Single step affinity chromatographic purification of human alpha-amylase from aspirated duodenal juice and its application in the measurement of pancreatic alpha-amylase synthesis rates in man.
Human alpha-amylase was purified from aspirated duodenal juice to electrophoretic homogeneity in a single step by affinity chromatography with the competitive inhibitor acarbose (IC50 = 1.22 mumol/l) as ligand. Duodenal juice was applied to an agarose resin to which acarbose had been coupled covalently via a 1.9 nm spacer group. Pure alpha-amylase, eluted with free acarbose, had a molecular mass of 55,000, and isoelectrofocusing revealed the presence of six isozymes with pI values of 7.3, 6.8, 6.7, 6.5, 6.4 and 6.3, all of which possessed amylase activity based on positive starch/iodine staining. The potential usefulness of this one-step purification procedure in the measurement of pancreatic alpha-amylase synthesis rates was evaluated in two control patients with non-pancreatic disease. Aspirated duodenal juice was obtained during a pulse/continuous intravenous 4 h infusion of [14C]leucine together with secretin and pancreozymin, and alpha-amylase purified using our protocol. Pancreatic alpha-amylase synthesis rates were determined from the rate of incorporation of [14C]leucine into alpha-amylase; values of 4.4 and 5.1 h were obtained for the two control patients.
Ogden
J M
JM
Gastrointestinal Clinic, Groote Schuur Hospital, University of Cape Town Medical School, Republic of South Africa.
O'Keefe
S J
SJ
Ehlers
M R
MR
Kirsch
R E
RE
Marks
I N
IN
D020909:Acarbose; D000328:Adult; D002846:Chromatography, Affinity / Q000379:methods; D004386:Duodenum; D006801:Humans; D007419:Intestinal Secretions / Q000032:analysis*; D008297:Male; D010179:Pancreas / Q000201:enzymology*; D010437:Peptic Ulcer / Q000201:enzymology; D014312:Trisaccharides / Q000494:pharmacology; D000516:alpha-Amylases / Q000096:biosynthesis / Q000302:isolation & purification*
D014312:Trisaccharides; D000516:alpha-Amylases; D020909:Acarbose