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.

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.

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.

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.

Corrections to the amino acid sequence of bovine chymotrypsinogen A.

Reinvestigation of the amino acid sequence of bovine chymotrypsinogen A suggests that the amino acid sequence at the N-terminus of the B-chain (residues 16-19) is -Ile-Val-Asn-Gly- rather than -Ile-Val-Gly-Asp- and that Ser-215 should be deleted.

The primary structure of porcine pancreatic elastase. The N-terminus and disulphide bridges.

1. The amino acid composition and N-terminal groups of purified elastase show that it is a single peptide chain of 234 residues. 2. The N-terminal sequence is Val-Val-Gly-Gly-Thr-Glu-. 3. The sequences around the four disulphide bridges were determined by using a ;diagonal' electrophoretic technique. 4. These four bridges are homologous with the four common to bovine trypsin and chymotrypsin. 5. Out of 83 residues of the elastase sequence so far determined, 43 are homologous with similar regions of trypsin and chymotrypsin. 6. The evolutionary ancestry of these enzymes is discussed.

Late ulnar neuropathy in the brain-injured adult.

Twenty-five brain-injured adults who were treated for tardy ulnar neuropathy during a 5-year period were studied. Two patients had bilateral involvement. The incidence of late ulnar neuropathy in this population was determined to be 2.5%. The ulnar neuropathy was always on the neurologically impaired side and associated with significant spasticity. Diagnosis was made when intrinsic atrophy was noted in the hand. No patient initiated a subjective complaint. Nerve conduction velocity measurements confirmed impingement of the ulnar nerve in the cubital canal in 16 cases. Twenty-one of the 27 (78%) elbows had moderate to severe heterotopic ossification causing impingement of the ulnar nerve. All patients were treated by anterior transposition of the ulnar nerve. Follow-up averaged 22.7 months. Twenty-three (85%) extremities had complete recovery of ulnar nerve function. Four patients had improved but incomplete recovery of function. Prolonged compression of the nerve led to incomplete recovery.

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

A 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.

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.

The presence of chondroitin sulfate in parotid secretory granules and saliva of the rat.

The presence of chondroitin sulfate in secretory granules of the rat parotid gland and its saliva was revealed by radioactive sulfate incorporation, followed by isolation and partial characterization of the sulfated species contained within the granules and in the parotid saliva. 35SO4 was incorporated into chromatographically identical macromolecular material both in vitro, in a gland-slice system followed by isolation of granule contents, and in vivo as measured in the pure parotid secretion following intravenous administration of 35SO4=. The majority of the 35SO4= label appeared in a peak in the region where the family of acidic proline-rich proteins elute from a DEAE-Sephadex A-50 column. Papain digestion freed the sulfated material from the bulk of protein present in this peak, leaving sulfate-labelled material that chromatographed on Sepharose CL6B as a single peak corresponding to a molecular weight of 13,000 daltons. The ratio of uronic acid to amino sugar in this sulfated peak was 0.56. The sulfated material was susceptible to degradation by chondroitinase AC. The presence of this chondroitin sulfate in secretory granules and saliva is consistent with previous suggestions that sulfated polyanions may play a role in formation and maturation of secretory granules.

Basic proline-rich proteins from human parotid saliva: relationships of the covalent structures of ten proteins from a single individual.

Eleven basic proline-rich proteins were purified from the parotid saliva of a single individual. The complete amino acid sequences of six of these were determined by conventional protein sequence methodology, bringing to nine the number of known primary structures of nonglycosylated basic proline-rich proteins from the same individual. The partial sequence of one additional protein is also reported. All of the basic proline-rich proteins studied contain segments with identical or very similar sequences, but with two possible exceptions, none of the proteins is derived from another secreted proline-rich protein. The amino acid sequences of nine nonglycosylated basic proline-rich proteins were compared with primary structures deduced from published nucleotide sequences of DNA coding for human parotid proline-rich proteins. The sequences align well, in general, but differences also exist pointing to the complexity of the genetics of these proteins. Seven secretory basic proline-rich proteins appear to be formed from three larger precursors by selective posttranslational proteolyses of arginyl bonds. One of the basic proline-rich proteins appears to derive from human acidic proline-rich proteins. The remaining two proteins studied do not conform to any DNA structure as yet reported. Two of the basic proline-rich proteins studied are phosphoproteins and exhibit abilities to inhibit hydroxyapatite formation in vitro.

Primary structure and possible origin of the non-glycosylated basic proline-rich protein of human submandibular/sublingual saliva.

Human submandibular/sublingual saliva contains one non-glycosylated basic proline-rich protein whereas parotid saliva contains multiple such components. The submandibular protein has a primary structure identical with the C-terminal segment [TZ] of the human parotid acidic proline-rich proteins that contain 150 amino acid residues (Mr 16,000). Northern-blot analyses of human parotid and submandibular glands revealed that mRNAs containing the HaeIII repeat sequence typical for acidic proline-rich proteins are expressed in both of these salivary glands whereas mRNAs for non-glycosylated basic proline-rich proteins containing a typical BstN1 repeat sequence are expressed in the parotid but not in the submandibular gland. Products of translation in vitro of mRNAs from human parotid and submandibular glands were also examined. Two immunoprecipitable bands with Mr 29,000 and 28,000 were obtained by translation of both parotid and submandibular mRNA. In the presence of microsomal membranes these proteins gave rise to proteins electrophoretically identical with the secreted acidic proline-rich proteins of Mr 16,000. These proteins were cleaved by kallikrein, giving rise to proteins with electrophoretic mobilities identical with those of a smaller acidic proline-rich protein with Mr 11,000 and peptide TZ. Additional immunoprecipitable bands with Mr ranging from 35,000 to 46,000 were seen when parotid mRNA was used for translation in vitro, and are believed to be precursors of the basic proline-rich proteins encoded by the BstN1 repeat type mRNA. Neither these bands nor a separate precursor for the basic non-glycosylated proline-rich protein was detected when submandibular mRNA was used for translation in vitro. It is suggested that the non-glycosylated basic proline-rich protein present in human submandibular saliva arises by cleavage of acidic proline-rich proteins.

Influence of circulating catecholamines on protein secretion into rat parotid saliva during parasympathetic stimulation.

Secretion of proteins by rat parotid glands in response to parasympathetic nerve stimulation was studied in vivo during pentobarbitone anaesthesia. Parasympathetic stimulation (3-10 Hz) via the auriculotemporal nerve resulted in a copious flow of saliva low in protein. In contrast, sympathetic stimulation (5 Hz) via the cervical sympathetic trunk evoked saliva low in volume but high in protein. Nevertheless, the specific concentrations of amylase and peroxidase (mg/mg protein) and the ratio of amylase to peroxidase remained constant. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis revealed a single, rapidly migrating protein band of unknown identity in proportionately greater amounts in parasympathetic saliva than in sympathetic saliva. Bilateral adrenalectomy led to reduced amylase and peroxidase secretion in response to parasympathetic stimulation both on a mg/ml and a mg/mg protein basis. SDS gel electrophoresis also demonstrated the decrease in specific amylase concentration following adrenalectomy. The ratio of amylase to peroxidase, however, was not significantly affected. Administration of 6-hydroxydopamine 17-72 h prior to adrenalectomy caused no further reduction in the secretion of amylase and peroxidase. Chronic sympathectomy of 2.5-4 months duration resulted in an increased protein secretion (mg/ml) by the parotid gland in response to parasympathetic stimulation. This increase was only slightly reduced by bilateral adrenalectomy. However, as observed in non-sympathectomized rats, adrenalectomy caused a significant reduction in the specific concentrations of both amylase and peroxidase, but did not affect the amylase to peroxidase ratios. We conclude that parasympathetic nerve stimulation of rat parotid glands after overnight starvation causes secretion of proteins in proportions similar to, but in significantly lower concentrations than those found in sympathetic saliva. Circulating catecholamines, however, influence the amount of amylase and peroxidase secreted by the rat parotid gland in response to parasympathetic nerve stimulation and account for most of the increased secretion of these enzymes following chronic sympathectomy.