Comparison of stored and secreted rat pancreatic digestive enzymes by mass spectrometry: alpha-amylase.
As part of a continuing effort to better understand the mechanisms of protein secretion, we compared the mass of pancreatic digestive enzymes, in resting and stimulated states, both in secretion and in the zymogen granule to determine whether their secretion is accompanied by chemical modification. Mass spectra were obtained applying the electrospray method on samples separated by reverse-phase HPLC. We report here our results for alpha-amylase (1,4-alpha-D-glucan glucanohydrolase EC 3.2.1.1). The data illustrate structural differences between states and compartments for this enzyme. Multiple isozymes were identified from the mass spectra, varying roughly from 52 to 60 kDa. On the basis of mass comparisons, not all of the products seen in the zymogen granule were found in secretion, nor were all secreted isoforms in the granule. Stimulation of protein secretion with a cholinergic agonist, led to time-dependent changes in the number and masses of isoforms in secretion, leaving only one of five resolvable forms in the granule. Only one form, 55.5 kDa, was found in all samples, granule and secretion. In addition to these differences, microheterogeneities of 400 Da or less were observed. The data suggest the differential or non-parallel release of different amylase forms and their chemical modification during the secretion process. As such, release appears to involve a third, intermediate compartment, between zymogen granule to ductal space, such as the cytoplasm, in which chemical modification takes place.
Young
M K
MK
Department of Chemistry, University of California, Riverside, CA 92521, USA.
Tseng
H C
HC
Fang
H
H
Liang
W
W
Rothman
S S
SS
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000201:enzymology*; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D005089:Exocytosis; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D016210:Methacholine Chloride / Q000494:pharmacology; D008970:Molecular Weight; D018721:Muscarinic Agonists / Q000494:pharmacology; D010179:Pancreas / Q000201:enzymology*; D010189:Pancreatic Juice / Q000201:enzymology; D051381:Rats; D017207:Rats, Sprague-Dawley; D000516:alpha-Amylases / Q000737:chemistry* / Q000378:metabolism
D004792:Enzyme Precursors; D007527:Isoenzymes; D018721:Muscarinic Agonists; D016210:Methacholine Chloride; D000516:alpha-Amylases