Structural characterization of adrenal chromogranin A and parathyroid secretory protein-I as homologs

We have isolated and purified adrenal chromogranin A (Ch A) for the purpose of making structural comparisons to parathyroid secretory protein-I (SP-I), because our earlier data indicated these two molecules may be the same protein. An improved purification step, using high-performance liquid chromatography (HPLC), has enabled us to demonstrate that both SP-I and Ch A consists of two species, one of approximately 72,000 Da and one of approximately 66,000 Da. The amino acid composition is the same for all four species. The difference in molecular mass is assumed to be due to carbohydrate content. Cyanogen bromide digestion of each of the four samples, followed by HPLC separation of the generated peptides, resulted in a chromatographic profile that was the same for each digest. Amino acid analysis of the eight peptide fragments obtained from each digest indicates that both species of Ch A and both species of SP-I yielded the same peptide mixtures following this cleavage reaction. One large (approximately 50,000 Da) CNBr peptide was obtained and seven smaller ones, one of which contains cysteine. The large fragment behaved similarly to the intact molecule in a radioimmunoassay. HPLC separation of tryptic digests of Ch A (72,000 Da) and SP-I (72,000 Da) also resulted in elution profiles that were very similar to each other. Amino acid analysis revealed 23 peptides common to each digest. Ch A contained four peptides ranging in size from 4 to 30 residues that were not observed in the SP-I digest. SP-I contained two peptides, each with about 30 residues, that were not found in the Ch A digest. Nothing unusual was noted in any of the uncommon peptides. Thus, both a chemical and an enzymatic digestion of these molecules followed by analysis of the peptides generated, indicates that SP-I and Ch A are nearly identical homologs.     

Purification of a skeletal growth factor from human bone

A skeletal growth factor was isolated and purified from demineralized human bone matrix. A dose of 6 micrograms/mL of the purified factor significantly increased the proliferation rate of embryonic chick bone cells in serum-free culture (292% of controls, p less than 0.0001) but had no effect on embryonic chick skin cells plated at the same initial density. The factor is sensitive to inactivation by trypsin and urea, but not by collagenase, 20% butanol, or 1% mercaptoethanol. It is also resistant to inactivation by heat (stable for 15 min at 75 degrees C) and extremes of pH (stable for 30 min at 4 degrees C from pH 2.5 to 10.0). Purification of the active factor by selective heat and acid precipitations, molecular sieve column chromatography, and preparative polyacrylamide gel electrophoresis provided a material that was homogeneous by the criteria of high-pressure liquid chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. The apparent molecular weight is 83 000. The purified factor increases bone cell proliferation at doses comparable to other mitogens: 0.3 microgram/mL (3.6 nM) significantly increases DNA synthesis to 231% of controls (p less than 0.001). The purified factor was also active on cultured embryonic chick bones, enhancing the growth rate of tibiae and femurs, as measured by increased dry weight (185% of controls, p less than 0.025) and [3H]proline incorporation (164% of control, p less than 0.001), respectively.     

Sulfation of porcine parathyroid secretory protein I. Detection of tyrosine sulfate

Secretory Protein I (SP-I) is an acidic glycoprotein that is stored and co-secreted with parathormone by parathyroid glands. It has been found to be chemically similar, if not identical, to chromogranin A of the adrenal medulla and to be present in most endocrine cells. In the present study, 35SO4 was shown to be incorporated into SP-I and several other proteins of porcine parathyroid tissue incubated in vitro. The predominant sulfated species secreted to the medium was SP-I. Up to 20% of the tyrosine residues in secreted SP-I were labeled with 35SO4. Both the cellular and secreted forms migrated on sodium dodecyl sulfate gels as a pair of proteins with apparent molecular weights of 82,000 and 78,000. The 82-kDa protein could be converted to the 78-kDa species by treatment with neuraminidase. Sulfate exists in SP-I as tyrosine sulfate based on the identification of this amino acid by thin layer electrophoresis following alkaline hydrolysis. Extracellular Ca2+ (3 mM) greatly suppressed the secretion of 35SO4-labeled SP-I without affecting the intracellular sulfation of the molecule or the secretion of a minor sulfated protein unrelated to SP-I. The ratio of incorporated 35SO4 to 3H-amino-acid was greater in secreted SP-I than in tissue SP-I, suggesting that much sulfation of this protein occurred during or just before secretion.     

Localization of putative gonadotrophin releasing hormone receptor protein in the anterior pituitary

Specific binding of a fully biologically active 125I-gonadotrophin releasing hormone (GnRH) to isolated anterior pituitary cells is time dependent, saturable and the concentration dependent binding curves exhibit positive cooperativity. Binding to intact or solubilized plasma membranes and an affinity purified GnRH receptor protein reveals in all instances multiple high affinity binding sites. Thus, GnRH receptor protein appears to be an intrinsic constituent of the cell membrane, and perhaps, other membranous organelles. To investigate the latter, the binding of 125I-GnRH to various subcellular fractions was studied and its affinity and time requirements determined. GnRH binding to plasma membranes and secretory granules was to multiple high affinity sites, while that to nuclei and microsomes was to a single high affinity site. Binding was 1.83 +/- 0.07, 0.78 +/- 0.04, 0.31 +/- 0.03 and 0.27 +/- 0.03 fmol micrograms-1 protein for isolated plasma membranes, secretory granules, microsomes and nuclei, respectively, after 30 min incubation with 10(-9) M GnRH. The magnitude of binding to microsomes did not change during the incubation period. It did not show any decrease (p greater than 0.05) in isolated nuclei and plasma membranes, except for the 24 h time period, when a significant drop (p less than 0.001) was seen. Binding to the secretory granule fraction culminated at 15 min and then decreased (p less than 0.001) steadily to a non-detectable level at 24 h. Thus GnRH receptor protein or its portion may be an integral part of some membranous particles in the anterior pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins

PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.     

Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology.

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.     

Insulin-dependent alterations of phorbol ester binding to adipocyte subcellular constituents. Evidence for the involvement of protein kinase C in insulin action

The binding of tritiated phorbol-12,13-dibutyrate (3H-PBu2) was employed to estimate the mass of protein kinase C associated with plasma membranes and cytosol isolated from untreated and insulin-treated adipocytes. Binding of 3H-PBu2 to both plasma membranes and cytosol was rapid, achieving a steady state within minutes. Treatment of cells with physiological concentration of insulin (0.67 nM) caused a 42% increase (from 0.92 +/- 0.08 to 1.30 +/- 0.12 pmol 3H-PBu2/mg protein, p less than 0.0001) and a 27% decrease (from 0.41 +/- 0.07 to 0.30 +/- 0.05 pmol 3H-PBu2/mg protein, p less than 0.020) in phorbol ester bound to cytosol and plasma membranes, respectively. The half-maximal concentrations of unlabelled PBu2 needed to displace 3H-PBu2 bound to cytosol from control and insulin-treated cells were 54 and 13 pM, respectively. These data indicate that insulin modifies protein kinase C in adipocytes.     

Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity

A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70-75 kd (reduced) and 62-66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 A diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.     

Retinal pigment epithelium lipofuscin proteomics

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.     

Localization of glutathione-insulin transhydrogenase (protein-disulfide interchange enzyme) in pancreas using immunocytochemistry, immunodiffusion, and enzymatic activity assay techniques

The localization of the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (GIT), in rat and mouse pancreas was studied by protein A-gold immunocytochemistry, immunodiffusion, and assay of enzymatic activity. Immunocytochemistry on tissue sections using antibody to GIT and protein A-gold complex indicated the presence of GIT in alpha and beta cells in islets as well as acinar cells. The beta cells in obese (ob/ob) hyperinsulinemic mice showed increased GIT immunoreactivity. In both alpha and beta cells, GIT immunoreactive sites were associated predominantly with secretory granules. In pancreas from rats injected with glibenclamide, the degranulated beta cells contained GIT immunoreactive sites on the cisternal surface of the rough endoplasmic reticulum (RER). In acinar cells, the RER, Golgi elements, condensing vacuoles, and zymogen granules possessed GIT immunoreactive sites as did mitochondria. Immunocytochemistry on sections of isolated subcellular fractions showed that GIT was associated with different membranes. The enzymatic activity of GIT was found in the following order: Golgi elements greater than mitochondria greater than microsomes greater than zymogen granules greater than cytosol. In Ouchterlony immunodiffusion tests, each subcellular fraction showed a precipitin band which was continuous with that of purified GIT, a result indicating the presence of immunologically identical GIT in all fractions.     

The activator of the Rhodospirillum rubrum PHB depolymerase is a polypeptide that is extremely resistant to high temperature (121 degrees C) and other physical or chemical stresses

Hydrolysis of native (amorphous) polyhydroxybutyrate (nPHB) granules isolated from different sources by soluble PHB depolymerase of Rhodospirillum rubrum in vitro requires the presence of a heat-stable compound (activator). The activator was purified and was resistant against various physical and chemical stresses such as heat (up to 130 degrees C), pH 1-12, dryness, oxidation by H2O2, reducing and denaturing compounds (2-mercaptoethanol, 5 M guanidinium-HCl) and many solvents including phenol/chloroform. The activator coding gene was identified by N-terminal sequencing of the purified protein, and the deduced protein showed significant homology to magnetosome-associated protein (Mms16) of magnetotactic bacteria. Analysis of the activation process in vitro showed that the activator acts on nPHB granules but not on the depolymerase. The effect of the activator could be mimicked by pretreatment of nPHB granules with trypsin or other proteases but protease activity of the purified activator was not detected. Evidence is shown that different mechanisms were responsible for activation of nPHB by trypsin and activator, respectively. PHB granule-associated protein (PhaP) of Ralstonia eutropha nPHB granules were cleaved by trypsin but no cleavage occurred after activator treatment. Hydrolysis of artificial protein-free PHB granules coated with negatively charged detergents (sodium dodecyl sulfate (SDS), cholate but not cetyltrimethyl-ammonium bromide (CTAB)) did not require activation and confirmed that surface layer proteins of nPHB granules are the targets of the activator rather than lipids. All experimental data are in agreement with the assumption that trypsin and the activator enable the PHB depolymerase to find and to bind to the polymer surface: trypsin by removing a portion of proteins from the polymer surface, the activator by modifying the surface structure in a not yet understood manner presumably by interaction with phasins of the proteinous surface layer of nPHB.     

Effects of a cytosolic protein on the interaction of rat pancreatic zymogen granules in vitro

Photon correlation spectroscopy has been used to study the kinetics of aggregation of isolated rat pancreatic zymogen granules in vitro by monitoring time-dependent changes in mean particle size derived from the photon count autocorrelation function, g2(tau). Isolated granules were stable in isotonic sucrose (pH 5.4-7.0). At pH 6.0 they maintained a mean diameter of 1225 +/- 18 nm with a polydispersity index of 0.199 +/- 0.007. The mean granule diameter showed a limited decrease (approx. 20%) with increasing pH within the range 5.4-7.0, but the polydispersity index was unaltered. At pH greater than 7.0 granule instability was indicated by a rapid reduction in total photon counts. In solutions of monovalent cations ([M+] greater than 10 mM) and divalent cations ([M2+] greater than 0.5 mM) zymogen granules aggregated at a rate dependent upon both ion and granule concentration. These effects were consistent with the bimolecular nature of the interaction mechanism and were clearly distinguishable from the limited size changes associated with osmolarity. At concentrations of Na+ or K+ salts greater than 50 mM granule aggregation was accompanied by anion-dependent solubilisation. A soluble protein fraction separated from the pancreatic acinar cell cytosol by gel filtration reduced the mean diameter and polydispersity index of zymogen granules suspended in isotonic sucrose, inhibited cation-induced aggregation and stabilised granules to solubilisation induced by raising pH greater than 7.0 or exposure to high ionic strength media. The inhibitory effects of this protein were apparent at concentrations less than or equal to 10 micrograms X ml-1 (i.e. at inhibitor: granule protein ratios less than 1:20) and could not be mimicked by bovine serum albumin, the Ca2+-binding proteins calmodulin and troponin C (less than or equal to 100 micrograms X ml-1), nor the highly negatively charged polymer polyglutamate (less than or equal to 10 micrograms X ml-1). Inhibitory activity was also absent from fractions of rat liver cytosol prepared identically to pancreatic acinar cytosol. These observations are consistent with the presence in pancreatic acinar cells of a specific cytosolic granule stabilisation factor (or factors) that normally restricts zymogen granule interaction and may therefore play an important role in the regulation of granule mobility and exocytosis.     

Inhibition of the lytic activity of perforin by lipoproteins

Cytoplasmic granules isolated from cytolytic T lymphocytes (CTL) lyse red blood cells or tumor cell lines in a nonspecific manner. The activity of highly purified granules was inhibited by human or rabbit serum at dilutions as high as 1/10,000. The main inhibitory activity of human serum was isolated by chromatography and was determined to be high density lipoprotein (HDL). HDL not only inhibited at a concentration of 70 ng/ml the lytic activity of isolated granules, but also of the purified, pore-forming protein perforin present in the granules. Purified low density lipoprotein was equally active. Because the CTL granule activity was inhibited by pure egg lecithin vesicles at a concentration equivalent to the phospholipid content of lipoproteins, the lipid portion of lipoproteins is the likely candidate for granule inactivation. Lipoproteins also decreased in a dose-dependent manner the cytotoxic activity of intact cytolytic T cells. However, cytotoxicity was not completely suppressed, and only in the case of CTL exhibiting low efficiency in killing their targets. It is proposed that lipoproteins inactivate perforin and may thereby inhibit a possible lysis of innocent bystander cells.     

Immunoelectron microscopic localization of dopamine beta-hydroxylase and chromogranin A in adrenomedullary chromaffin cells

Dopamine beta-hydroxylase (DBH) was purified from bovine adrenal medullae. Rabbit IgG raised against DBH inhibited its activity by 80%. In an immunoblot analysis, the IgG specifically recognized two subunits of DBH the 72 and 75 KD components. Chromogranin A (CGA) also was purified from bovine adrenal medullae, and rabbit IgG against CGA recognized this chromogranin A in the immunoblot analysis. The intracellular distribution of DBH and CGA in bovine chromaffin cells was determined quantitatively by immunoelectron microscopy using post-embedding protein A-gold technique. DBH and CGA were localized exclusively on chromaffin granules. The binding of gold particles to these granules was saturable. The maximum number of gold particles bound to the granules roughly corresponded to the number of DBH or CGA molecules in the granules estimated biochemically. DBH was observed evenly in the periphery and in the dense matrix of the chromaffin granules.     

Ultrastructural localization of rat Clara cell 10 KD secretory protein by the immunogold technique using polyclonal and monoclonal antibodies

Using a monoclonal antibody and affinity-purified polyclonal antiserum against a 10 KD protein isolated from rat pulmonary lavage, we have localized the protein within Clara cells by a post-embedment protein A-gold technique. The gold particles were localized over the secretory granules of rat Clara cells. Ultrastructural immunolocalization was abolished when the primary antibodies were previously absorbed with purified 10 KD protein. Other pulmonary cells, including type II pneumocytes and ciliated cells, were negative with this technique. These results demonstrate the presence of the 10 KD protein in the secretory granules of the Clara cell and support the concept that this protein constitutes a specific and unique secretory product of Clara cells.     

Morphometry of feline neutrophil granule genesis

During neutrophil granule genesis, the formation of primary granules is generally thought to be limited to the promyelocyte stage; whereas synthesis of secondary granules is thought to occur only at the myelocyte stage. This hypothesis was tested morphometrically in feline neutrophils that are known to contain both granule types. Marrow specimens obtained from six cats were stained with peroxidase for identification of neutrophil primary granules and counterstained with periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) for identification of secondary granules. By regression analysis using arithmetic models, numbers of cytoplasmic granules in 311 cells were correlated with the degree of nuclear chromatin condensation, which was shown to be an adequate parameter for cell maturation. Promyelocytes and myelocytes had similar mean numbers of peroxidase-positive granules per unit area. A significant increase (p less than or equal to 0.0001) in the numbers of peroxidase-positive granules was noted between the metamyelocyte and the mature neutrophil stage, despite the lack of peroxidase activity in endoplasmic reticulum and Golgi lamellae. By contrast, a significant increase of peroxidase-negative granules between the metamyelocyte and the mature neutrophil stage was not clearly established with these methods. The increase in peroxidase-positive granules may indicate continued production of peroxidase-containing granules and/or redistribution of peroxidase among lysosomal organelles in late feline neutrophils.     

Assessment of human natural killer and lymphokine-activated killer cell cytotoxicity against Toxoplasma gondii trophozoites and brain cysts

Because previous work has suggested that NK cells may be important in host resistance against the intracellular parasite Toxoplasma gondii we examined whether human NK cells and lymphokine-activated killer (LAK) cells have activity against trophozoites and cysts of this organism in vitro. A method to radiolabel Toxoplasma trophozoites with 51Cr was developed and direct cytotoxic activity was determined by using modifications of the standard 51Cr release assay. Viability of 51Cr-labeled trophozoites assessed by both methylene blue staining and trypan blue exclusion was greater than 90%. Significantly more 51Cr was released by anti-Toxoplasma antibody and C than by antibody in the absence of C. Incubation of trophozoites with freshly isolated human NK cells or NK cells activated with either rIL-2 or rIFN-alpha did not result in significant release of 51Cr (specific lysis was 0 to 2.3%). In contrast, the average specific lysis of radiolabeled trophozoites by LAK cells was significant (specific lysis was 7.8% +/- 1.1, p less than 0.01). In a series of separate experiments, preincubation of radiolabeled trophozoites with heat-inactivated normal or Toxoplasma antibody-positive human serum increased the cytotoxicity of LAK cells from a mean specific lysis of 15% +/- 4.5 to 39% +/- 8.5, respectively (p less than 0.05), as assessed by 51Cr release. Because previous work has shown that radioisotope release from parasites may be nonspecific, separate experiments were performed to determine the cytotoxicity of LAK cells against antibody-coated trophozoites by using ethidium bromide-acridine orange staining to assess effector cell damage. LAK cells had a mean specific lysis of 51% against antibody-coated trophozoites by ethidium bromide-acridine orange staining. Preincubation with heat-inactivated Toxoplasma-antibody positive human serum did not increase activity of rIL-2-activated NK cells against 51CR-labeled trophozoites. Neither human NK cells (freshly isolated or activated by rIL-2 or rIFN-alpha) nor LAK cells were cytotoxic for purified preparations of cysts of Toxoplasma isolated from the brains of chronically infected mice.     

Isolation and characteristics of bovine pituitary secretory granules

Secretory granules containing primarily growth hormone and prolactin were isolated from bovine anterior pituitaries. Marker enzyme analysis and electron microscopy indicated that the secretory granule fraction did not contain measureable amounts of other intracellular organelles. Such isolated granules were resistant to a variety of chemical and physical challenges including variations in osmolarity, ionic strength, EGTA, sonication, boiling, etc. The only treatments that were found to routinely result in granules lysis were alkaline pH and 0.5% SDS. Nonspecific leakage of both growth hormone and prolactin was less than 9% of total hormone pool even after a 60-min incubation. The release of prolactin but not growth hormone could be increased by lowering the free calcium concentration. Conversely, 10(-5) M ionophore A23187 caused a decrease in nonspecific hormone leakage. This raises the possibility that a nonexocytosis secretory pathway might be involved in pituitary hormone release. The initial secretory granule fraction was further purified using discontinuous sucrose gradient ultracentrifugation to yield a subfraction highly enriched in prolactin granules. These granules had the same stability characteristics as the original secretory granule fraction. The use of such granules should prove useful in our efforts to understand how calcium regulates cellular secretion.     

Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase.

The outer membrane (OM) of Fibrobacter succinogenes was isolated by a combination of salt, sucrose, and water washes from whole cells grown on either glucose or cellulose. The cytoplasmic membrane (CM) was isolated from OM-depleted cells after disruption with a French press. The OM and membrane vesicles isolated from the extracellular culture fluid of cellulose-grown cells had a higher density, much lower succinate dehydrogenase activity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles different from those of the CM. The OM from both glucose- and cellulose-grown cells and the extracellular membrane vesicles from cellulose-grown cultures exhibited higher endoglucanase, xylanase, and acetylesterase activities than the CM and other cell fractions. Endoglucanase 2 was absent from the isolated OM fractions of glucose- and cellulose-grown cells and from the extracellular membrane vesicles of cellulose-grown cells but was present in the CM and intracellular glycogen granule fractions, while endoglucanase 3 was enriched in the OM. Cellobiosidase was located primarily in the periplasm as previously reported, while cellobiase was mainly present in the glycogen granule fraction of glucose-grown cells and in a nongranular glycogen and CM complex in cellulose-grown cells. The cellobiase was not eluted from glycogen granules by cellobiose, maltose, and maltotriose nor from either the granules or the cell membranes by nondenaturing detergents but was eluted from both glycogen granules and cell membranes by high concentrations of salts. The eluted cellobiase rebound almost quantitatively when diluted and mixed with purified glycogen granules but exhibited a low affinity for Avicel cellulose. Thus, we have documented a method for isolation of OM from F. succinogenes, identified the OM origin of the extracellular membrane vesicles, and located glycanases and cellobiase in membrane and glycogen fractions.     

Substance P-like immunoreactivity in rat and cat carotid bodies: light and electron microscopic studies

Substance P-immunoreactive (SP-1) structures in the carotid bodies of rats and cats were examined with the light and electron microscopes. In both species SP-I varicose nerve fibers were located singly in the interstitial connective tissue in close association with blood vessels. They were small unmyelinated fibers enveloped in a common Schwann cell sheath with other SP-negative fibers. Some of SP-I fibers contained large dense-cored granules and small clear vesicles in addition to microtubules and mitochondria and probably represented nerve fiber varicosities. The latter often were found incompletely invested by Schwann cell sheaths. SP-fibers were found occasionally in the envelopes of supporting cells at the periphery of parenchymal cell groups. However, none of the nerve terminals making synaptic contacts with glomus cells exhibited SP-like immunoreactivity. In cat carotid bodies some glomus cells showed moderate to intense SP-like immunoreactivity. The intense SP-I glomus cells displayed numerous dense-cored vesicles of 85 to 140 nm in diameter and frequently showed synaptic contacts with SP-negative nerve terminals. In rat carotid bodies we were unable to detect consistent SP-immunoreactivity in glomus cells. Our results do not favor the hypothesis that SP is a neurotransmitter/modulator in the chemoreceptor afferents synapsing on glomus cells in either the cat or rat carotid body. However our results support the hypothesis that SP in cat glomus cells may play a role in the modulation of chemoreceptor activity.