alpha 2-Macroglobulin-proteinase complexes in cystic fibrosis serum. Analysis by monoclonal antibodies and receptor-mediated endocytosis by normal human fibroblasts

alpha 2-Macroglobulin complexed to proteinases activated during clotting of cystic fibrosis and control sera was quantitated with the complex-specific monoclonal antibody F2B2 . Similar amounts of alpha 2-macroglobulin complexes (between 40 and 90 micrograms/ml) were generated in cystic fibrosis and control sera. Endocytosis of the complexes by normal human fibroblasts was compared to the amount of complexes detected by the F2B2 -radioimmunoassay. Normal uptake was observed with 13 out of 14 cystic fibrosis sera. One cystic fibrosis serum showed strongly reduced endocytosis of the complexes. Complexes isolated from this serum on immobilized F2B2 failed to inhibit binding of purified alpha 2-macroglobulin-trypsin to its receptor, demonstrating deficient receptor-binding of these complexes. The low uptake complexes could not be distinguished from complexes isolated from control or other cystic fibrosis sera by isoelectric focusing, rate electrophoresis or SDS-polyacrylamide gel electrophoresis.     

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 Close Linkage of Human Amylase Loci

HUMAN α-amylase (α-1,4-glucan 4-glucanohydrolase, E.C. 3.2.1.1) is primarily in the pancreas and salivary glands and can be detected in serum and urine (UAmy) as well as in saliva (SaAmy) and duodenal secretions. The SaAmy locus (Amy₁) has at least three mutant alleles with the combined frequency of electrophoretic variant phenotypes totalling approximately 0.01 in Caucasian Americans¹. Subsequently, UAmy was shown to be a convenient source of pancreatic amylase. The pancreatic amylase locus (Amy₂) has at least two mutant alleles in Caucasian and Afro-Americans². The variant UAmy Ss is common in Caucasians whereas UAmy Sp has been found only in Afro-Americans; the frequency of variant phenotypes is 0·086 in each population².     

Amylase in the thyroid gland

Summary Amylase activity detected in thyroid extracts was significantly higher than that of normal sera. A starch film technique revealed the existence of amylase activity in the follicular lumen and on the follicular epithelia. By electrophoretic analysis of thyroid extracts, 4 bands of amylase activity were observed, one being of the same mobility as parotid and the other 3 more anodic. Amylase extracted from the thyroid appeared in the same position as pancreatic or parotid amylase on Sephadex G75 gel filtration. The possibility is discussed that the thyroid may synthesize amylase of salivary type, which is secreted from the follicular epithelia into the follicular lumen, where it may be transformed into anionic forms.     

Amylase in the thyroid gland

Amylase activity detected in thyroid extracts was significantly higher than that of normal sera. A starch film technique revealed the existence of amylase activity in the follicular lumen and on the follicular epithelia. By electrophoretic analysis of thyroid extracts, 4 bands of amylase activity were observed, one being of the same mobility as parotid and the other 3 more anodic. Amylase extracted from the thyroid appeared in the same position as pancreatic or parotid amylase on Sephadex G75 gel filtration. The possibility is discussed that the thyroid may synthesize amylase of salivary type, which is secreted from the follicular epithelia into the follicular lumen, where it may be transformed into anionic forms.     

Purification and properties of an alpha-amylase inhibitor from wheat.

Four inhibitors of alpha-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-exchange chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60--90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract.     

α2-Macroglobulin-proteinase complexes in cystic fibrosis serum Analysis by monoclonal antibodies and receptor-mediated endocytosis by normal human fibroblasts

α₂-Macroglobulin complexed to proteinases activated during clotting of cystic fibrosis and control sera was quantitated with the complex-specific monoclonal antibody F₂B₂. Similar amounts of α₂-macroglobulin complexes (between 40 and 90 μg/ml) were generated in cystic fibrosis and control sera. Endocystosis of the complexes by normal fibroblasts was compared to the amount of complexes detected by the F₂B₂-radioimmunoassay. Normal uptake was observed with 13 out of 14 cystic fibrosis sera. One cystic fibrosis serum showed strongly reduced endocytosis of the complexes. Complexes isolated from this serum on immobilized F₂B₂ failed to inhibit binding of purified α₂-macroglobulin-trypsin to its receptor, demonstrating deficient receptor-binding of these complexes. The low complexes could not be distinguised from complexes isolated from control or other cystic fibrosis sera by isoelectric focusing, rate electrophoresis or SDS-polyacrylamide gel electrophoresis.     

A novel type of human alpha-amylase produced in lung carcinoid tumor

A novel type of alpha-amylase was detected in a lung carcinoid tissue after surveying the cDNA library constructed from this tumor mRNA. Nucleotide sequence analysis showed that the amylase expressed in this carcinoid tumor has 13 and 6 amino acid substitutions when compared with salivary amylase (Amy1) and pancreatic amylase (Amy2), respectively. The nucleotide sequence homologies of cDNAs between this carcinoid amylase and amy1, amy2 are 97.5% and 98.2%, respectively. The nucleotide sequence comparison strongly suggests that this new amylase is the product of the amy3 gene that has been detected in human genome [Emi et al., Gene 62 (1988) 229-235]     

Serum pancreatic lipase activity in cystic fibrosis.

Patients with cystic fibrosis have been found to have abnormal serum concentrations of immunoreactive trypsin and abnormal activities of pancreatic isoamylase. A study was undertaken to discover whether activity of pancreatic lipase is also altered in cystic fibrosis. Serum from 23 patients with cystic fibrosis was assayed for immunoreactive trypsin and pancreatic lipase. Median serum pancreatic lipase activity was significantly lower in patients with cystic fibrosis than in controls, as was immunoreactive trypsin concentration (p less than 0.0001). Some patients had supranormal lipase concentrations but these were not always associated with absence of malabsorption. Serum pancreatic lipase activity is considerably changed in cystic fibrosis.     

Primary structure of human pancreatic alpha-amylase gene: its comparison with human salivary alpha-amylase gene

We have determined the entire structure of the human pancreatic alpha-amylase (Amy2) gene. It is approx. 9 kb long and is separated into ten exons. This gene (amy2) has a structure very similar to that of human salivary alpha-amylase (Amy1) gene [Nishide et al. Gene 41 (1986a) 299-304] in the nucleotide sequence and the size and location of the exons. The major difference lies in the fact that amy1 has one extra exon on the 5' side. Other differences are at the 5' border of exon 1 and the 3' border of exon 10. The close similarity of these two genes, as compared with mouse pancreatic and salivary amylase genes, suggests that during evolution, the divergence into the two amylase genes may have occurred after the divergence of mice and man.     

Immunological relationships and post-translational modifications of human salivary amylase (Amy1) and pancreatic amylase (Amy2) isozymes

Electrophoretic phenotypes of human salivary amylase (Amy₁) and pancreatic amylase (Amy₂) consist of complex isozyme patterns which may result from post-translational modifications of the primary products of the amylase loci. Biochemical separation of the two molecular weight families of salivary amylase and development of a new electrophoretic system have allowed the identification of complete isozyme patterns corresponding to variant alleles in Amy ₁ and Amy₂ heterozygotes. Further, immunological studies show no nonidentities among salivary isozymes and among pancreatic isozymes, which is to be expected if each series is derived from a single gene product. Both results support the hypothesis that the primary products of the amylase loci undergo post-translational modifications. Salivary and pancreatic amylase appear to be immunologically identical.This investigation was supported in part by PHS Research Grant GM-19178.Supported by PHS Training Grant DE 119.Supported by PHS Training Grant GM 1056.     

Further studies on bovine serum amylase. 2. Affinity electrophoresis

The polymorphism of bovine serum amylase, which is controlled by the Ami locus, has previously only been demonstrated by starch gel electrophoresis. The addition of maltose to starch gels has been demonstrated to inhibit any subsequent separation of the Ami isozymes by starch gel electrophoresis. When electrophoresis was conducted in a support medium in the absence of starch no polymorphic variation was detected amongst samples from animals of different Ami phenotypes. The addition of starch to agarose gels has been shown to facilitate the subsequent detection of the Ami polymorphism by agarose/starch gel electrophoresis. The electrophoretic resolution of the Ami isozymes has been demonstrated to depend upon differences in affinity for starch rather than differences in net charge. The starch gel electrophoretic separation of the Ami isozymes is. therefore, another example of affinity electrophoresis. All the Ami amylases have been shown to share a common isoelectric point of pH 3.5.     

Interpretation of polymorphic DNA patterns in the human alpha-amylase multigene family.

Previous molecular studies have clearly shown that the human amylase locus has a very complicated structure. Multiple salivary and pancreatic amylase genes are present on haplotypes with variable numbers of genes. To study the population heterogeneity, human genomic DNA from family members and random individuals was digested with a number of different restriction enzymes and hybridized with probes representing various parts of the human pancreatic amylase cDNA. The complex patterns obtained were, in most cases, compatible with predictions from the restriction enzyme maps of cloned human amylase genes. With some enzymes deviations from the predicted intensities of the bands associated with the pancreatic amylase gene AMY2A were observed. These findings can be explained by unequal homologous crossovers between AMY2A and AMY1A, resulting in haplotypes with one gene less or one gene more than the haplotypes described thus far. Moreover, a very complicated TaqI polymorphism was found that can be explained by homologous crossovers between different salivary amylase genes. Because some salivary amylase genes have an inverted orientation with respect to the others, these data provide evidence for the occurrence of intrachromosomal, homologous crossovers, as proposed by us previously (P. C. Groot et al., 1990, Genomics 8: 97-105).     

Additional evidence for the close linkage of amy-1 and amy-2 in the mouse

The enzyme amylase is produced in large quantities in two mammalian tissues, the pancreas and the parotid salivary gland. A substantial body of biochemical and genetic evidence is consistent with the existence of distinct genes encoding salivary and pancreatic amylases, but testcrosses in mice have indicated that the two putative loci must be very closely linked. We have studied crosses between two pairs of inbred mice that differ with respect to electrophoretic mobility of salivary and pancreatic amylases. Among 343 potentially recombinant chromosomes examined, no recombinants were found. Our data sets an upper limit of 0.87 cM (P = 0.95) for the distance between the salivary and pancreatic loci.     

Amylase heterogeneity

Serum amylase activity comprises two isoenzymes which differ in their electrophoretic mobility. The first is produced by the salivary gland, the second by the pancreas.Urine amylase is filtered from the plasma by the glomerules and the number of its isoenzymes with their respective activities correspond to those in the serum.In 11 out of 120 cases of children a duplication of the serum pancreatic fraction was detected. The same trait was also found in one of the parents of each of these eleven children, suggesting that an autosomal codominant mode of inheritance may be involved.In diabetic children an increased serum amylase activity in the first (i.e. the salivary) fraction was detected, in five other children out of the group of 120, this first salivary amylase fraction was missing completely.Director: Prof. MUDr. Z. Brunecký, C. Sc. This paper was partially read on the 6th Biochemical days, 10th to 12th June, 1965, in Hradec Králové.     

The Amylase gene cluster on the evolving sex chromosomes of Drosophila miranda.

On the basis of chromosomal homology, the Amylase gene cluster in Drosophila miranda must be located on the secondary sex chromosome pair, neo-X (X2) and neo-Y, but is autosomally inherited in all other Drosophila species. Genetic evidence indicates no active amylase on the neo-Y chromosome and the X2-chromosomal locus already shows dosage compensation. Several lines of evidence strongly suggest that the Amy gene cluster has been lost already from the evolving neo-Y chromosome. This finding shows that a relatively new neo-Y chromosome can start to lose genes and hence gradually lose homology with the neo-X. The X2-chromosomal Amy1 is intact and Amy2 contains a complete coding sequence, but has a deletion in the 3''-flanking region. Amy3 is structurally eroded and hampered by missing regulatory motifs. Functional analysis of the X2-chromosomal Amy1 and Amy2 regions from D. miranda in transgenic D. melanogaster flies reveals ectopic AMY1 expression. AMY1 shows the same electrophoretic mobility as the single amylase band in D. miranda, while ectopic AMY2 expression is characterized by a different mobility. Therefore, only the Amy1 gene of the resident Amy cluster remains functional and hence Amy1 is the dosage compensated gene.     

Inheritance of a Parotid Secretory Protein in Mice and Its Use in Determining Salivary Amylase Quantitative Variants

Among inbred strains of mice, a major protein, PSP, produced and secreted by the parotid glands, shows variation in electrophoretic mobility and in the peptides produced by cyanogen bromide treatment. This variation is inherited as a single Mendelian factor with two alleles showing co-dominant expression. In genetic crosses, it segregates independently from the amylase complex and is closely linked to the agouti locus on chromosome 2. The protein ratios between amylase and PSP in saliva, obtained by scanning of electrophoretic gel separations, were found to reflect genetic differences in salivary amylase production in strains YBR/Cv and C3H/As.     

The Pancreozymin-Secretin Test After Partial Gastrectomy

The administration of pancreozymin and secretin to eight control patients and 18 patients after partial gastrectomy caused no rise in serum amylase levels above normal. Four postgastrectomy patients had unexplained elevations of basal serum lipase levels but in only one did the lipase increase above the basal level after stimulation.Duodenal aspirates from the control group and from one patient with a Billroth I gastrectomy showed a wide range of values for volume, bicarbonate and amylase content. This would appear to limit the diagnostic value of the test. The two-tube method used for collecting duodenal contents proved difficult.     

Interstrain Variation in Amylase Gene Copy Number and mRNA Abundance in Three Mouse Tissues

Amylase expression in strain YBR differs in several respects from the standard mouse phenotype. The synthesis of salivary amylase is elevated twofold in YBR mice and the synthesis of pancreatic amylase is reduced to one-half the normal rate. We have compared the concentrations of amylase mRNA in the parotid, liver and pancreas of YBR mice with those in strains A/J and C3H. We observed differences in amylase mRNA abundance which can account for the levels of amylase protein synthesis in the parotid and pancreas of these strains. Unexpectedly, the concentration of amylase mRNA in the liver of YBR mice was also higher than in the other strains. Since liver amylase is transcribed from the same gene as parotid amylase, duplication of the Amy-1 locus could account for the elevated mRNA concentration in both tissues. Quantitative analysis of genomic DNA by Southern blotting provided direct evidence for duplication of Amy-1 in strain YBR.