obtain maximum chymotrypsin yields (approximately 1% of the wet weight of the pancreas). The activation was initiated by immobilized bovine trypsin that was removed by filtration. Then chymotrypsinogens in the homogenate were activated by mouse trypsin. After completed activation homogeneous chymotrypsins (one anionic and one cationic form) could be isolated in an one step analytical affinity chromatographic separation, using soybean trypsin inhibitor bound in Sepharose as a protease specific adsorbent. The end products were characterized by isoelectric focussing, amino acid composition, enzymatic parameters, molar extinction coefficient, and the number of polypeptide chains. Hereby, the existence of two chymotrypsinogen loci in the mouse genome could be demonstrated. Differences in structure and function between the corresponding enzymes from the two strains were found. This allelomorphism was verified in the crossing of the off-spring.
D000483:Alleles; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002918:Chymotrypsin / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D002919:Chymotrypsinogen / Q000378:metabolism*; D005796:Genes; D051379:Mice; D008815:Mice, Inbred Strains; D010179:Pancreas / Q000201:enzymology*; D013045:Species Specificity; D014357:Trypsin / Q000378:metabolism*
D002919:Chymotrypsinogen; D002918:Chymotrypsin; D014357:Trypsin