Immobilisation and activity of human α-amylase in the acquired enamel pellicle

https://doi.org/10.1016/j.archoralbio.2004.01.005Get rights and content

Abstract

Amylase is an important salivary component and structural element of the acquired enamel pellicle. Aim of the study was to establish a method for precise and direct determination of pellicle bound amylase activity in order to analyse kinetics and activity of the immobilised enzyme. Six bovine enamel slabs (5 mm diameter) were fixed on individual maxillary trays and worn by five subjects for different times (3, 30 and 120 min) on buccal and palatal sites on different days. Slabs were removed from the trays and rinsed with aqua dest. Afterwards, pellicle bound amylase activity was determined directly with a photometric method using 2-chloro-4-nitrophenyl-4-O-β-d-galactopyranosylmaltotriosid (GalG2CNP) as substrate yielding the coloured product chloronitrophenolate (CNP). All investigated pellicles exhibited immobilised amylase activity. Mean activity was 1.39±1.87 mU/cm2 (n=87, range 0.14–11.5 mU/cm2). Product formation of CNP by immobilised amylase was linear over time. Pellicle bound amylase showed a Michaelis type kinetic (Km=3.3×10−3 M). Immobilised activity on buccal surfaces ranged between 0.25 and 11.1 mU/cm2 (palatal slabs: 0.14–3.06 mU/cm2). Thirty minutes pellicles formed on buccal sites exhibited significantly higher immobilised amylase activity (2.85±3.65 mU/cm2) than palatal ones (0.63±0.32 mU/cm2). Amylase activity showed great intraindividual variability when comparing same positions on different days. Conclusion: Pellicle bound amylase activity can be determined directly with GalG2CNP and shows a Michaelis Menten kinetic. Enzyme activity of the amylase immobilised in the in situ pellicle reveals great intra- and interindividual differences.

Introduction

Enamel surfaces in the oral cavity become immediately coated with a proteinaceous film known as acquired enamel pellicle.1., 2. The enamel pellicle is of great physiological and pathophysiological significance. It serves as lubricant preventing abrasive damage, forms a barrier and buffer to demineralising agents, is a reservoir for remineralising electrolytes and has antimicrobial activity.1., 3., 4., 5.

The beneficial effects of the enamel pellicle will partly be counteracted when dental plaque is developing by selective binding of planktonic bacteria of the oral fluids to certain proteins of the pellicle.6

In human oral fluid α-amylase is the most abundant enzyme, mainly secreted by the parotid-gland. The function of the secreted soluble amylase is starch digestion. Amylase, as other salivary proteins, is able to form complexes with the high-molecular-weight mucin MG1. These complexes could act as reservoir for pellicle precursors. This may not exclude that amylase by itself is a precursor for pellicle formation.7

Additionally, amylase-sIgA-complexes in the pellicle are binding sites for Streptococcus sanguis and Streptococcus gordonii which are pioneer bacteria for formation of plaque.8., 9., 10. Oral streptococci yield an amylase binding protein A gene.11 This fact emphasises the special role of the pellicle bound amylase in the plaque formation process.

Previous studies utilising immunological and electrophoretic methods demonstrated that amylase is a component of the enamel pellicle. There is information on molecular weight12 and immunoreactivity13 of the pellicle bound salivary amylase. However, activity of α-amylase immobilised in the enamel pellicle has not been investigated until now.

In general, enzymes immobilised to insoluble carriers tend to show diffusion limited activity and thus lack zero-order kinetics.14 As a consequence, the turn-over of substrate is not linear with incubation time or amount of catalyst and thus the exact determination and calculation of enzyme activity-units are impossible.

In 1913, amylase was the first enzyme-activity determined in laboratory medicine for diagnostic purposes, e.g. acute pancreatitis. Since then, it has been the enzyme with by far the greatest number of methods for assaying its catalytic activity. The numerous amyloclastic methods based on polysaccharide digestion mostly lack linearity, due to the high-molecular-weight substrate. The polysaccharides are cleaved successively to smaller products, which themselves are substrates with different turn-over rates. Low-molecular substrates are maltooligosaccharides, which are mostly attached to a chromogene. Since the corresponding methods often need auxiliary enzymes to produce the chromophore, these assays exhibit a lag-phase of minutes before showing a constant reaction rate.15., 16.

Amylase activity has been determined in saliva of different origin and from different regions of the oral cavity.17 Pellicle-bound amylase has been determined immunologically on synthetic hydroxylapatite, on enamel powder and on natural enamel surfaces.18., 19. These data give no information concerning catalytic activity of pellicle bound amylase in the immobilised state. Up to now, catalytic activity of pellicle immobilised amylase has only been measured after partial desorption of amylase from artificial pellicles formed on hydroxylapatite or on enamel powder.19., 20., 21., 22. However, amylase activity on in situ formed enamel pellicles has not been measured quantitatively and has not been characterised kinetically until now.

Aim of the present in situ study was to investigate if and to which extent pellicle bound amylase shows activity. For this purpose a method for precise and direct determination of pellicle bound amylase activity was established.

Section snippets

Subjects and specimens

Five healthy subjects, members of the laboratory staff, with physiological salivary flow rate participated in the study. Visual oral examination was carried out by an experienced dentist. The subjects showed no signs of gingivitis or caries. Informed written consent had been given of the subjects.

Cylindrical enamel slabs (diameter 5 mm, 19.63 mm2 surface area) were prepared from labial surfaces of bovine incisors of 2-year-old cattle. The enamel surface was polished by wet grinding with abrasive

Activity and kinetics of pellicle immobilised amylase

All in situ formed pellicle samples investigated yielded immobilised α-amylase activity. When regarding all pellicle samples, the immobilised amylase activity varied over a wide range (0.14–11.5 mU/cm2). Mean activity amounted to 1.39±1.87 mU/cm2 (n=87).

Pellicle immobilised amylase hydrolysed the substrate GalG2CNP at a constant rate over an incubation period of 80 min (Fig. 1).

The pellicle-immobilised salivary amylase incubated with varying concentrations of the substrate GalG2CNP showed a

Discussion

In the present study, a method for determination of pellicle immobilised α-amylase activity was established which is based on the hydrolysis of GalG2CNP.16 The substrate is most suitable for this purpose due to its low weight, its specifity and sensitivity. With the assay, fast stoichiometric determination of immobilised as well as of free amylase activity is possible.

A composition of the amylase reaction mixture not fully optimal for maximum activity was chosen, since decreased NaSCN (50 mmol/l

Conclusion

A novel method for precise and direct determination of α-amylase adsorbed in the in situ formed pellicle layer was established using GalG2CNP as substrate. Pellicle bound α-amylase shows a Michaelis Menten kinetic. The immobilised enzyme activity shows great intra- and interindividual differences. Furthermore, it can be concluded that in the oral cavity salivary amylase is immediately adsorbed by integration in the acquired pellicle which is formed on enamel surfaces. It is assumed that

References (27)

  • D. Bradway et al.

    Formation of salivary-mucosal pellicle: the role of transglutaminase

    Biochem. J.

    (1992)
  • T. Lie

    Morphologic studies on dental plaque formation

    Acta Odontol. Scand.

    (1979)
  • I. Iontecheva et al.

    Human salivary mucin MG1 selectively forms heterotypic complexes with amylase, proline-rich proteins, statherin, and histatins

    J. Dent. Res.

    (1997)
  • Cited by (0)

    View full text