Patricia J. Keller

The basic proline-rich proteins in human parotid saliva from a single subject

Eleven Chromatographic fractions of basic proline-rich proteins isolated from parotid saliva from a single donor showed similar but not identical chemical compositions; proline, glycine and glutamic acid-glutamine accounted for 70–80 per cent of the total residues in the proteins. The basic amino acids, lysine and arginine, accounted for a further 10 per cent. All the proteins were in freshly-collected saliva and did not result from post-secretory proteolysis. The most basic of the proline-rich proteins, I-B9, was purified and characterized. The amino terminal residue was serine and the carboxyl terminal residue arginine. The molecular weight determined by quantitative end-group analyses was 9000–9500. Protein I-B9 was resistant to hydrolysis by collagenase and Staphylococcus aureus protease, whereas papain and subtilisin extensively degraded it. I-B9 fragments from clostripain and elastase digestions were isolated for use in sequence determination. Basic proline-rich proteins, acidic proline-rich proteins and proline-rich glycoprotein accounted for 23, 30 and 17 per cent, respectively of the total protein in the parotid saliva.

The isoenzymes of human parotid amylase

Abstract The number and proportion of isoenzymes of α-amylase in the parotid saliva of a single subject have been studied. Five amylolytic bands were observed on disc-gel electrophoresis of samples containing approximately 120 μg amylase. Two additional bands were revealed at higher amylase concentrations. Limited storage of parotid saliva at 4 ° did not alter the banding pattern, nor did treatment of saliva and gel with 5 m m EDTA. Photocatalyzed polymerization was not a factor in isoenzyme formation. Densitometric scans of gels indicate that the relative proportions of amylase isoenzymes remain constant in a single individual over an extended period of time. Prolonged storage of human parotid saliva at 4 ° resulted in quantitative differences in the proportion of isoenzymes. Crystalline amylase was prepared from human parotid saliva, and was found to contain the same number of enzymatic forms present in freshly collected saliva. No evidence of dimers or higher polymers was gained on Bio-Gel filtration columns. All of the isoenzymes were retarded well beyond the elution volume expected for molecules of their reported size. Bio-Gel P-100 fractionates crystalline preparations into two families; the A family, containing isoenzymes 5, 3, and 1 and the B family, containing isoenzymes 4, 2, and two forms designated “z.” Exploratory experiments have been carried out with the respective families including comparison of their amino acid composition, free sulfhydryl groups, and sugar content. Peptide maps of pepsin hydrolysates have been compared. No differences were observed in the amino acid composition or in the number of free sulfhydryl groups per molecule. The major difference observed was in the carbohydrate content. The A family appears to contain 8 moles of neutral sugar and 4 moles of glucosamine per mole of enzyme whereas the B family contained no detectable carbohydrate. Only minor differences were noted in the peptide maps of the respective families. Attempts to fractionate the isoenzymes of the B family on QAE-Sephadex appear to confirm the observations made with whole parotid saliva that storage at pH 8–9 promoted the conversion of isoenzyme 4 to 2 and z.

Mytilus byssal threads as an environmental marker for metals

Abstract The byssal apparatus of the marine mussel, Mytilus, anchors this bivalve to a substratum by means of collagen threads and attachment discs. Byssal threads and discs have been examined for their metal content from a variety of environments in Scotland, Israel and the U.S.A. Significant concentrations of zinc, iron and copper have been found, which reflect the geochemical nature of the environment. The attachment disc generally contains higher concentrations than the threads. High concentrations of calcium, magnesium, sodium and potassium together with a wide variety of other elements present in trace amounts have also been found. These include silver, gold and the radionuclide, uranium. Metal analyses of threads and discs harvested from glass plates suspended in experimental aquaria indicate that the metals are transported through the tissues rather than adsorbed onto the surface of the threads and discs.

Preparation and some properties of human pancreatic amylase including a comparison with human parotid amylase

A method for the preparation of α-amylase (α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) from human pancreatic juice is described. Procedures utilizing (NH4)2SO4 fractionation, ion-exchange chromatography and gel filtration resulted in an amylase preparation of high purity (2085 maltose units per mg) and good yield (59%). Electrophoresis on polyacrylamide disc gels yielded characteristic isoenzyme patterns with up to six bands on anionic gels. Comparisons with crystalline human parotid amylase indicated that the two principal human amylases are closely related enzymes but also exhibit organ specific variation. The two enzymes yielded the same amino acid composition and similar but not identical peptide maps. Each appears to consist of a single polypeptide chain. At neutral pH both amylases exhibited the same action pattern. Differences were noted in molecular weight, gel filtration profile, carbohydrate content and the accessibility of reactive sulfhydryl groups. A both high and low temperature extremes pancreatic amylase was more labile than the parotid enzyme. A less compact configuration is indicated for pancreatic amylase. A relationship is postulated between the number of sulfhydryl groups in the amylase molecule and the mode of action. Amylases possessing 1 sulfhydryl group, namely those from human pancreatic juice, human parotid saliva and rat parotid saliva, exhibited the same type of action pattern whereas porcine pancreatic amylase, with 2 sulfhydryls, was different.

Changes in Rat Parotid Salivary Proteins Induced by Chronic Isoproterenol Administration

Changes in the rat parotid gland and its secretion, brought about by chronic isoproterenol administration, were studied. In addition to the expected enlargement, morphological and biochemical analyses of the glands showed evidence of changes in the secretory components. Chromatographic and electrophoretic experiments revealed both qualitative and quantitative changes in the secretory proteins.

The protein composition of rat parotid saliva and secretory granules.

Rat parotid saliva was collected by surgical cannulation of the ducts and stimulation with pilocarpine; The secreted salivary proteins were resolved on columns of DEAE-Sephadex into five major Fractions, I-V, which were characterized by polyacrylamide disc gel electrophoresis, amino acid analyses and enzymatic assay. Rat parotid secretory granules were isolated by density gradient centrifugation and lysed in hypotonic buffers. Granule content proteins were resolved and examined by the same techniques as for secreted proteins. In both experiments, Fraction I contained RNAase and a major unidentified protein, M1, Fraction II contained the isoenzymes of amylase; DNAase was present in Fraction III and, to a lesser degree, in Fraction IV. The proportions of the enzyme-containing peaks were the same in saliva and granule contents. Fractions IV and V contain proteins of unknown function; Fraction IV contains exceptionally high levels of glutamic acid, glycine and proline in its protein moieties and approx. 6-8% neutral sugars.

An electrophoretic analysis of the protein components of human parotid saliva.

Abstract Fresh samples of parotid saliva from one subject were analyzed by complementary anionic and cationic polyacrylamide disc gel electrophoresis systems. Nineteen protein components were evidenced with Amido Black, whereas a total of 30 discrete components were revealed by the more sensitive dye, Coomassie Brilliant Blue. The polyacrylamide gel patterns were qualitatively reproducible and have been described by grouping bands into regions designated A or A′, B or B′, C and D. Regions A and A′ contain protein and neutral carbohydrate. Regions B and B′ contain all of the α-amylase activity of parotid saliva, in the form of four or more isoenzymes. The dominant B-band also possesses acidic carbohydrate material. Region C contains five acidic components one of which contains acidic carbohydrate. Five cationic components are consistently seen in Region D.

The isolation of some basic proline-rich proteins from human parotid saliva.

Abstract A protein preparation corresponding to the core-protein described by Levine, Ellison and Bahl (1973) was isolated. Chromatographed on columns of the cation-exchange adsorbent SP-Sephadex, it comprised a family of proline-rich basic proteins which have similar but not identical amino acid composition and contain little or no carbohydrate.

Human pancreatic proteins: Amylase, proelastase, and trypsinogen

Abstract Alpha-amylase exists in human pancreatic juice in multiple molecular forms. Up to six isoenzymes have been demonstrated by polyacrylamide disc gel electrophoresis and starch-digestion zymograms. Five of the isoenzymes account for most of the amylase present, but analysis of higher amylase loads reveals the sixth form. The isoenzymes are not artefacts due to proteolysis nor to photocatalyzed polymerization of sample gels prior to electrophoresis. Comparison of the isoenzymes of human pancreatic amylase with those of human parotid amylase on polyacrylamide disc gels shows no common bands. Both human amylases are retarded on columns of Bio-Gel P-150 beyond the elution volume expected for molecules of their reported sizes, the pancreatic enzyme being held slightly longer than the parotid enzyme. Proelastase has been demonstrated among the more basic proteins. The proelastase is activated by bovine trypsin to a specific enzymatic activity approximately onesixth that of crystalline porcine elastase. A pro-ATEEase is closely associated with the proelastase fraction. A human trypsinogen was isolated from the anionic protein fraction. It can be activated by bovine trypsin, porcine enterokinase, or spontaneously during prolonged incubations. The specific enzymatic activity of the human trypsin studied is lower than that of the bovine enzyme.

Isolation and characterization of the basic proline-rich proteins from rat parotid saliva

Five fractions of basic proline-rich proteins were isolated from rat parotid saliva, obtained by surgical cannulation of the ducts. The purification procedures employed DEAE-Sephadex to isolate a heterogeneous break-through fraction containing the basic proline-rich proteins, followed by gel filtration on Sephadex G-200 to separate the high molecular weight glycoprotein, fraction A, from the other basic proline-rich proteins which were resolved into four additional fractions, SP-1 to SP-4, by ion exchange chromatography on SP-Sephadex. The proteins differed in their amino acid composition and content of neutral and amino sugars. All the proteins were characterized by a high proportion of proline (approx. 40 mol per cent) and glycine (11-23 mol per cent). Four of the fractions were also enriched in glutamic acid/glutamine (19-26 mol per cent). The exception was fraction SP-4, which contained lower levels of glutamic acid/glutamine and has no counterpart in human basic proline-rich proteins. Fraction A, the basic glycoprotein, was heavily glycosylated (59 mol per cent), whereas SP-2 and SP-4 were less glycosylated. Fractions SP-1 and SP-3 contained low levels of neutral and amino sugars. Basic proline-rich proteins constitute a smaller percentage of the total protein in rat parotid saliva than they do in human parotid saliva (10.5 versus 40 per cent). Rat basic glycoprotein fraction constitutes less than 1 per cent whereas the human glycoprotein fraction constitutes 17 per cent. Rat basic proline-rich proteins appear to be larger and less basic than most of the human basic proteins, and they resolve into fewer protein fractions (4 versus 9) with SP-Sephadex chromatography.