Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions.

A fraction of secretion granules has been isolated from rabbit parotid by a procedure which was found to be especially effective in reducing contamination resulting from aggregation and/or cosedimentation of granules with other cell particulates. The fraction, representing 15 percent (on the average) of the total tissue amylase activity, was homogeneous as judged by electron microscopy and contaminated to exceedingly low levels by other cellular organelles as judged by marker enzymatic and chemical assays. Lysis of the granules was achieved by their gradual exposure to hypotonic NaHCO3, containing 0.5 mM EDTA. The content and the membranes separated by centrifugation of the granule lysate were characterized primarily by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis which indicated that the content was composed of a limited number of molecular weight classes of polypeptides of which three bands (having approximate mol wt 58,000, 33, 000, and 12,000) could be considered major components. The gel profile of the membrane subfraction was characterized by 20-30 Coomassie brilliant blue-staining bands of which a single species of mol wt 40,000 was the conspicuous major polypeptide. Two types of experiments employing gel electrophoretic analysis were carried out for identifying and assessing the extent of residual secretory protein adsorbed to purified granule membranes: (a) examination of staining and radioactivity profiles after mixing of radioactive secretion granule extract with nonradioactively labeled granule membranes and (b) comparison of gel profiles of secretion granule extract and granule membranes with those of unlysed secretion granules and secretory protein dischraged from lobules in vitro or collected by cannulation of parotid ducts, the last two samples being considered physiologic secretory standards. The results indicated that the membranes were contaminated to a substantial degree by residual, poorly extractable secretory protein even though assays of membrane fractions for a typical secretory enzyme activity (amylase) indicated quite through separation of membranes and content. Hence, detailed examination of membrane subfractions for residual content species by gel electrophoresis points to the general unity and sensitivity of this technique as a means for accurately detecting a defined set of polypeptides occurring as contaminants in cellular fractions or organelle subfractions.     

Ultrastructural studies on the secretory cycle of the neurosecretory cells and the formation of herring bodies in the paraventricular nucleus of the rat

Summary The ultrastructure of the neurosecretory cells in the paraventricular nucleus of the normal male rat was studied by electron microscopy during various functional states. Four morphologically distinct types of neurosecretory cells were observed. It appears that they do not represent different classes of cells but different phases of secretory activity of a single cell type. The perikarya of the neurosecretory cells show a definite cycle of formation and transportation of secretory granules. We have designated the phases of this cycle as: (1) phase of synthesis, (2) phase of granule production, (3) phase of granule storage and (4) phase of granule transport. The neurosecretory granules appear to be moved in bulk into the axons, forming a large axonal swelling filled with granules as a result of one cycle in the neurosecretory process. Thus it may be postulated that a secretory cycle in the perikaryon of the neurosecretory cell seems to result in the formation of a Herring body in its axon, and that its content is then conveyed to the posterior pituitary.     

Isolation and characterization of neurosecretory granules of the rat posterior pituitary gland

Summary Neurosecretory granules (NSG) of rat posterior pituitary glands were prepared by differential centrifugation techniques mainly according to the procedure as described by Barer, Heller and Lederis (1963). As revealed by electron microscopy, the recovery of neurophysin and the contents of enzymes, purified NSG were obtained in a pellet at 30 000 g/60 min (0.44 M sucrose). Eighteen h after injection of (³⁵S) cysteine into the supraoptic nucleus 60% of the recovered radioactivity in the neural lobe was found in the NSG, whereas 20% was found in the final supernatant (100 000 g/120 min). Sixteen days after injection the NSG and the final supernatant fraction contained fairly equal amount of (³⁵S) cysteine (approximately 40%). It is suggested that after a period of intragranular maturation neurophysin is extruded into an extragranular pool of neurosecretory material.With the use of conventional polyacrylamide-gel electrophoresis it was shown that the predominating proportion of radioactivity in the NSG after a hypothalamic injection of (³⁵S) cysteine was located within the neurophysin fraction A and in fraction B. Fraction B is suggested to be partly bound to the NSG membranes. When the NSG soluble and NSG insoluble proteins, obtained after lysis of NSG, were separated on polyacrylamide gels in the presence of sodium dodecylsulphate, the highly radioactive soluble protein was shown to consist of two components with average molecular weights of 12 300 and 14 600. Most of the proteins in the lysate were found in the NSG membranes, though less radioactive. A component with a mol.wt. of 37 000 was enriched in the membrane fraction. At longer times after isotope injection the high mol.wt. proteins, particularly those of the NSG membranes, contained increased amounts of radioactivity.Abbreviations NSG neurosecretory granule - NSM neurosecretory material - SON supraoptic nucleus The present investigation was supported by grants from Svenska Livförsäkringsbolags nämnd för medicinsk forskning from Svenska Sällskapet för Medicinsk forskning, from the Medical Faculty, University of Göteborg, and from the Swedish Medical Research Council (B 72-12X-2543-04A).We are indebted to Mrs. Marie-Louise Eskilsson, Mrs. Wally Holmberg and Mrs. Ulla Svedin for technical assistance, and to Miss Gull Grönstedt for careful secreterial work.     

Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.     

Glycoprotein synthesis in the adult rat pancreas. IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Glycoprotein synthesis in the adult rat pancreas: IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5–10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50–100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and $\text{N-}\text{acetylglucosamine}$. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes < smooth microsomes < zymogen granules.Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptide was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [³H]glucosamine or [³H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules.Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB³H₄. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

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.     

Release in vitro of neurohypophysial proteins from neural lobe tissue slices and from isolated neurosecretory granules of the rat

Summary The release of neurophysin from neural lobe tissue slices and isolated neurosecretory granules of the rat was studied at various time intervals after injection of (³⁵S) cysteine into the supraoptic nucleus. For hours after isotope injection the release of radioactive neurophysin from neural lobe tissue was increased by depolarizing concentration of potassium in the presence of calcium ions. Fourteen and 30 days after isotope injection the release of radioactive neurophysin was relatively decreased in a medium of high potassium concentration which might be explained by the heterogeneity of the pool of neurophysin within the neural lobe. Four h after isotope injection the spontaneous release of neurophysin from neural lobe tissue was higher in dehydrated rats than in controls, and the neural lobes of these animals did not respond with an increased release of radioactive neurophysin when exposed to high potassium concentration.Eighteen h after isotope injection the predominating proportion of neurophysin-bound radioactivity was found in the neurosecretory granule fraction, whereas 14 days after injection a fairly equal amount of radioactivity was found in this fraction and in the soluble protein fraction. This indicates that with time an increasing amount of radioactive neurophysin passes from an intragranular to an extragranular pool.The spontaneous release of radioactive neurophysin from isolated neurosecretory granules was higher, and the increase of release upon exposure to an ATP-regenerating system was higher 14 days after isotope injection than 18 h after injection. This may imply that the neurosecretory material undergoes an intragranular maturation process to become more easily releasable. The release of radioactive neurophysin was inhibited in the presence of AMP and EDTA, which demonstrates the dependence of the release process of an ATPase and, probably, of calcium ions.Neurosecretory granules from dehydrated rats possessed a higher spontaneous release than those of control rats 18 h after injection, which may indicate an enhanced intragranular maturation process of the neurosecretory material due to osmotic stimulation.Abbreviations NSG neurosecretory granule - NSM neurosecretory material The present work was supported by grants from Carl-Bertel Nathhorsts vetenskapliga stiftelse, from Svenska Livförsäkrings bolags nämnd för medicinsk forskning, from Överläkare Albert Wallins fond and from the Medical Faculty of the University of Göteborg. I am most indebted to Miss Gull Grönstedt for careful secreterial work.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : II. Lipids

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to ~20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.     

Isolation and compositional analysis of secretion granules and their membrane subfraction from the rat parotid gland

Summary A secretory granule fraction has been isolated from rat parotid by discontinuous gradient centrifugation using hyperosmotic sucrose-Ficoll solutions of low ionic strength. The secretion granule fraction comprises 25% of the total tissue -amylase activity and is judged to be of high purity, both morphologically and by its low level of contamination by enzyme activities associated with other organelles.Secretion granules were lysed by capitalizing on their lability in KCl-containing media, and the low density granule membranes were separated from residual organelle and soluble contaminants by flotation in a sucrose gradient. Residual, poorly extractable secretory contaminants of the granule membrane subfraction were selectively removed by a saponin- (10 g/ml) Na₂SO₄ (0.3m) wash, apparently with negligible disruption of granule membrane structure. Based on detailed consideration of the extent of contamination by residual mitochondria and incompletely removed secretory polypeptides, it is possible to estimate that 95% of the protein associated with the purified secretion granule membrane is bona fide granule membrane protein. Further analyses indicate that -glutamyltransferase constitutes a marker enzymatic activity shared by granule membranes and the apical domain of the plasma membrane.Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretograms of radio-iodinated granule membrane polypeptides are characterized by 20–25 radioactive bands of which 5–6 are suggested to be glycoproteins by virtue of their binding of concanavalin A. The limited polypeptide composition of the secretion granule membrane (in comparison to membranes of other cellular compartments) and the high phospholipid-protein ratio (4.4 mg/mg) may reflect the functional specialization of this storage container for secretory proteins.     

Phosphorylation of a chromaffin granule-binding protein in stimulated chromaffin cells

A procedure was devised to determine whether in the stimulated chromaffin cell phosphate is incorporated into specific proteins ("chromobindins") that bind to chromaffin granule membranes in a Ca2+-dependent manner. Cells were preincubated with 32P-labeled orthophosphate, then challenged with secretory stimuli. A postmicrosomal supernatant fraction was prepared from the cells and incubated with unlabeled chromaffin granule membranes in the presence of 5 mM Ca2+. Proteins that bound to the membranes were isolated by centrifugation and examined for 32P content by electrophoresis and autoradiography. Stimulation by carbamylcholine, nicotine, 56 mM K+, or 2 mM Ba2+ led to the incorporation of 32P into a 37-kDa protein that had previously been characterized as a substrate for protein kinase C in vitro (chromobindin 9, or CB9; Summers, T. A., and Creutz, C. E. (1985) J. Biol. Chem. 260, 2437-2443). Incorporation of 32P into this protein was dependent on extracellular Ca2+ and followed a time course that paralleled secretion of catecholamines, returning to base-line levels after 30 min, when secretion terminated. 32P was also incorporated into a 58-kDa protein that may be tyrosine hydroxylase and into an unidentified 28-kDa protein in response to cell stimulation, but neither of these proteins bound to granule membranes in a Ca2+-dependent manner. Treatment of cells with phorbol 12,13-dibutyrate, an activator of protein kinase C, led to 32P incorporation into the 37-kDa protein that was only 30% of the level obtained with nicotinic stimulation, suggesting that additional kinases may be involved in phosphorylating this protein in the stimulated cell.     

Isolation and protein composition of membranes of neurosecretory vesicles and plasma membranes from the neural lobe of the bovine pituitary gland.

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential ultracentrifugation. 2. Neurosecretory vesicles were isolated by sucrose-gradient ultracentrifugation and membranes were obtained after hypo-osmotic lysis of the particles. 3. A method is described for the isolation of a preparation of purified neuronal plasma membranes by using a fraction enriched in nerve endings as a starting material. 4. The purity of the subcellular fractions was estimated by enzyme assays and by examination with the electron microscope. 5. On the basis of the results it was estimated that neuronal plasma membranes constitute more than 30% of the protein of the nerve endings and neurosecretory vesicles more than 45% of the total amount of protein in the homogenate. 6. The proteins of membranes of neurosecretory vesicles and of plasma membranes were solubilized by means of sodium dodecyl sulphate. Polyacrylamide-gel electrophoresis of such preparations showed that both membranes contained a large number of proteins, including three glycoproteins.     

FINE STRUCTURE OF THE NEUROHYPOPHYSIS OF THE OPOSSUM (DIDELPHIS VIRGINIANA)

The neurohypophysis of the opossum (Didelphis virginiana) was studied by electron microscopy in order to amplify Bodian''s classic light microscopic observations in which he demonstrated a definite lobular pattern. The lobule of the opossum neurohypophysis is divided into three regions: a hilar, a palisade, and a septal zone. The hilar portion contains bundles of nerve fibers, the extensions of the hypothalamo-hypophyseal tract containing neurofilaments but few neurosecretory granules. In the opossum, pituicytes have a densely fibrillar cytoplasm. Herring bodies are prominent in the hilar region. They are large bodies packed with neurosecretory granules that have been described as end bulb formations of axons. From the hilar region, axons fan out into a palisade zone where the nerve terminals packed with neurosecretory granules, mitochondria, and microvesicles abut upon basement membranes. The neurosecretory granules are similar to those present in the neurohypophysis of other mammals, except for an occasional huge granule of distinctive type. Material morphologically and histochemically resembling glycogen occurs as scattered particles and as aggregates within nerve fibers. The septal zone, containing collagen, fibroblasts, and numerous small capillaries, is separated from the adjacent glandular tissue by a basement membrane.     

Neurosecretory granule formation in ligated axons: additional arguments for a local differentiation from a Golgi apparatus extension

The sorting domain for the different types of granules and small synaptic vesicles in neurosecretion is still largely a matter of debate. Some authors state that an exocytotic process has to precede granule formation. In previous studies, we favoured the idea that neurosecretory packages in terminals are assembled from axonal reticulum membranes simply by differentiation at the axon ending, the axonal reticulum being an extension of the Golgi apparatus. By ligating bovine splenic nerve, a de novo differentiation can be induced. After ligation, granules and granulo-tubular complexes appear. They were immunoreactive for SV2, VMAT2 and synaptobrevin II, which are all known to be highly enriched in large dense granules. Previously the granulo-tubular structures have already been recognized as precursor stadia of neurosecretory granules.It is concluded that at a de novo differentiation, a sorting out and aggregation is taking place of molecules typical for large dense granules. The small dense granules and tubules can be considered unripe, precursor forms of the large dense granules. All this occurs in the absence of signs of exocytosis. The present findings corroborate the view that granule formation occurs via local differentiation at an axon ending.     

Distribution of calmodulin and calmodulin-binding proteins in bovine pituitary: association of myosin light chain kinase with pituitary secretory granule membranes

Calcium is necessary for secretion of pituitary hormones. Many of the biological effects of Ca²⁺ are mediated by the Ca²⁺-binding protein calmodulin (CaM), which interacts specifically with proteins regulated by the Ca²⁺-CaM complex. One of these proteins is myosin light chain kinase (MLCK), a Ca²⁺-calmodulin dependent enzyme that phosphorylates the regulatory light chains of myosin, and has been implicated in motile processes in both muscle and non-muscle tissues. We determined the content and distribution of CaM and CaM-binding proteins in bovine pituitary homogenates, and subcellular fractions including secretory granules and secretory granule membranes. CaM measured by radioimmunoassay was found in each fraction; although approximately one-half was in the cytosolic fraction, CaM was also associated with the plasma membrane and secretory granule fractions. CaM-binding proteins were identified by an ²⁵¹-CaM gel overlay technique and quantitated by densitometric analysis of the autoradiograms. Pituitary homogenates contained nine major CaM-binding proteins of 146, 131, 90, 64, 58, 56, 52, 31 and 22 kilodaltons (kDa). Binding to all the bands was specific, Cat+-sensitive, and displaceable with excess unlabeled CaM. Severe heat treatment (100°C, 15 min), which results in a 75% reduction in phosphodiesterase activation by CaM, markedly decreased ²⁵¹I-CaM binding to all protein bands. Secretory granule membranes showed enhancement for CaM-binding proteins with molecular weights of 184, 146, 131, 90, and 52000. A specific, affinity purified antibody to chicken gizzard MLCK bound to the 146 kDa band in homogenates, centrifugal subcellular fractions, and secretory granule membranes. No such binding was associated with the granule contents. The enrichment of MLCK and other CaM-binding proteins in pituitary secretory granule membranes suggests a possible role for CaM and/or CaM-binding proteins in granule membrane function and possibly exocytosis.     

Large-scale purification of calf pancreatic zymogen granule membranes.

A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl- conductance, a Cl-/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5-10 mg membrane protein from approximately 1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl- channels and regulatory proteins, since they contain fewer proteins than plasma membranes.     

Subcellular localization and some properties of lipoxygenase activity in human blood platelets.

Lipoxygenase activity was measured in human platelet subcellular fractions. From a sonicated platelet preparation, a granule fraction, mixed membranes (surface and intracellular) and cytosol fractions were separated by differential centrifugation. With respect to activities in the sonicated preparation, the lipoxygenase was slightly enriched in both the cytosol and mixed-membrane fractions and consistently de-enriched in the granule fractions. Approx. 65% and 20% of the total cell enzyme activity were found in the cytosol and mixed membranes respectively, with only 8% present in the granule fraction. Additionally we measured the lipoxygenase activity in purified surface- and intracellular-membrane subfractions prepared from the mixed membranes by free-flow electrophoresis. There was a slight enrichment in activity in the intracellular membrane fraction compared with that in the mixed membranes, and a depletion of activity in the surface membranes. Characterization of the enzyme activity, i.e. time course, pH-dependence, Ca2+-dependence, Vmax. and Km for arachidonic acid, and the carbon-position specificity for this acid, failed to reveal any significant differences between the membrane-bound and soluble forms of the lipoxygenase. These findings suggest that in human platelets the same lipoxygenase is associated with the membranes as in the cytosol and that the membrane-bound activity predominates in intracellular membrane elements.     

ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS : I. Isolation of a Secretory Granule Fraction

A method has been devised for the isolation of a secretory granule fraction from isolated rat islets of Langerhans. The islets were homogenized in buffered sucrose, and the homogenate was separated into nuclear, mitochondrial, secretory granule, and microsomal fractions by differential centrifugation. The secretory granule fraction was purified by differential centrifugation in discontinuous sucrose density gradients. A greater degree of purification could be achieved by the use of two successive gradients of this type, although the final yield was greatly reduced. Biochemical and morphological characterization of the fractions was obtained; the secretory granule fraction contained both insulin and glucagon. The limiting membranes of the granules remained intact and the general appearance of the granules was similar to that seen within the whole islet cells.     

Identification of G-proteins in rat parotid gland plasma membranes and granule membranes: presence of distinct components in granule membranes

We have identified by immunoblotting and ADP-ribosylation by cholera toxin and pertussis toxin the presence of Mr 43 and 46 KDa G_s, and 39 and 41 KDa G_i;.. subunits in rat parotid gland plasma membranes but not in granule membranes. A Mr 28 KDa polypeptide that served as substrate for ADP-ribosylation by both cholera toxin and pertussis toxin was present exclusively in granule membranes. Photoaffinity crosslinking of [-³²P]GTP showed the presence of high molecular weight GTP-binding proteins (Mr 160,100 KDa) in granule membranes. Six low molecular weight GTP-binding proteins (Mr 21–28 KDa) were differentially distributed in both plasma membranes and granule membranes. The present study identifies various GTP-binding proteins in rat parotid gland plasma membranes and granule membranes, and demonstrates the presence of distinct molecular weight GTP-binding proteins in granule membranes. These granule-associated GTP-binding proteins may be involved in secretory processes.     

Studies of cytochrome b-245 translocation in the PMA stimulation of the human neutrophil NADPH-oxidase

The human neutrophil respiratory burst, activated by phorbol 12-myristate 13-acetate (PMA), results from specific receptor-ligand binding and activation of the NADPH-oxidase in the plasma membrane. The role of granule membrane constituents has been elucidated with neutrophils disrupted by nitrogen cavitation and then fractionated in Percoll gradients to resolve four postnuclear fractions: cytoplasm, light membranes or gamma fraction (site of the NADPH-oxidase), a light granule (beta) fraction containing putative constituents of the NADPH-oxidase (cytochrome b-245 and an associated flavoprotein), and a fraction of heavy granules. Cytochrome b-245 is localized to two pools of specific granules within the beta fraction as assessed by differing sedimentation in narrow Percoll gradients and translocates upon PMA-stimulation from one of these specific granule sub-pools to the plasma membrane where it exhibits no change in its midpoint redox potential. Translocation of cytochrome b-245 parallels O2-production initiated by PMA stimulation as assessed in the time course of each activity. The finding of increased amounts of the b cytochrome in cytoplast membranes relative to plasma membranes of unstimulated cells suggests that the cytoplasts, devoid of granules yet capable of O2-generation upon PMA-stimulation, are useful in assessing post-translocation events in the activation pathway of the NADPH-oxidase. These data support the hypothesis that translocation of NADPH-oxidase components from an intracellular granular pool contributes to respiratory burst expression.