Structural differences between the isoenzymes of human parotid α-amylase

Keller, Patricia J.; Kauffman, Dorothy L.; Allan, Barbara J.; Williams, Betsy L.

doi:10.1021/bi00802a006

Cited by 115 publications

(31 citation statements)

References 30 publications

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“…Human parotid a-amylase, for example, is synthesized as a group of five isozymes, three of which are glycosylated and two of which are not glycosylated. These aamylase isozymes are indistinguishable on the basis of their enzymic activities and they are all secreted to the cell exterior (19). Secreted proteins that are not glycosylated frequently possess a putative glycosylation site.…”

Section: Discussionmentioning

confidence: 99%

Evidence for Precursor Forms of the Low Isoelectric Point α-Amylase Isozymes Secreted by Barley Aleurone Cells

Jacobsen¹,

Bush²,

Sticher³

et al. 1988

Plant Physiol.

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Gibberellin-treated barley (Hordeum vulgare L.) aleurone cell protoplasts have been shown previously to contain two a-amylase isozymes which are not secreted (JV Jacobsen, JA Zwar, PM Chandler 1985 Planta 13: 430438). This report shows that these intracellular forms are immunochemically related to the low isoelectric point but not the high isoelectric point group of a-amylase isozymes and that they arise by new synthesis like the secreted forms. Pulse-chase studies show that the intracellular isozymes are precursors to the secreted isozymes. Conversion of the intra-to the extracellular forms involves decreases in isoelectric points with no change in size detectable by SDS-PAGE. The precursor isozymes were also detected in aleurone layer homogenates but they were unstable. They could be stabilized by various treatments including heating the homogenate to 70°C for 10 minutes indicating that the instability was enzymically mediated. Using purified radioactive precursor isozymes, it was shown that instability did not involve inactivation but the conversion to secreted forms. The nature of the covalent modification associated with conversion was not determined but available data indicate that it does not involve glycosylation.two forms shows differential sensitivity to monensin and Ca2" which led Akazawa and Hara-Nishimura (1) to propose that there may be differential intracellular sorting and transport of the two forms and that this may be based on post-translational glycosylation and the nature of the side chain added. Monensin and Ca2" also differentially affect the secretion of the low pl2 and high pI groups of barley aleurone a-amylase (17, 23) indicating that perhaps there are similarities in the mechanisms of aamylase secretion between the two tissues.The goals of the experiments reported in this paper were to determine whether barley aleurone a-amylase isozymes are posttranslationally modified during intracellular transport and to establish the nature of any such covalent change. Our experiments were prompted by the finding that barley aleurone protoplasts contain forms oflow pI a-amylase which are not secreted (16). We present evidence that these intracellular forms of aamylase are precursors of the secreted forms of the enzyme. We also show that the modification results in a lowering of isozyme pI without significantly affecting the size of the enzyme. We conclude that this post-translational modification is not brought about by glycosylation, although the precise nature of the covalent change remains to be identified. MATERIALS AND METHODSThe cereal aleurone layer is a specialized tissue which synthesizes and secretes hydrolytic enzymes into the starchy endosperm of germinating grain. Although details of the regulation of enzyme synthesis by GA3 are beginning to be understood (12)(13)(14), far less is known about the mechanisms of intracellular transport and secretion of the inducible proteins. a-Amylase is probably synthesized on the endoplasmic reticulum (9, 17), and exocytosis probably occurs v...

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Section: Discussionmentioning

confidence: 99%

Evidence for Precursor Forms of the Low Isoelectric Point α-Amylase Isozymes Secreted by Barley Aleurone Cells

Jacobsen¹,

Bush²,

Sticher³

et al. 1988

Plant Physiol.

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“…Eight additional isoforms in the same pI range were found, each exhibiting a mobility change of 3 kDa in apparent molecular mass. Although this 3-kDa difference between glycosylated and non-glycosylated amylase was originally reported using a one-dimensional electrophoresis system (48,49), the resolution into eight different isoforms was made possible through the use of this 2-DE approach.…”

Section: Fig 2 Two-dimensional Gel Electrophoresis Of Salivary Glanmentioning

confidence: 99%

Identification of Protein Components in Human Acquired Enamel Pellicle and Whole Saliva Using Novel Proteomics Approaches

Yao¹,

Berg²,

Costello³

et al. 2003

Journal of Biological Chemistry

210

183

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Precursor proteins of the acquired enamel pellicle derive from glandular and non-glandular secretions, which are components of whole saliva. The purpose of this investigation was to gain further insights into the characteristics of proteins in whole saliva and in vivo formed pellicle components. To maximize separation and resolution using only micro-amounts of protein, a two-dimensional gel electrophoresis system was employed. Protein samples from parotid secretion, submandibular/sublingual secretion, whole saliva, and pellicle were subjected to isoelectric focusing followed by SDS-PAGE. Selected protein spots were excised, subjected to "in-gel" trypsin digestion, and examined by mass spectrometry (MS). The data generated, including peptide maps and tandem MS spectra, were analyzed using protein data base searches. Components identified in whole saliva include cystatins (SA-III, SA, and SN), statherin, albumin, amylase, and calgranulin A. Components identified in pellicle included histatins, lysozyme, statherin, cytokeratins, and calgranulin B. The results showed that whole saliva and pellicle have more complex protein patterns than those of glandular secretions. There are some similarities and also distinct differences between the patterns of proteins present in whole saliva and pellicle. MS approaches allowed identification of not only well characterized salivary proteins but also novel proteins not previously identified in pellicle.Human teeth are exposed to whole saliva (WS), 1 consisting mainly of secretions derived from three pairs of major salivary glands, which comprise parotid, submandibular, and sublingual glands. Protein components that have been identified in all of the major glandular secretions are proline-rich proteins (acidic, basic, and glycosylated families), amylase, statherin, histatins, lysozyme, lactoferrin, lactoperoxidase, and secretory IgA (1-10), whereas cystatins and mucins have been identified in submandibular/sublingual secretions (9, 11-13). However, detailed understanding of the protein composition in WS is still limited because of the lack of knowledge about proteins in other contributors to whole saliva such as secretions from minor salivary glands and gingival crevicular fluid. In addition, little is known about modifications that occur on proteins during or after secretion into the oral cavity.The acquired enamel pellicle (EP) is a protein film thought to result from the selective adsorption of precursor proteins present in WS onto tooth surfaces. Because of its intimate contact with enamel surfaces, the EP plays an important role in maintaining tooth integrity by controlling the mineral solution dynamics of enamel. At its interface with the oral environment, the EP exerts selectivity on bacterial attachment and is involved in the initial stages of plaque formation (14). Because of the limiting amount of proteins that can be harvested from EP formed in vivo, previous investigations have utilized sensitive but indirect approaches such as enzymatic assays and immunologic detectio...

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“…W h e n separated by electrophoresis in p o l y a c r y l a m i d e gel, p a r o t i d saliva n o r m a l l y exhibits a basic p a t t e r n of 6 isoenzymes with the occasional occurrence of a d d i t i o n a l isoamylases migrating faster towards the a n o d e ( K a u f f m a n et al, 1970). These "fast isoamylases", whose n u m b e r increases with age (Arglebe et al, 1976), are p r o b a b l y d e a m i d a t i o n p r o d u c t s of the basic isoamylases (Keller et al, 1971).…”

Section: Km Doering Et Almentioning

confidence: 99%

?Fast isoamylases? in the parotid saliva of children with cystic fibrosis and heterozygous carriers

et al. 1977

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On electrophoresis, parotid saliva always exhibits a basic pattern of 6 isoamylases. Additional faster migrating isoamylases occur in varying numbers. These "fast isoamylases" are generated, at least in part, by deamidation. Compared with juvenile and adult controls, a significantly greater number of "fast isoamylases" was found in the parotid saliva of children with cystic fibrosis and their healthy heterozygous parents. A shift in the equilibrium between amidation and deamidation is discussed in terms of its possible connection with the metabolic defect responsible for cystic fibrosis.

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Structural differences between the isoenzymes of human parotid α-amylase

Cited by 115 publications

References 30 publications

Evidence for Precursor Forms of the Low Isoelectric Point α-Amylase Isozymes Secreted by Barley Aleurone Cells

Evidence for Precursor Forms of the Low Isoelectric Point α-Amylase Isozymes Secreted by Barley Aleurone Cells

Identification of Protein Components in Human Acquired Enamel Pellicle and Whole Saliva Using Novel Proteomics Approaches

?Fast isoamylases? in the parotid saliva of children with cystic fibrosis and heterozygous carriers

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