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Biomedical subjects

S D Freedman

Publications and source records attributed to S D Freedman.

At least 37 records · Page 2Linked to original sources
PubMed1993

Regulated secretory proteins in the exocrine pancreas aggregate under conditions that mimic the trans-Golgi network.

Fifteen pancreatic secretory proteins, including seven serine-endoproteinases (isoenzyme forms of trypsinogen, chymotrypsinogen and proelastase), four metallo-exoproteinases (isoenzymic forms of procarboxypeptidase A and procarboxypeptidase B), amylase, lipase, and two forms of carboxyl ester lipase were observed to aggregate under conditions of acidic pH (5.5) and calcium that mimic the trans-Golgi network. Subsequent neutralization of the pH resulted in disruption of protein aggregates and solubilization of pancreatic (pro)enzymes. In the absence of secretory granule membranes, granule contents display an "intrinsic" property for reversible, pH-dependent aggregation under conditions of mild acidification.

Amylases↗
PubMed1993

GP2, the homologue to the renal cast protein uromodulin, is a major component of intraductal plugs in chronic pancreatitis.

Protein plug obstruction of the pancreatic duct is one of the early events in chronic pancreatitis yet little is known about its pathogenesis. GP2, a protein in the exocrine pancreas, is a glycosyl phosphatidylinositol-anchored protein that is cleaved from the zymogen granule membrane and secreted into pancreatic juice. Since its homologue, uromodulin, is involved in renal cast formation, we asked the question whether GP2 might play a similar role in plug formation in chronic pancreatitis. The protein composition of intraductal plugs from patients with noncalcific chronic pancreatitis was examined. Plugs purified from pancreatic juice obtained by endoscopic cannulation were analyzed by SDS-PAGE. A 97-kD protein was found not only to be a reproducible constituent but also enriched within intraductal plugs. This protein was confirmed as GP2 by its localization to zymogen granule membranes, its isoelectric point, and by Western blotting. Although the pancreatic stone protein was identified in plugs, it was not a major reproducible component. These results demonstrate that GP2 is an integral component of plugs in pancreatic juice and suggest that GP2 may play a role in pancreatic plug formation that is analogous to the role played by uromodulin in the pathogenesis of renal casts.

Cytoplasmic Granules↗
PubMed1993

Reversible pH-induced homophilic binding of GP2, a glycosyl-phosphatidylinositol-anchored protein in pancreatic zymogen granule membranes.

GP2, the major zymogen granule membrane (ZGM) protein in the pancreas, is linked to the lumenal leaflet of the lipid bilayer via a glycosyl-phosphatidylinositol (GPI) moiety. We demonstrate that the peptide domain of GP2 (pGP2, approximately 75 kDa), purified from pancreatic ZGMs after phospholipase C cleavage, shows pH- and calcium-dependent self-association into sedimenting complexes. This homophilic binding process is progressive as pH is reduced from 7.0 to 5.5 and calcium is increased from 0 to 10-20 mM. This self-association reaction is temperature-dependent, optimal between 20 and 37 degrees C, progressively reduced below 20 degrees C, and eliminated at 10 degrees C. The reaction is reversible as a function of pH and abolished in the presence of nonionic detergents. Specificity in the homophilic reaction is demonstrated by the exclusion of heterologous proteins (globin, serum albumin, and IgG) from sedimenting complexes. At pH 5.5 in the presence of 20 mM calcium, oligomeric structures (approximately 300 kDa) consistent with tetrameric complexes were observed by gel filtration chromatography and elliptical structures (14-18 nm), frequently arranged in variegated clusters, were observed in the electron microscope by negative staining techniques. The pH- and calcium-dependent self-association observed for GP2 may represent an important mechanism by which GPI-anchored membrane proteins engage in homotypic binding reactions to establish highly functional membrane (micro)- domains targeted to regulated secretory compartments in polarized epithelial cells.

Animals↗
PubMed1992

GP-2/THP gene family encodes self-binding glycosylphosphatidylinositol-anchored proteins in apical secretory compartments of pancreas and kidney.

A family of homologous genes is shown to encode GP-2, the major glycosylphosphatidylinositol (GPI)-linked glycoprotein of pancreatic zymogen granule membranes, and Tamm-Horsfall protein (THP), a GPI-linked glycoprotein associated with apical vesicles in kidney thick ascending limb of Henle (TALH) cells. The C-terminal regions of GP-2 (Asp54-Phe530) and THP (Asp175-His644) from rat show 53% identity, 86% similarity, and 26 conserved cysteine residues including one epidermal growth factor motif. The unique N-terminal domain of rat THP (unique-THP, Pro29-Gln174) shows four conserved epidermal growth factor motifs, three in tandem and one in reverse orientation. GP-2 homologues are observed in a wide variety of epithelial cells, several of which contain highly regulated secretory processes. GP-2 released from zymogen granule membranes with phosphatidylinositol phospholipase C reacts with anti-cross-reactive determinant antibody (anti-CRD), confirming the GPI nature of the pancreatic homologue. In contrast, GP-2 and THP, released endogenously from pancreas and kidney, respectively, do not react with anti-cross-reactive determinant antibody, suggesting alternative enzymatic mechanisms for their physiological release. Globular domains of GP-2 and THP, but not albumin, show pH- and ion-dependent self-association in vitro. The GP-2/THP family appears to represent a newly discovered class of GPI-anchored proteins, which may utilize pH- and ion-dependent self-association mechanisms for establishing membrane (micro)domains targeted to intracellular secretory compartments.

Amino Acid Sequence↗
PubMed1991

A single gene encodes membrane-bound and free forms of GP-2, the major glycoprotein in pancreatic secretory (zymogen) granule membranes.

GP-2, a 75-kDa glycoprotein, was isolated from dog pancreatic zymogen granule membranes (ZGMs). In a carbohydrate-shift strategy, N-terminal and internal peptide sequences were obtained on glycosylated and deglycosylated forms of GP-2, respectively, by gas-phase sequencing. Sets of mixed oligonucleotides and the polymerase chain reaction were used to obtain a double-stranded cDNA probe, which was used to isolate overlapping cDNA clones from a dog pancreatic cDNA library. The sequence of these clones revealed an open reading frame that encodes a protein of 509 amino acids, eight N-linked oligosaccharide attachment sites, and an N-terminal signal sequence absent from the mature form of GP-2 associated with ZGMs. The C terminus shows a 20-residue hydrophobic transmembrane domain preceded by a decapeptide containing potential phosphatidylinositol-glycan attachment sites. GP-2 completely released from ZGMs by exogenous phospholipase C showed similar immunochemical properties and electrophoretic mobilities compared to the form associated with ZGMs. A similar form of GP-2 was released from zymogen granules permeabilized with saponin and incubated in the absence of added phospholipase C. Kinetic analysis of GP-2 release at 0 degrees C and 37 degrees C suggested the presence of a granule enzyme responsible for endogenous release of GP-2 to granule contents and into the apical medium. The data indicate that GP-2 is a phosphatidylinositol-glycan-linked membrane protein released from the membrane of mature zymogen granules by an enzymatic mechanism. The cDNA structure presented here thus encodes both membrane-bound and free forms of GP-2.

Amino Acid Sequence↗
PubMed1983

Calmodulin-stimulated protein kinase activity from rat pancreas.

Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein S6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co-factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When 125I-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM-PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calcium-calmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.

Animals↗
PubMed1982

Hormone-induced protein phosphorylation. I. Relationship between secretagogue action and endogenous protein phosphorylation in intact cells from the exocrine pancreas and parotid.

We undertook studies to determine whether secretagogue action on the exocrine pancreas and parotid is accompanied by phosphorylation of proteins in intact cells. For this purpose, rat pancreatic, and parotid lobules were preincubated with 32Pi for 45 min at 37 degrees C, washed, and then incubated at 37 degrees C in the presence or absence of secretagogues that effect discharge through different second messengers. Among a variety of polypeptides exhibiting enhanced phosphorylation in pancreatic lobules upon a 30-s incubation in the presence of the secretagogues carbamylcholine, cholecystokinin octapeptide, or secretin, one species with an Mr of 29,000 was especially notable for three reasons: (a) its enhanced level of phosphorylation was dependent on the dose of secretagogue used and was still apparent after incubation for 30 min at 37 degrees C; (b) an analogous phosphorylated polypeptide was observed in isoproterenol-stimulated parotid lobules; and (c) in both tissues its selective dephosphorylation was observed upon termination of stimulation by administration of atropine to carbamylcholine-stimulated pancreatic lobules and propranolol to isoproterenol-stimulated parotid lobules. These results suggest that the phosphorylation of one protein with an Mr of 29,000 is closely correlated both temporally and in a dose-dependent fashion with secretagogue action in both the exocrine pancreas and parotid.

Animals↗
PubMed1982

Hormone-induced protein phosphorylation. II. Localization to the ribosomal fraction from rat exocrine pancreas and parotid of a 29,000-dalton protein phosphorylated in situ in response to secretagogues.

In the preceding paper, we demonstrated that the endogenous phosphorylation of a protein with a molecular weight of 29,000 was enhanced by various secretagogues in rat pancreatic and parotid lobules, the phosphorylation of this protein correlating both temporally and in a dose-dependent fashion with secretory protein discharge. In the present study, we established a specific methodology to characterize this phosphoprotein. Once established, this 29,000-dalton phosphoprotein was then followed selectively and quantitatively throughout subcellular fractionation procedures. Analysis of two-dimensional polyacrylamide gels demonstrated that proteins with similar mobilities (Mr 29,000; pl greater than 8.4) were affected by cholecystokinin octapeptide and isoproterenol in rat pancreatic and parotid lobules, respectively, suggesting that the same 29,000-dalton phosphoprotein was covalently modified in both tissues. Cellular fractionation studies using differential velocity and sucrose density gradient centrifugation revealed that the 29,000-dalton phosphoprotein copurified with the rough microsomal fraction of pancreas and was highly enriched in ribosomal fractions of both pancreas and parotid. Electrophoresis in two dimensions confirmed that the 29,000-dalton polypeptide that was resolved directly from stimulated cells and from ribosomal fractions exhibited a common mobility, and apparent identity of the species was strongly suggested when the 29,000-dalton polypeptides from both sources were compared by peptide mapping following limited digestion with Staphylococcus aureus V8 protease. This phosphoprotein was tentatively identified as ribosomal protein S6 after analysis by pH 8.6/4.2 two-dimensional PAGE.

Animals↗
PubMed1982

Hormone-induced protein phosphorylation. III. regulation of the phosphorylation of the secretagogue-responsive 29,000-dalton protein by both Ca2+ and cAMP in vitro.

In the preceding papers, we demonstrated that the endogenous phosphorylation of a 29,000-dalton protein is stimulated in response to secretagogue application to intact cells from the rat exocrine pancreas and parotid and dephosphorylated upon termination of secretagogue action. One- and two-dimensional gel analysis of 32Pi-labeled pancreatic and parotid lobules as well as their respective subcellular fractions revealed that the same protein was covalently modified in both tissues and was localized to the ribosomal fraction. To identify the intracellular second messengers which may mediate or modulate the phosphorylation of the 29,000-dalton protein in intact cells, the effects of Ca2+, cAMP, and cGMP on the endogenous phosphorylation of this protein were assessed in subcellular fractions from the rat pancreas and parotid. Our results demonstrate that the phosphorylation of the 29,000-dalton polypeptide may be regulated by both Ca2+ and cAMP in the pancreas and in the parotid. No cGMP-dependent protein phosphorylation was found in either tissue. As in the in situ phosphorylation studies, the Ca2+- and cAMP-dependent phosphorylation of this same protein was localized to the ribosomal fraction. The cAMP-dependent protein kinase activity was found primarily in the postmicrosomal supernatant in contrast to the Ca2+-dependent protein kinase that appeared to be tightly associated with the substrate in addition to being present in the postmicrosomal supernatant. The data suggest that, in cells from the exocrine pancreas and parotid, secretagogues may regulate the phosphorylation of the 29,000-dalton protein through Ca2+ and/or cAMP.

Animals↗
PubMed1982

Aspergillus endophthalmitis in a drug abuser.

In a 31-year-old man, metastatic Aspergillus endophthalmitis developed after intravenous cocaine injection. His reluctance to admit to drug abuse resulted in delay of diagnostic and curative vitrectomy. Metastatic osteomyelitis also developed, underscoring the need for careful surveillance for other systemic manifestations of fungemia. We believe this to be the first published case in which useful vision was salvaged in an eye afflicted by metastatic Aspergillus endophthalmitis.

Adult↗
PubMed1980

Binding of horseradish peroxidase-alpha-bungarotoxin to axonal membranes at the node of Ranvier.

The binding of horseradish peroxidase (HRP)-labeled alpha-bungarotoxin (alpha-BuTx) was investigated in rat sciatic nerve. Activity was found to be localized to the axolemma of myelinated nerve fibers at the nodes of Ranvier. Activity was also seen in other regions of the axolemma where the myelin sheath was separated from the axon by enzymatic treatment. Pretreatment of nerves with native alpha-BuTx or curare blocked the binding of HRP-alpha-BuTx to the axonal membranes. This study demonstrates binding of alpha-BuTx to axonal membranes although the nature and significance of the toxin receptor is uncertain.

Animals↗
PubMed1980

Kawasaki disease in healthy young adult.

This report describes a 26-year-old woman who fulfills the criteria for the diagnosis of Kawasaki disease or mucocutaneous lymph node syndrome, an acute febrile illness that usually afflicts young children. The diagnosis is made in persons with fever lasting 5 or more days when four of the following criteria are met: bilateral injection of ocular conjunctivae; the involvement of the mucous membranes of the upper respiratory tract consisting of any combination of the following--redness and fissuring of lips; "strawberry tongue," or erythema of the pharynx; involvement of the peripheral extremities characterized in the early stages by an indurative erythematous rash of palms and soles followed by membranous desquamation; polymorphous nonvesicular truncal exanthem; and acute nonsuppurative enlargement of cervical lymph nodes. An added stipulation is that the illness must not be attributable to a known disease process.

Adult↗
PubMed1979

Stimulation of Ca2+-dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles.

Synaptic vesicles have a Ca(2+)-dependent protein kinase system that may play a role in mediating Ca(2+)-stimulated neurotransmitter release and vesicle function. Calcium's ability to initiate norepinephrine release and protein phosphorylation in synaptic vesicle preparations was shown to be stimulated by the presence of an endogenous heat-stable vesicle protein fraction. The heat stability and characteristics of this endogenous vesicle fraction were similar to those of calmodulin (Ca(2+)-dependent regular protein) isolated from rat and bovine brain. Calmodulin, like endogenous heat-stable vesicle factor, restored calcium's ability to stimulate vesicle neurotransmitter release and protein kinase activity. Calmodulin-like vesicle protein and purified calmodulin were also equally effective in stimulating cyclic nucleotide-dependent phosphodiesterase, further indicating that these two proteins are functionally equivalent. Depolarization-dependent Ca(2+) uptake in intact synaptosomes simultaneously stimulated release of neurotransmitter and phosphorylation of particular synaptic vesicle proteins that were shown in the isolated vesicle preparation to be dependent on Ca(2+) and calmodulin. The results suggest that calcium's effects on neurotransmitter release and presynaptic nerve terminal protein phosphorylation may be mediated by endogenous calmodulin-like proteins.

Animals↗