Dorothy L. Kauffman

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.

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.

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.

Isolation and partial characterization of two populations of secretory granules from rat parotid glands

SummaryA method is described for the isolation of two populations of secretory granules from rat parotid glands utilizing differences in their sedimentation characteristics. The granule preparations were analyzed for homogeneity by electron microscopy and chemical analyses. The soluble contents of both types of granules were obtained by hypotonic lysis, and the proteins compared by SDS-PAGE and ion exchange-gel filtration chromatography. Both populations of secretory granules appear to have the same protein composition as that of the parotid saliva. The secretory granules with the smaller apparent buoyant density became labelled with radioactive leucine earlier than the heavier granules when a pulse of this amino acid was supplied to a gland slice system. The lighter granules appear to represent an earlier stage in maturation.

The existence of glycosylated and non-glycosylated forms of human submandibular amylase

Abstract Amylase was isolated from human submandibular-sublingual saliva by BioGel P-2 filtration, ammonium sulphate precipitation and DEAE-Sephadex chromatography. The purified enzyme was separated into isoenzyme families A and B on Bio-Gel P-100 and the families were compared with the corresponding families of human parotid amylase. The respective enzymes exhibited identical specific enzymatic activities and isoenzyme patterns on polyacrylamide disc gels. Family A of human submandibular amylase contained the equivalent of 6 moles of covalently linked neutral sugar per mole of enzyme as compared to 7 moles/mole for the parotid family A. Family B contained no carbohydrate. The proportion of submandibular amylase that is glycosylated (25 per cent) is the same as in the parotid. All α-amylases tested exhibited an unusual absorption spectrum with a maximum at 405 nm when reacted with the phenol-sulphuric acid reagent. The A families of both human parotid and submandibular amylases have an additional maximum at 485 nm, typical of glycoproteins. The B families do not exhibit the second maximum.

Demonstration of a class of proteins loosely associated with secretory granule membranes

It is shown, in this study, that rat secretory granule membrane preparations, as prepared by the method of Amsterdam et al. [(1971) J. Cell Biol. 50, 187-200], contain a protein fraction which is removed by washing in isotonic medium. This fraction contains unusually high levels of Pro, Gly and Glx, and appears to label rapidly if the rats are pulsed with [14-c] amino acids prior to removal of the glands. The fraction, which may represent specifically adsorbed secretory protein(s) or peripheral membrane protein, is significant to investigators using this model system to study secretory phenomena.

Relationship of the basic glycoprotein to the basic proline-rich proteins in human parotid saliva

Abstract The relationship was investigated by chemically deglycosylating the glycoprotein and comparing the protein moiety so obtained with non-glycosylated proline-rich proteins. Deglycosylation of the purified salivary basic proline-rich glycoprotein resulted in three protein moieties. After purification, these fractions were compared biochemically to the non-glycosylated basic proline-rich proteins by amino acid composition, cationic and sodium dodecyl sulphate gel electrophoreses, end groups and peptide mapping. The findings indicate that none of the basic proline-rich proteins are precursor proteins to the basic glycoprotein.

Alignment of Amino Acid and DNA Sequences of Human Proline-rich Proteins

Human proline-rich proteins (PRPs) constitute a complex family of salivary proteins that are encoded by a small number of genes. The primary gene product is cleaved by proteases, thereby giving rise to about 20 secreted proteins. To determine the genes for the secreted PRPs, therefore, it is necessary to obtain sequences of both the secreted proteins and the DNA encoding these proteins. We have sequenced most PRPs from one donor (D.K.) and aligned the protein sequences with available DNA sequences from unrelated individuals. Partial sequence data have now been obtained for an additional PRP from D.K. named II-1. This protein was purified from parotid saliva by gel filtration and ion-exchange chromatography. Peptides were obtained by cleavage with trypsin, clostripain, and N-bromosuccinimide, followed by column chromatography. The peptides were sequenced on a gas-phase protein sequenator. Overlapping peptide sequences were obtained for most of II-1 and aligned with translated DNA sequences. The best fit was obtained with clones containing sequences for the allele PRB4M (Lyons et al., 1988). However, there was not complete identity of the protein amino acid sequence and the DNA-derived sequences, indicating that II-1 is not encoded by PRB4M. Other PRPs isolated from D.K. also fail to conform to any DNA structure so far reported. This shows the need to obtain amino acid sequences and corresponding DNA sequences from the same person to assign genes for the PRPs and to determine the location of the postribosomal cleavage points in the primary translation product.