Effect of substrate size on immunoinhibition of amylase activity

Abstract

Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10-fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one.

Accurate quantitation of salivary and pancreatic amylase activities in human plasma by microchip electrophoretic separation of the substrates and hydrolysates coupled with immunoinhibition

“…The oligosaccharides migrated in order of their molecular weights (data not shown). Several methods for the determination of amylase activity involving maltooligosaccharides as sub- strates have been developed over the last few decades [6,[18][19][20][21][22][23][24]. The pattern of cleavage of synthesized maltooligosaccharides by each a-amylase isoenzyme is known to be different [20].…”

“…Several analytical methods for each individual amylases have been reported based on electrophoresis [8][9][10][11][12], column chromatography [13,14], and radioimmunoassaying [15][16][17]. Recently, various-length maltooligosaccharides have been employed for amylase assaying as substrates [6,[18][19][20][21][22][23][24], and a wheat germ inhibitor [19] and a mAb [6,24] have also been introduced to isoamylase assay-ing to distinguish between P-AMY and salivary amylase (S-AMY). Although this colorimetric analysis involving oligosaccharides as enzyme substrates with mAb to inhibit S-AMY has become a widely used method for the determination of P-AMY in human blood for the diagnosis of acute pancreatitis in the clinical laboratory, it is time-and sampleconsuming, and is not adaptable to automated analyzers.…”

“…Several methods for the determination of amylase activity involving maltooligosaccharides of defined chain length coupled to a chromophore as substrates have been reported [6,[18][19][20][21][22][23][24]; however, these methods are complex because of the necessity of auxiliary enzymes such as a-glucosidase and glucoamylase [22][23][24] or stimulants for chromophore release from maltooligosaccharides such as potassium thiocyanate and sodium acetate [20,21]. With this proposed analysis method involving microchip electrophoresis with a commercial chip without any surface treatment, direct determination of blood amylase hydrolysis could be performed by substrate/hydrolysate separation in a microchannel without chromophore release from maltooligosaccharides.…”

Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin

“…Fd and FNR are plastid-localized redox components, and we sought to test their role in the cyclase reaction. Antibodies are known to inhibit enzymatic reactions by steric interference [ 27 ], and antibodies against FNR have previously been shown to be effective FNR inhibitors [ 28 ]. Therefore, we used antibodies against either FNR (α-FNR) or Fd (α-Fd) to test for inhibition of cyclase activity in assays using well-established barley plastid fractions prepared according to Bollivar et al [ 23 ].…”