Amylase John A. Williams Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, Michigan 48109-0622 Email: [email protected] Version 1.0, April 24, 2017 [DOI: 10.3998/panc.2017.04] Gene Name: AMY1, AMY2 1. Overview (General) in other tissues or tumors; the major salivary and pancreatic amylase proteins are very similar (47). α-Amylase (1,4-α-D-glucan-glucanohydrolase, EC Salivary amylase initiates carbohydrate digestion 3.2.1.1) is the primary digestive enzyme acting on in the mouth and pancreatic amylase is the main starch or glycogen and is present in plants, enzyme for luminal digestion of carbohydrate in animals, bacteria and fungi. Starch from plants is the small intestine. Human pancreatic α-amylase a high molecular weight polymer of glucose. It is is a protein of 57 KDa for which the cDNA predicts made up of amylose, a straight-chain α-1,4 linked a protein of 512 amino acids (54). This includes a polymer of about 105units and amylopectin, a signal sequence; amylase isolated from human branched chain polymer with α1,4 linked glucose pancreatic juice has 496 amino acids (15). In with branch points made up of α-1,6 linkages that various species the reported molecular weight for contains about 106 units of glucose. Glycogen amylase is 50-57 kDa and consists of a single from animals is similar to amylopectin in structure chain protein with one carbohydrate (some but is smaller. α-Amylase cleaves the α-1,4 species have an isoform with none) and an linkage when it is not next to a branch point or isoelectric point of 7.1. (39). Human pancreatic terminal glucose residue. Thus its products are juice amylase has no sugar groups and exists as maltose (2 glucose residues), maltotriose (3 two isoforms of pI 7.2 and 6.6 termed HPA I and glucose residues) and α-limit dextrins (5-6 HPA II (15). Five disulfide bridges have been residues which contain a branch point). In described in porcine pancreatic amylase (35) and vertebrates, these are further cleaved to most species have one or two free sulphydryl monosaccharides by the intestinal brush border groups. Salivary amylase is coded for by the enzymes isomaltase and maltase which hydrolyze AMY1 gene and pancreatic amylase by AMY2; a the α-1,6 and α-1,4 bonds respectively (3). third form present in some tumors is termed Studies show that hydrolysis and not AMY2B (33, 56, 57). Amylase is a multigene monosaccharide absorption is the rate limiting family with multiple genes and pseudogenes on step in complex carbohydrate absorption in chromosome 1 in humans and chromosome 3 in humans (20). mice (19, 48). Interestingly, individuals from agricultural societies that consume high starch In animals α-amylase occurs in pancreas, parotid, diets have a higher copy number of salivary serum, urine and occasionally in smaller amounts amylase genes (37) and a high copy number 1 This work is subject to an International Creative Commons Attribution 4.0 license. leads to more protein and increased perception of digestion but prevents too rapid absorption of oral starch (26, 42). glucose. After binding to brush border proteins amylase is internalized into enterocytes restoring The amylase protein contains three domains glucose uptake (12). termed A, B, and C starting at the amino terminal with C being a globular domain of unknown 2. Pancreatic Function function. The active site is located in a cleft between the A and B domains (7). Calcium and Amylase Localization, Regulation and chloride ions bind to the A domain and may Deficiency stabilize the active site. The active center Pancreatic amylase is similar to other secretory contains 5 subsites which bind different glucose proteins and is synthesized in the rough residues in the substrate (43). Kinetic evidence endoplasmic reticulum. It then moves through the supports two additional carbohydrate binding sites Golgi apparatus and is stored in secretory (2). The rate of starch digestion also depends on granules until the time it undergoes regulated the structure of the starch which can be affected secretion. Immunostaining reveals its by storage or cooking (13). Studies of the concentration in the granules (Figure 1). As with enzymatic mechanism have also benefited from other pancreatic enzymes, the amount of amylase the existence of a large family of plant α-amylase in the pancreas is regulated by the amount of inhibitory proteins (5, 49). The best studied of dietary substrate (6, 17, 46). Rodents which eat a these are from beans and the structure of the carbohydrate rich diet synthesize more amylase enzyme-inhibitor complex has been determined which is the most abundant pancreatic digestive (41). More potent inhibitors have been derived enzyme in these species. This effect occurs at from pseudooligosaccarides of which one, the the transcriptional level and the effect is mediated pseudotetrasacharide, acarbose has been in large part by insulin. With diabetes the approved for clinical use. The crystal structure of pancreatic amylase falls, but can recover pig amylase complexed with acarbose has been following insulin treatment (24, 51). This is reported (16). These pseudooligosaccaride especially prominent in rats where in experimental inhibitors, in contrast to the proteinaceous diabetes pancreatic amylase falls to only a few inhibitors, block all glucosidases including percent of normal. A 30 base pair insulin intestinal brush border enzymes as well as responsive element was identified in the mouse amylase (44). Amy2-2 gene (22). The fact that mice express more than one functional Amy2 gene explains Multiple pancreatic enzymes including amylase why total pancreatic amylase does not fall in are found absorbed to the surface of the duodenal diabetes as much as it does in rats. mucosa although they could be washed off with high salt (3). Amylase, however, was shown to Healthy volunteers are able to absorb nearly specifically bind to N-linked oligosaccharides of 100% of a starch load. However, in chronic brush border glycoproteins and that binding pancreatitis with loss of functioning pancreatic enhanced amylase activity (27). Specific tissue, only about 90% is digested and absorbed; glycoproteins binding amylase have been with complete inhibition of amylase this is reduced identified as sucrase-isomaltase (SI) and SGLT1 to 80-85% (25). In the latter case up to 20% of (4). This binding enhanced the activity of SI as carbohydrate reaches the ileum and the rise of well as that of amylase but inhibited the activity of plasma GIP and C peptide from proinsulin is SGLT1. The authors proposed that this enhances blocked. Patients with isolated amylase 2 deficiency have been reported with carbohydrate population studied and the gold standard used malabsorption and its resulting symptoms (30, (10, 52). A higher specificity is obtained when a 45). The patients evaluated have intact mRNA higher cutoff point is used such as 5 or even 9 but no amylase protein. This is similar to times the ULN, but at the cost of lower sensitivity. physiological pancreatic amylase deficiency at Utilizing assays to inhibit salivary amylase with birth where lack of symptoms is in part due to low wheat germ inhibitor or monoclonal antibodies dietary starch requiring amylase and in part the (50) or removing it with immobilized monoclonal action of salivary amylase. Amylase deficiency antibody (32) increase specificity somewhat and may be underreported due to mild symptoms. helps eliminate a contribution from tumors or other organs which primarily produce salivary amylase, but these tests are not always available. The combination of serum lipase with amylase is generally available and helps when serum amylase levels have already peaked and declined. A rise in serum amylase can also be due to macroamylassemia where amylase is bound to plasma protein (usually IgA or IgM) but this only occurs in only 1-3 % of normal humans. Amylase is also elevated in a variety of surgical, traumatic and neoplastic diseases (10, 38, 52). Renal disease can reduce amylase filtration and enhance plasma levels, but this increase is usually small as glomerular filtration is only a small portion of amylase clearance (38). A chronic increase in multiple serum pancreatic enzymes including amylase in otherwise healthy Figure 1. Immunoflourescent localization of amylase in subjects without pancreatic disease was mouse pancreas (green). Nuclei are stained blue with DAPI described by Gullo in 1996 and is now termed benign pancreatic hyperenzymemia or Gullo’s Serum Amylase as a Measure of syndrome (18). Advanced imaging techniques Pancreatic Function in Pancreatitis may uncover a pancreatic abnormality in some of Determination of serum amylase has long been a these patients. mainstay in the diagnosis of acute pancreatitis. Serum amylase in normal individuals is about half It is also important to be aware that pancreatitis salivary and half pancreatic form and each exists can occur without increased serum amylase in several isoforms all of which contribute to total especially with alcoholic pancreatitis or recurrent amylase. Because of the existence of different chronic pancreatitis (34). In some of these cases, assays and standards, the cutoff for acute lipase is elevated. Thus, while amylase is still one pancreatitis is usually expressed related to the of the most useful tests in diagnosing acute range of normal for the particular clinical pancreatitis, it should be carried out in conjunction laboratory. Levels greater than three times the with other tests (1) along with an understanding of upper limit of normal (ULN) are usually taken as amylase metabolism. indicative of pancreatitis. However, the diagnostic specificity is only 70-90% depending on the 3 Amylase secretion as a measure of autoanalyzer technique. Recently both types of regulated pancreatic or parotid secretion assays have been used to determine human Measurement of amylase secretion has become duodenal amylase with high correlation (14).