Radioimmunoassay for human salivary amylase

A radioimmunoassay (RIA) for human salivary amylase was developed. Human salivary amylase was purified from parotid saliva by a combination of Sephadex gel filtration and cation exchange chromatography. Purified salivary amylase was used both as the standard antigen and for the generation of ¹²⁵I-labeled amylase. Antibody to salivary amylase was raised in New Zealand white rabbits and used in a nonequilibrium double-antibody procedure for the RIA. The RIA was sensitive (10 ng/ml) and specific, displaying a limited cross-reactivity for pancreatic amylase (1%, $\text{w}\text{w}$). Analysis of patient sera by RIA shows that salivary amylase constitutes approximately 60% of the total serum amylase, that the salivary amylase found in the serum of patients with Sjögren's syndrome and macroamylasemia is immunologically indistinguishable from that of normal persons, and that salivary amylase can be evaluated by RIA in the serum of patients with pancreatitis.     

Evidence for amylase in avian salivary glands

Four glands of the house sparrow, chicken and turkey were examined histologically and for their content of amylase. These were the external and intermediate mandibular glands, the maxillary gland and glandula anguli oris of the sparrow and the anterior and posterior mandibular, maxillary and anguli oris glands of the chicken and turkey. Amylase was determined by a starch substrate slide method and by biochemical assay. General morphology and mucopolysaccharide staining are described. All four glands of the sparrow demonstrated significant amylolytic activity by the assay. In the external mandibular and anguli oris glands this activity could be traced to mucous and seromucous cells of origin by means of the starch substrate slide procedure. None of the glands of the chicken or turkey displayed significant amylolytic activity.     

Polymorphism of human salivary amylase

Summary Variation of human salivary amylase isoenzymes was investigated by isoelectric focusing on thin-layer polyacrylamide gel. Preliminary studies indicate a genetic polymorphism.     

Isoenzyme variation of human salivary amylase

Summary Variation of human salivary amylase was investigated by immunofixation electrophoresis. Preliminary data disclosed 16 different isoamylase variants in a total frequency of about 15%. More than 1 gene locus may determine the salivary isoamylase pattern.

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Zusammenfassung Die Variabilität der menschlichen Speichelanalyse wurde durch Immunfixationselektrophorese untersucht. Eine vorläufige Auswertung ergab 16 verschiedene Isoamylase-Varianten mit einer Gesamthäufigkeit von ungefähr 15%. Es sieht so aus, als ob mehr als 1 Genlocus das Speichelisoamylase-Muster determinieren würde.

     

Variation in gene copy number and polymorphism of the human salivary amylase isoenzyme system in Caucasians

Summary The polymorphic patterns of human salivary amylase of a large number of individuals of Caucasian origin were determined by using isoelectric focusing and polyacrylamide gel electrophoresis. Nine different salivary amylase protein variants were found; three of them are recorded for the first time and their heredity is shown. Some of the variants are encoded by haplotypes expressing three allozymes. Most variants display low frequencies. Analysis of the relative intensities of variant-specific isozyme bands, combined with segregation analysis, show that extensive quantitative variation is present in the population. The numbers of salivary amylase genes in some families showing quantitative variation at the protein level have been estimated by the polymerase chain reaction. We present evidence that quantitative variations in amylase protein patterns do not always reflect variations in gene copy number but that other mechanisms are also involved.     

Modification of human salivary amylase using dextran T-70

Summary Human salivary amylase was immobilized to soluble dextran T-70. On comparing the thermal stabilities of the immobilized and free enzyme it was found that the former was much more stable. It was also found that immobilization to the dextran resulted in the enzyme having a broader pH range and an increased temperature optimum.     

Evidence for gene conversion in the amylase multigene family of Drosophila pseudoobscura

The alpha-amylase (Amy) multigene family in Drosophila pseudoobscura is located on the third chromosome, which is polymorphic for more than 40 inverted gene arrangements. The number of copies in this family ranges from one to three, depending on the arrangement in question. A previous study of the three Amy genes from the Standard (ST) arrangement suggested either that duplicated copies (Amy2 and Amy3) are functionally constrained or that they are undergoing gene conversion with Amy1. In order to elucidate further the pattern of molecular evolution in this family, we cloned and sequenced four additional Amy genes, two from the Santa Cruz (SC) and two from the Chiricahua (CH) gene arrangement. Of the two alternatives, only the hypothesis of gene conversion is supported by the sequence analysis. The homogenization effect of gene conversion has been strongest in SC, whose copies differ by only two nucleotides, less noticeable in ST, and negligible in the CH. Furthermore, the action of gene conversion is apparently localized, occurring only in the coding region. Interestingly, these results concur with the findings of other workers for the duplicated Amy genes in the Drosophila melanogaster group. Thus, the occurrence of gene conversion in the Amy multigene family seems to be a common feature in the Drosophila species studied so far.      

Codon bias differentiates between the duplicated amylase loci following gene duplication in Drosophila

We examined the pattern of synonymous substitutions in the duplicated Amylase (Amy) genes (called the Amy1- and Amy3-type genes, respectively) in the Drosophila montium species subgroup. The GC content at the third synonymous codon sites of the Amy1-type genes was higher than that of the Amy3-type genes, while the GC content in the 5'-flanking region was the same in both genes. This suggests that the difference in the GC content at third synonymous sites between the duplicated genes is not due to the temporal or regional changes in mutation bias. We inferred the direction of synonymous substitutions along branches of a phylogeny. In most lineages, there were more synonymous substitutions from G/C (G or C) to A/T (A or T) than from A/T to G/C. However, in one lineage leading to the Amy1-type genes, which is immediately after gene duplication but before speciation of the montium species, synonymous substitutions from A/T to G/C were predominant. According to a simple model of synonymous DNA evolution in which major codons are selectively advantageous within each codon family, we estimated the selection intensity for specific lineages in a phylogeny on the basis of inferred patterns of synonymous substitutions. Our result suggested that the difference in GC content at synonymous sites between the two Amy-type genes was due to the change of selection intensity immediately after gene duplication but before speciation of the montium species.     

Metal binding characteristics of human salivary and porcine pancreatic amylase

With the exception of calcium very little is known about metal binding characteristics of either human salivary or porcine pancreatic amylase. In order to learn more about these protein-metal binding interactions, calcium-free human salivary and porcine pancreatic amylase [P(protein)] were obtained by carboxymethylcellulose chromatography of the partially purified proteins. Because these proteins acquired small amounts of calcium after further preparatory studies, they were dialyzed against 1 mM EDTA, pH 7.4, at 22 degrees C, which removed essentially all acquired calcium. The calcium-free amylases were then subjected to equilibrium dialysis against copper or zinc solutions with or without added glycine. The experimental data were fitted to appropriate mathematical equations, and binding constants of the metal complexes were calculated. Both human salivary and porcine pancreatic amylase were found to have two metal ion binding sites, only one of which was selective for calcium. Copper or zinc appeared to bind to the second site forming the species CuCaLP (or ZnCaP), where L, a ligand, is the glycine anion. Neither copper nor zinc displaced calcium from human salivary amylase, although copper bound to both binding sites in human salivary apoamylase to form the species Cu2L2P in which the amylase molecule appeared to form a bridge between the two copper atoms. In the case of the zinc-human salivary apoamylase system, the experimental data could not be analyzed quantitatively since the protein formed an insoluble complex species. Copper displaced calcium from porcine pancreatic amylase and formed a mixed ligand species similar to that formed with human salivary apoamylase. Zinc bound to both metal binding sites of porcine pancreatic apoamylase, forming species ZnP and Zn2P, although it did not displace calcium from the protein. While calcium in amylase is known to be critical for its amylolytic activity, little is known about the function of either zinc or copper in amylase albeit both of these metals are important in biological systems.     

Flow-injection-type biosensor system for salivary amylase activity

The authors aim to establish a method that can quantitatively evaluate vital reactions to stress. We have been examining the correlation between stress and salivary amylase activity in order to verify its validity as a stress index. In order to quantify human stress, which changes over time, the relationship between stress and salivary amylase activity must be verified by fast and repeated analysis of salivary amylase activity. Standard biosensors are designed such that the enzyme immobilized on an electrode (enzyme electrode) and the substrate-dependent activity is measured. The reverse approach of measuring the alpha-amylase-dependent activity was adopted. We fabricated an amylase activity analytical system. Maltopentaose was selected as a substrate for alpha-amylase and a flow-injection-type device was used to supply maltopentaose continuously. alpha-Glucosidase, having relatively low enzyme activity, was immobilized on a pre-activated membrane so that it could be enclosed in a pre-column, Glucose oxidase, having higher enzyme activity, was immobilized on a working electrode so that it could function as an amperometric biosensor. A saliva-collecting device was fabricated to make saliva pretreatment unnecessary. As a result, an amylase activity analytical system was fabricated that enabled us to measure salivary amylase activity from 0 to 30 kU/l, with an R(2) value of 0.97. The time-course changes in the salivary amylase activities for 1 week were 5.1%, and the initial sensitivity remained nearly constant. Through this study, we were able to verify the possible development of the amylase activity analytical system.     

Restriction fragment length polymorphism detected by human salivary amylase cDNA

Summary The plasmid clone which contains human salivary amylase cDNA was used to detect restriction fragment length polymorphisms (RFLPs). After double digestion with Pst 1 and Bam H1, a polymorphism with two alleles was observed. In Japanese, frequencies of these alleles, tentatively called 5.7kb and 6.5kb fragment alleles, are 0.55 and 0.45, respectively.     

Evidence for posttranslational modification and gene duplication of Campylobacter flagellin.

A gene encoding a flagellin protein of Campylobacter coli VC167 has been cloned and sequenced. The gene was identified in a pBR322 library by hybridization to a synthetic oligonucleotide probe corresponding to amino acids 4 to 9 of the N-terminal sequence obtained by direct chemical analysis (S. M. Logan, L. A. Harris, and T. J. Trust, J. Bacteriol. 169:5072-5077, 1987). The DNA was sequenced and shown to contain an open reading frame encoding a protein with a molecular weight of 58,945 and a length of 572 amino acids. The deduced amino acid sequence was identical to the published N-terminal amino acid sequence of VC167 flagellin and to four internal regions whose partial sequences were obtained by direct chemical analysis of two tryptic and two cyanogen bromide peptides of VC167 flagellin. The C. coli flagellin protein contains posttranslationally modified serine residues, most of which occur within a region containing two 9-amino-acid repeating peptides separated by 34 unique amino acids. Comparisons with the sequences of flagellins from other bacterial species revealed conserved residues at the amino- and carboxy-terminal regions. Hybridization data suggest the presence of a second flagellin copy located adjacent to the first on the VC167 chromosome.     

Expression, characterization, and biochemical properties of recombinant human salivary amylase

Human salivary amylase, a major component of human salivary secretions, possesses multiple functions in the oral cavity. It is the only enzyme in saliva capable of degrading oligosaccharides, which are used by the oral microflora for nutritional purposes. In order to understand its role in disease processes such as caries, we have undertaken the structure-function analyses of amylase. In this regard, the nonglycosylated human salivary amylase was expressed in a baculovirus expression system. The native and the recombinant amylases exhibit similar biochemical as well as biophysical properties. Unlike recombinant human pancreatic amylase, recombinant human salivary amylase is not glycosylated when expressed in a baculovirus system as determined from the crystal structure determination of the recombinant enzyme. Therefore, this system is suitable for further structure-function work without resorting to enzymatic removal of the carbohydrate chain. Details of the expression, purification, and biophysical properties will be presented.