Accumulation of cystine from glutathione-cysteine mixed disulfide in cystinotic fibroblasts; blockade by an inhibitor of gamma-glutamyl transpeptidase

Cystinotic and normal skin fibroblasts in tissue culture were treated with varying concentrations of reduced glutathione, oxidized glutathione and glutathione-cysteine mixed disulfide, substrates of gamma-glutamyl transpeptidase, the catabolic enzyme of the gamma-glutamyl cycle. Cystine accumulated more rapidly and to a greater extent from the glutathione-cysteine mixed disulfide in cystinotic than in normal cells. Inhibition of gamma-glutamyl transpeptidase activity by serine in a borate buffer partially blocked this accumulation of cystine. Reduced glutathione and oxidized glutathione have lesser effects on cystine accumulation. Stored cystine in cystinotic tissues may derive in part from glutathione-cysteine mixed disulfide via transpeptidation.     

Not all secretory granules are created equal: Partitioning of soluble content proteins

Secretory granules carrying fluorescent cargo proteins are widely used to study granule biogenesis, maturation, and regulated exocytosis. We fused the soluble secretory protein peptidylglycine alpha-hydroxylating monooxygenase (PHM) to green fluorescent protein (GFP) to study granule formation. When expressed in AtT-20 or GH3 cells, the PHM-GFP fusion protein partitioned from endogenous hormone (adrenocorticotropic hormone, growth hormone) into separate secretory granule pools. Both exogenous and endogenous granule proteins were stored and released in response to secretagogue. Importantly, we found that segregation of content proteins is not an artifact of overexpression nor peculiar to GFP-tagged proteins. Neither luminal acidification nor cholesterol-rich membrane microdomains play essential roles in soluble content protein segregation. Our data suggest that intrinsic biophysical properties of cargo proteins govern their differential sorting, with segregation occurring during the process of granule maturation. Proteins that can self-aggregate are likely to partition into separate granules, which can accommodate only a few thousand copies of any content protein; proteins that lack tertiary structure are more likely to distribute homogeneously into secretory granules. Therefore, a simple "self-aggregation default" theory may explain the little acknowledged, but commonly observed, tendency for both naturally occurring and exogenous content proteins to segregate from each other into distinct secretory granules.     

Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma

The distribution of the glutathionyl moiety between reduced and oxidized forms in rat plasma was markedly different than that for the cysteinyl moiety. Most of the glutathionyl moiety was present as mixed disulfides with cysteine and protein whereas most of the cysteinyl moiety was present as cystine. Seventy percent of total glutathione equivalents was bound to proteins in disulfide linkage. The distribution of glutathione equivalents in the acid-soluble fraction was 28.0% as glutathione, 9.5% as glutathione disulfide, and 62.6% as the mixed disulfide with the cysteinyl moiety. In contrast, 23% of total cysteine equivalents was protein-bound. The distribution of cysteine equivalents in the acid-soluble fraction was 5.9% as cysteine, 83.1% as cystine, and 10.8% as the mixed disulfide with the glutathionyl moiety. A first-order decline in glutathione occurred upon in vitro incubation of plasma and was due to increased formation of mixed disulfides of glutathione with cysteine and protein. This indicates that plasma thiols and disulfides are not at equilibrium, but are in a steady-state maintained in part by transport of these compounds between tissues during the inter-organ phase of their metabolism. The large amounts of protein-bound glutathione and cysteine provide substantial buffering which must be considered in analysis of transient changes in glutathione and cysteine. In addition, this buffering may protect against transient thiol-disulfide redox changes which could affect the structure and activity of plasma and plasma membrane proteins.     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

Inactivation of human placenta glutathione S-transferase by SH/SS exchange reaction with biological disulfides

The oxidized glutathione inhibited the activity of glutathione S-transferase purified from human placenta just through competitive inhibition. On the other hand, cystine and cystamine inactivated the activity by pseudo first-order in low concentrations, accompanying the stoichiometric incorporation of the radioactivity of [14C]-cystine to the enzyme protein until a half mole per one subunit. This and the protective effect of glutathione analogues suggested that the SH/SS exchange reaction occurred between the disulfide and the SH group near the glutathione binding site of the enzyme to form a mixed disulfide.     

Inhibitor studies with adenohypophyseal granule membrane ATPase. Evidence for a membrane environment which modulates sensitivity to inhibitors

The limiting membranes of pituitary growth hormone and prolactin secretory granules contain a Mg2+-ATPase sensitive to anions. This enzyme is in many ways similar to mitochondrial ATPase. The enzyme was potently inhibited by oligomycin (Ki 6.5 X 10(-9) M), and was much more sensitive to the inhibitor than pituitary mitochondrial ATPase (Ki 2.7 X 10(-7) M). In contrast, the enzyme activity of intact secretory granules was only sparingly inhibited by oligomycin (maximal inhibition close to 30% at 5 X 10(-4) M). However, oligomycin (5 microM) did diminish to basal levels the enhanced granule ATPase activity observed in the presence of a stimulatory anion (25 mM sodium sulfite). Other compounds known to inhibit the proton translocating mitochondrial ATPase were also tested for their ability to inhibit the secretory granule ATPase. A similar pattern of limited inhibition in granules and greater sensitivity in isolated membranes was seen with the inhibitors N,N-dicyclohexylcarbodiimide and efrapeptin. In contrast, tri-n-butyltin chloride was a potent inhibitor of the ATPase of intact granules, and the susceptibility of the enzyme to inhibition by this compound was less after isolation of membranes. These observations suggest that pituitary secretory granule membrane ATPase may have a proton pumping function similar to that of the mitochondrial enzyme. In addition, the data imply that the inhibitor binding site(s) may be masked, inaccessible, or ineffective in intact granules, but exposed (or activated) in isolated membranes. The greater sensitivity of granule ATPase to tri-n-butyltin chloride, in contrast to the greater sensitivity of membrane ATPase to the other inhibitors, indicates that the tin compound may be effective at a membrane site(s) distinct from the others, or that the mechanism of inhibition is different.     

Sulfhydryl oxidase from egg white. A facile catalyst for disulfide bond formation in proteins and peptides.

Both metalloprotein and flavin-linked sulfhydryl oxidases catalyze the oxidation of thiols to disulfides with the reduction of oxygen to hydrogen peroxide. Despite earlier suggestions for a role in protein disulfide bond formation, these enzymes have received comparatively little general attention. Chicken egg white sulfhydryl oxidase utilizes an internal redox-active cystine bridge and a FAD moiety in the oxidation of a range of small molecular weight thiols such as glutathione, cysteine, and dithiothreitol. The oxidase is shown here to exhibit a high catalytic activity toward a range of reduced peptides and proteins including insulin A and B chains, lysozyme, ovalbumin, riboflavin-binding protein, and RNase. Catalytic efficiencies are up to 100-fold higher than for reduced glutathione, with typical K(m) values of about 110-330 microM/protein thiol, compared with 20 mM for glutathione. RNase activity is not significantly recovered when the cysteine residues are rapidly oxidized by sulfhydryl oxidase, but activity is efficiently restored when protein disulfide isomerase is also present. Sulfhydryl oxidase can also oxidize reduced protein disulfide isomerase directly. These data show that sulfhydryl oxidase and protein disulfide isomerase can cooperate in vitro in the generation and rearrangement of native disulfide pairings. A possible role for the oxidase in the protein secretory pathway in vivo is discussed.     

The secretory lectin ZG16p mediates sorting of enzyme proteins to the zymogen granule membrane in pancreatic acinar cells

The recently established in vitro assay of condensation-sorting of pancreatic enzymes to the zymogen granule membrane (ZGM) (Dartsch, H., R. Kleene, H. F. Kern: In vitro condensation-sorting of enzyme proteins isolated from rat pancreatic acinar cells. Eur. J. Cell Biol. 75, 211-222 (1998)) was used to study the involvement of a novel secretory lectin, ZG16p, in the binding of aggregated proteins to ZGM. In isolated zymogen granules the lectin is predominantly associated with the membrane and can be removed to a large extent by bicarbonate treatment at pH 11.5. In the in vitro assay in which secretory proteins aggregate at pH 5.9 but only those bound to ZGM are sedimented into the pellet, ZG16p is significantly enriched in this pellet fraction, shown both by biochemical and fine structural analysis. Pretreatment of ZGM with anti-ZG16p antibody before their addition to the assay inhibits binding to the membrane by about 50%. Similarly, removal of ZG16p or prevention of its interaction with glycosaminoglycans (GAGs) in the submembranous matrix of ZGM by sodium bicarbonate treatment or chondroitinase digestion of ZGM also inhibits the binding efficiency of secretory proteins to ZGM to about the same extent. We conclude that ZG16p may act as a linker molecule between the submembranous matrix on the luminal side of ZGM and aggregated secretory proteins during granule formation in the TGN.     

Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat beta-cell tumour.

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.     

Retrieval and reuse of pituitary secretory granule proteins

The pituitary contains professional secretory cells, devoting a large fraction of their energy to the synthesis of hormones that are stored for secretion in response to a complex mixture of inputs. Ba2+, a substitute for Ca2+, and phorbol ester, a mimic for diacylglycerol, have a synergistic effect on exocytosis. By using these secretagogues, we developed a paradigm in which phorbol ester potentiation of Ba2+-evoked exocytosis produces a robust secretory response in multiple pituitary cell types. Because cells subjected to this stimulatory paradigm remain healthy despite their greatly reduced hormone content, we used this paradigm to study the fate of granule membrane proteins. We examined the turnover of peptidylglycine alpha-amidating monooxygenase (PAM), a membrane enzyme involved in the final maturation of many peptides, and VAMP2, a vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). The stability of recently synthesized PAM was increased by sustained exocytosis. Biotinylation studies established that the appearance of integral membrane PAM at the plasma membrane was stimulated along with hormone secretion. PAM biotinylated on the cell surface undergoes cleavage to yield soluble peptidylglycine-alpha-hydroxylating monooxygenase that can then be secreted in a regulated fashion. Consistent with a kiss-and-run or cavicapture mode of secretion (Taraska, J. W., Perrais, D., Ohara-Imaizumi, M., Nagamatsu, S., and Almers, W. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2070-2075), biotinylated prolactin was also retained by the cells and later released in response to secretagogues. Thus, pituitary cells can retrieve and reuse components of the machinery involved in the final stages of exocytosis (the SNAREs) as well as soluble and membrane granule proteins.     

Organellar proteomics: analysis of pancreatic zymogen granule membranes

The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.     

Features of a Spatially Constrained Cystine Loop in the p10 FAST Protein Ectodomain Define a New Class of Viral Fusion Peptides

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusion proteins. With ectodomains of only ∼20–40 residues, it is unclear how such diminutive fusion proteins can mediate cell-cell fusion and syncytium formation. Contained within the 40-residue ectodomain of the p10 FAST protein resides an 11-residue sequence of moderately apolar residues, termed the hydrophobic patch (HP). Previous studies indicate the p10 HP shares operational features with the fusion peptide motifs found within the enveloped virus membrane fusion proteins. Using biotinylation assays, we now report that two highly conserved cysteine residues flanking the p10 HP form an essential intramolecular disulfide bond to create a cystine loop. Mutagenic analyses revealed that both formation of the cystine loop and p10 membrane fusion activity are highly sensitive to changes in the size and spatial arrangement of amino acids within the loop. The p10 cystine loop may therefore function as a cystine noose, where fusion peptide activity is dependent on structural constraints within the noose that force solvent exposure of key hydrophobic residues. Moreover, inhibitors of cell surface thioreductase activity indicate that disruption of the disulfide bridge is important for p10-mediated membrane fusion. This is the first example of a viral fusion peptide composed of a small, spatially constrained cystine loop whose function is dependent on altered loop formation, and it suggests the p10 cystine loop represents a new class of viral fusion peptides.     

A subclass of proteins and sulfated macromolecules secreted by AtT-20 (mouse pituitary tumor) cells is sorted with adrenocorticotropin into dense secretory granules

The AtT-20 cell, a mouse pituitary tumor line that secretes adrenocorticotropin and beta-endorphin, sorts the proteins it externalizes into two exocytotic pathways. Cells that are labeled with [35S]methionine or [35S]sulfate can be shown to transport three acidic polypeptides (65,000, 60,000, and 37,000 mol wt) and at least two sulfated macromolecules into storage secretory granules. When the cells are stimulated by the secretagogue 8-bromo-cAMP, these polypeptides are coordinately secreted with mature adrenocorticotropin into the culture medium. In contrast, a completely different set of secreted polypeptides and sulfated macromolecules does not enter a storage form and is transported to the cell surface more rapidly. Their secretion from the cells is constitutive and does not require the presence of secretagogues. These molecules, like a viral membrane glycoprotein described previously (Gumbiner, B., and R. B. Kelly, 1982, Cell, 28:51-59) are not found in isolated secretory granules and therefore must reach the cell surface in a different exocytotic vesicle. The segregation of a subclass of secretory macromolecules into the secretory granules, despite the existence of another potential secretory pathway, suggests that these molecules have specific functions related to regulated hormone secretion or storage. Presumably all of the proteins secreted by the regulated secretory granule pathway share some common property that targets them to the secretory granule.     

S-METHYLTHIO-CYSTEINE AND CYSTAMINE ARE POTENT STIMULATORS OF THIOL PRODUCTION AND GLUTATHIONE SYNTHESIS

The effects of methylthio-cysteine disulfide (MT-Cy) and cystamine (CAM) on the thiol production and glutathione content of a human T cell line (CEM-SS) have been investigated. MT-Cy per se and CAM in the presence of cystine greatly enhanced thiol production and glutathione content of cells while cystine alone exerted no or slight influence in the first hours. The MT-Cy- or CAM-induced extracellular SH-generation was observed both in a complete nutrient medium and even more in SH-free D-PBS. The acid-soluble thiol level and glutathione content of cells elevated markedly (up to 5–6 fold in two hours) when incubating cells in complete medium. Inhibition of glutathione synthesis by DL-buthionine (S,R)-sulfoximine did not alter the MT-Cy- or CAM-induced extracellular thiol production indicating that glutathione synthesis is not involved in this effect. The results suggest that MT-Cy easily enters the cells thus accelerating the thiol cycle in SH-poor medium while CAM promotes cystine uptake into the cells. Phenylalanine and leucine inhibited both MT-Cy- and CAM-dependent thiol production in D-PBS most effectively suggesting the involvement of the L membrane transport system in these effects. © 1998 Elsevier Science Inc.     

Calmodulin-Binding Proteins in Chromaffin Cell Plasma Membranes

Abstract: Calmodulin-binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin-binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine- to 10–fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X-100 and applied to a calmodulin-affinity column in the presence of calcium. Several major calmodulin-binding proteins ( 240, 105 , and 65 kilodaltons) were eluted by an EGTA-containing buffer. ¹²⁵I-Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin-binding proteins in common ( 65, 60, 53 , and 50 kilodaltons), as well as unique calmodulin-binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65–kilodalton calmodulin-binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two-dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65–kilodalton calmodulin-binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin-binding proteins isolated from these membranes, they recognized a 65–kilodalton protein. These results indicate that an immunologically identical calmodulin-binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.     

Glutathione excretion in response to heterologous protein secretion in Saccharomyces cerevisiae

Glutathione is excreted in a dose-dependent, non-stoichiometric fashion from Saccharomyces cerevisiae cells expressing and secreting Bovine Pancreatic Trypsin Inhibitor (BPTI), a small, disulfide-bonded protein. Glutathione excretion commences 40 hours following induction of BPTI synthesis. Expression of several secretory proteins with varying disulfide and cysteine contents results in glutathione excretion with no apparent requirement for protein disulfide content. Glutathione excretion is also triggered by overexpression of Kar2p/BiP, a native ER-resident protein-folding chaperone, indicating that the response is a general one not restricted to overexpression of thiol-containing heterologous proteins. Functional vesicular transport is not required at the time of glutathione excretion, and glutathione excretion requires the presence of molecular oxygen. These data are consistent with a delayed oxidative stress response potentiated by earlier heterologous secretion, but are inconsistent with secretory transport of glutathione spent as oxidizing equivalents for disulfide-bond formation in the endoplasmic reticulum.     

Astrocytes provide cysteine to neurons by releasing glutathione

Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine<-->cysteine + cysteine-glutathione disulfide. Conditioned medium from neuron-enriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS.     

Cystine knot mutations affect the folding of the glycoprotein hormone alpha-subunit. Differential secretion and assembly of partially folded intermediates

The common glycoprotein hormone alpha-subunit (GPH-alpha) contains five intramolecular disulfide bonds, three of which form a cystine knot motif (10-60, 28-82, and 32-84). By converting each pair of cysteine residues of a given disulfide bond to alanine, we have studied the role of individual disulfide bonds in GPH-alpha folding and have related folding ability to secretion and assembly with the human chorionic gonadotropin beta-subunit (hCG-beta). Mutation of non-cystine knot disulfide bond 7-31, bond 59-87, or both (leaving only the cystine knot) resulted in an efficiently secreted folding form that was indistinguishable from wild type. Conversely, the cystine knot mutants were inefficiently secreted (<25%). Furthermore, mutation of the cystine knot disulfide bonds resulted in multiple folding intermediates containing 1, 2, or 4 disulfide bonds. High performance liquid chromatographic separation of intracellular and secreted forms of the folding intermediates demonstrated that the most folded forms were preferentially secreted and combined with hCG-beta. From these studies we conclude that: (i) the cystine knot of GPH-alpha is necessary and sufficient for folding and (ii) there is a direct correlation between the extent of GPH-alpha folding, its ability to be secreted, and its ability to heterodimerize with hCG-beta.     

Human beta-adrenergic receptors. Simultaneous purification of beta 1- and beta 2-adrenergic-receptor peptides.

Monoclonal antibodies to insulin secretory granule membranes were obtained following immunization of mice with granule membranes purified from a rat transplantable insulinoma. The specificities of the antibodies were investigated by using binding assays with different insulinoma subcellular fractions, by indirect immunofluorescence studies with intact and permeabilized cells, and by immunoblotting of granule membrane proteins fractionated by SDS/polyacrylamide-gel electrophoresis. Fifty-six antibodies were characterized initially, and 21 representative cell lines were cloned. The antibodies fell into four categories: (1) binding preferentially to secretory granules, and reacting with a component of approx. 80,000 Da on immunoblots (antigen designated SGM 80); (2) binding preferentially to secretory granules, and reacting with components of approx. 110,000 and 50,000 Da on immunoblots (antigen designated SGM 110); (3) binding preferentially to secretory granules but unreactive on immunoblots; (4) binding to membrane antigen(s) with a widespread intracellular distribution which included granules and plasma membranes. The antigens SGM 80 and SGM 110 were studied in more detail and both were shown to be integral membrane glycoproteins with antigenic determinants located on the internal face of the secretory granule membrane. These antigens were also present in normal rat islets of Langerhans and similar components were detected by immunoblotting in secretory granules from anterior pituitary and adrenal medulla. Proteins which were immunologically related to SGM 80 and SGM 110, but distinct in molecular size, were also identified in liver. It is concluded that secretory granules contain specific components which are restricted in subcellular location but widespread in tissue distribution. The antibodies obtained will be valuable reagents in the further investigation of the biogenesis and turnover of insulin secretory granules.     

An atypical protein disulfide isomerase from the protozoan parasite Leishmania containing a single thioredoxin-like domain

In higher eukaryotes, secretory proteins are under the quality control of the endoplasmic reticulum for their proper folding and release into the secretory pathway. One of the proteins involved in the quality control is protein disulfide isomerase, which catalyzes the formation of protein disulfide bonds. As a first step toward understanding the endoplasmic reticulum quality control of secretory proteins in lower eukaryotes, we have isolated a protein disulfide isomerase gene from the protozoan parasite Leishmania donovani. The parasite enzyme shows high sequence homology with homologs from other organisms. However, unlike the four thioredoxin-like domains found in most protein disulfide isomerases, of which two contain an active site, the leishmanial enzyme possesses only one active site present in a single thioredoxin-like domain. When expressed in Escherichia coli, the recombinant parasite enzyme shows both oxidase and isomerase activities. Replacement of the two cysteins with alanines in its active site results in loss of both enzymatic activities. Further, overexpression of the mutated/inactive form of the parasite enzyme in L. donovani significantly reduced their release of secretory acid phosphatases, suggesting that this single thioredoxin-like domain protein disulfide isomerase could play a critical role in the Leishmania secretory pathway.