Isolation of a purified mitochondrial fraction from viable clonal insulin-producing cells (RINm5F) by percoll density gradient centrifugation

Summary A Percoll density gradient was employed for selecting large numbers of viable insulin-producing RINm5F cells. Homogenates of these cells were then subjected to gradient centrifugation and two clearly visible bands were obtained. The light fraction was essentially composed of mitochondria banded at a density of about 1.06 g/ml. The heavier fraction banded at 1.09 to 1.10 g/ml and contained lysosomes and a small number of secretory granules. The distribution of Percoll particles was restricted to the extracellular space and there was no adsorption to any membrane structures. The distribution pattern of marker enzymes for the mitochondria and lysosomes was similar to that of normal pancreatic β-cells. With the use of a Percoll density gradient it was thus possible to isolate a purified mitochondrial fraction from viable RINm5F cells. This work was supported by the Swedish Medical Research Council (03x-4, 12x-562, 12x-6240), the Swedish Diabetes Association, the Nordic Insulin Foundation, Syskonen Svenssons Foundation, and ?ke Wiberg’s Foundation. Per-Olof Berggren is a recipient of a postdoctoral fellowship from the Swedish Medical Research Council.     

Atrial natriuretic factor-like peptide and its prohormone within single cell organisms.

The present investigation was designed to determine if the atrial natriuretic peptide hormonal system is present within single cell organisms. Paramecium multimicronucleatum were examined with 3 sensitive and specific radioimmunoassays which recognize the N-terminus [amino acids 1-98; proANF(1-98)], the midportion of the N-terminus [amino acids 31-67; proANF(31-67)] and C-terminus (amino acids 99-126; ANF) of the 126 amino acid atrial natriuretic factor (ANF) prohormone. ProANF(1-98), proANF(31-67), and ANF-like peptides were all present within these unicellular organisms at concentrations of 460 +/- 19 pg/ml, 420 +/- 15 pg/ml, and 14.5 +/- 2 pg/ml, respectively. These concentrations are similar to their respective concentrations in the plasma of the rat (Rattus norvegicus). These results suggest that even single cell organisms contain the atrial natriuretic peptide-like hormonal system.     

Isolation of pig thyroid lysosomes. Biochemical and morphological characterization.

Open thyroid follicles were prepared by mechanical disruption of pig thyroid fragments through a metal sieve. This procedure allowed preparation of thyroid-cell material depleted of colloid thyroglobulin. Open thyroid follicles were used to prepared a crude particulate fraction, which contained lysosomes, mitochondria and endoplasmic reticulum. These organelles were subfractionated by isopycnic centrifugation on iso-osmotic Percoll gradients. A lysosomal peak was identified by its content of acid hydrolases: acid phosphatase, cathepsin D, beta-galactosidase and beta-glucuronidase. The lysosomal peak was well separated from mitochondria and endoplasmic reticulum. The lysosomal peak, from which Percoll was removed by centrifugation, was taken as the purified lysosome fraction (L). Lysosomes of fraction L were purified 45-55-fold (as compared with the homogenate) and contained about 5% of the total thyroid acid hydrolase activities. Electron microscopy showed that fraction L was composed of an approx. 90% pure population of lysosomes, with an average diameter of 220 nm. Acid hydrolase activities were almost completely (80-90%) released by an osmotic-pressure-dependent lysis. Thyroglobulin was identified by polyacrylamide-gel electrophoresis as a soluble component of the lysosome fraction. In conclusion, a 50-fold purification of pig thyroid lysosomes was achieved by using a new tissue-disruption procedure and isopycnic centrifugation on Percoll gradient. The presence of thyroglobulin indicates that the lysosome population is probably composed of primary and secondary lysosomes. Isolated thyroid lysosomes should serve as an interesting model to study the reactions whereby thyroid hormones are generated from thyroglobulin and released into the thyroid cells.     

A rapid procedure for the isolation of lysosomes from kidney cortex by Percoll density gradient centrifugation

Highly pure lysosomes were isolated from buffalo(Bubalus bubalis) kidney cortex by a procedure involving differential and isopycnic Percoll density gradient centrifugations. Arylsulphatase, N-acetyl-Β-glucosamindase and cathepsin D in the lysosomal preparation were 26–45-fold enriched over the homogenate. The purified lysosomes contained less than 0·06% of mitochondrial, microsomal and peroxisomal marker enzymes. In the electron micrographs the particles appeared as large dense granules of size 0·3-1·9 μm with no apparent structural features belonging to mitochondria or microsomes. The isolation procedure was also found to be suitable to obtain highly pure lysosome particles from renal cortex of other sources such as rat, lamb and beef. No ultracentrifugation steps were involved in the procedure     

Subpopulations of endosomes generated at sequential stages in the endocytic pathway of asialoganglioside-containing ferrite ligands in rat liver

Subpopulations of endosomes generated at different stages of the endocytic pathway were isolated by a high-gradient magnetic separation followed by a Percoll density gradient centrifugation. Rat livers were perfused for 5 min with asialoganglioside (ASG)-containing ferrite particles and chased at 37 degrees C. At various times after the internalization, the endocytic vesicles containing ferrite particles were isolated by the magnetic separation. Isolated fractions contained endosomes until 15-min perfusion, after which most of the particles were transported to lysosomes. The endosomal fractions isolated after the 5- or 15-min perfusions were further analyzed by 30% Percoll density gradient centrifugation. The endosomes after 5-min perfusion showed peaks around the density of 1.05 g/ml (peak I) and 1.07 g/ml (peak Is), both of which contained asialoglycoprotein receptors. In the 15-min perfusion, another peak of endosomes (peak II) was observed at the higher density of 1.09 g/ml without the receptors, in addition to peak I. These endosomes had their own characteristic proteins. Some proteins were common in the subgroups of endosomes. These results suggest that the endosome I containing the ligands and the receptors was first produced after endocytosis and, through the endosome is, was scissioned into the endosome II containing the ligands. The endosome II was then fused with primary lysosomes for proteolytic cleavage of ligands.     

Internalization of β-adrenergic receptor binding sites: Involvements of lysosomal enzymes

Lysosomal enzymes were detected in a highly purified preparation of frog erythrocytes. Pretreatment of intact erythrocytes with lysosomotropic drugs reduced the number of soluble β-receptors in isoproterenol-treated cells, whereas the level of membrane-bound receptors in these cells was unaffected. Subcellular fractionation by Percoll gradient centrifugation revealed that one species of lysosomes (density: 1.15 g/ml), contained a fraction of membrane-bound β-adrenergic receptors. This fraction of membrane-bound receptors was markedly increased when desensitized cells were pretreated with chloroquine. Thus the internalized receptors appear to be delivered to lysosomes and released from the endocytic vesicles by the lysosomal enzymes.     

Two new hormones: prohormone atrial natriuretic peptides 1-30 and 31-67 circulate in man

Two peptides consisting of amino acids 1-30 and 31-67 of the N-terminal end of the prohormone of atrial natriuretic factor (pro ANF) which vasodilate aortas in vitro, lower blood pressure in vivo, and have natriuretic properties were found to circulate in 54 normal human volunteers. The mean circulating concentration of pro ANF 1-30 was 1861 +/- 87 pg/ml (SEM) while pro ANF 31-67 mean concentration was 1478 +/- 71 pg/ml versus a level of 67 +/- 3 pg/ml for atrial natriuretic factor (ANF). In chronic renal failure their mean concentrations increased to 40,484 +/- 6,929 pg/ml (SEM), 108,566 +/- 16,888 pg/ml, and 348 +/- 81 pg/ml for pro ANFs 1-30 and 31-67 and ANF respectively. Since pro ANF 1-30 and pro ANF 31-67 circulate in man and have physiologic effects they meet the criteria of two new hormones.     

Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor

We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides.     

Isolation of highly purified rat liver lysosomal membranes using two Percoll gradients

Normal rat liver lysosomal membranes in the form of membrane vesicles have been purified using Percoll density gradient centrifugation. Lysosomes (density = 1.111) were purified approximately 63 +/- 12-fold (mean +/- standard deviation, n = 5) using a gradient of Percoll made isotonic with sucrose and buffered to pH 7.0. These lysosomes were then exposed to 10 mM methionine methyl ester, pH 7.0, the uptake of which resulted in swelling and breakage of the lysosomes with subsequent vesicle formation. These vesicles (density = 1.056) were further separated from residual mitochondrial and plasma membrane enzyme activities using a second Percoll density gradient. Marker enzyme analysis and electron microscopy indicated that the lysosomal membranes were essentially free of both beta-hexosaminidase, a soluble lysosomal enzyme, and contaminating organelles. The specific activity of lysosomal ATPase in the lysosomal membranes was fourfold greater than in the intact lysosomes.     

Determinants of natriuretic peptide gene expression

The cardiac natriuretic peptides (NP) atrial natriuretic factor or peptide (ANF or ANP) and brain natriuretic peptide (BNP) are polypeptide hormones synthesized, stored and secreted mainly by cardiac muscle cells (cardiocytes) of the atria of the heart. Both ANF and BNP are co-stored in storage granules referred to as specific atrial granules. The biological properties of NP include modulation of intrinsic renal mechanisms, the sympathetic nervous system, the rennin-angiotensin-aldosterone system (RAAS) and other determinants, of fluid volume, vascular tone and renal function. Studies on the control of baseline and stimulated ANF synthesis and secretion indicate at least two types of regulated secretory processes in atrial cardiocytes: one is stretch-stimulated and pertussis toxin (PTX) sensitive and the other is Gq-mediated and is PTX insensitive. Baseline ANF secretion is also PTX insensitive. In vivo, it is conceivable that the first process mediates stimulated ANF secretion brought about by changes in central venous return and subsequent atrial muscle stretch as observed in acute extracellular fluid volume expansion. The second type of stimulation is brought about by sustained hemodynamic and neuroendocrine stimuli such as those observed in congestive heart failure.     

Microtubule-associated distribution of specific granules and secretion of atrial natriuretic factor in primary cultures of rat cardiomyocytes

A close spatial relationship between specific granules containing atrial natriuretic factor (ANF) and microtubules was demonstrated in primary cultures of neonatal rat cardiac myocytes. For the detection of specific granules and microtubules, the myocytes were double immunolabelled with antibodies against -ANF and -tubulin and examined by conventional fluorescence or laser scanning confocal microscopy. In addition, the ultrastructural distribution of specific granules was demonstrated by electron microscopy. In the atrial myocytes, ANF was stored in numerous specific granules that were mainly localized in the perinuclear sarcoplasm. In the ventricular myocytes, however, a minority of the cells (10%) exhibited limited ANF immunoreactivity after 4 days in culture. Microtubules were present throughout the sarcoplasm of the myocytes. They were most densely packed in the perinuclear regions. Depolymerization of the microtubules with nocodazole was followed by dispersal of ANF immunostaining both in the atrial myocytes and in the ventricular myocytes exhibiting ANF immunoreactivity. When the microtubules were allowed to recover, the perinuclear distribution of specific granules, as seen in non-treated myocytes, reappeared. Measurements of secreted immunoreactive ANF by radioimmunoassay revealed that the secretion of ANF from atrial myocytes into the medium was significantly reduced following nocodazole treatment, whereas a similar decrease in secretion from ventricular myocytes was not observed. These findings indicate that ANF-containing specific granules are closely associated with microtubules within the myocytes. It is suggested that secretion of ANF from the atrial myocytes, in contrast to the ventricular myocytes, is microtubule-dependent.     

Developing Dictyostelium cells contain the lysosomal enzyme alpha- mannosidase in a secretory granule

The prespore vesicle (PSV) is an organelle which secretes spore coat proteins and gal/galNAc polysaccharides from prespore cells of Dictyostelium. By combining the techniques of protein A-gold immunocytochemistry and ricin-gold affinity cytochemistry we have demonstrated colocalization of the lysosomal enzyme alpha-mannosidase with gal/galNAc polysaccharides in prespore vesicles and the spore coat. To determine the origin of prespore vesicles a series of pulse- chase experiments were performed. Cells were labeled with [35S]methionine or [35S]sulfate at different times during development and allowed to differentiate in the presence of unlabeled methionine or sulfate for various periods of time. The cells were homogenized and intracellular organelles were separated using Percoll density gradient centrifugation. The distribution of [35S]methionine-labeled alpha- mannosidase and [35S]sulfate-labeled glycoproteins in the Percoll gradients was determined. It was found that prespore vesicles contained protein which was previously found in lysosomes. Newly labeled protein also entered these vesicles. The data suggest that developing Dictyostelium cells either restructure preexisting lysosomes into prespore vesicles or transport protein between these two organelles. We propose that secretory granules and lysosomes may have a common biosynthetic origin and may be evolutionarily related.     

Purification and properties of cytoplasmic granules from cytotoxic rat LGL tumors

To evaluate the role of NK cell granules in the lytic activity of NK cells, cytoplasmic granules of rat NK tumors were purified by centrifugation of the cell homogenates in a Percoll gradient. Analysis of such gradients showed a band of light-scattering material near the bottom of the tube; assay of gradient fractions for lytic activity against SRBC showed a potent lytic activity giving a sharp peak in this region. Complete lysis of SRBC was achieved with less than 1 microgram/ml protein of the most active fractions. Examination in the electron microscope showed that a pool of fractions containing lytic activity consisted of pure cytoplasmic granules showing similar morphology to those found in the LGL tumors. The lytic band was associated with a peak in the activity of four different lysosomal enzymes. Analysis of Percoll gradient fractions showed that marker enzymes for mitochondria, plasma membrane, and cytosol were well separated from this activity peak. Analysis of the Percoll gradient fractions by SDS gel electrophoresis showed that this granule fraction was free of contamination of proteins from other parts of the gradient. The granules contained major protein bands of 62, 58, 30, 29, and 28 kilodaltons. In addition to protein, the purified granule fractions contain hexose and uronic acid, but no nucleic acids or phospholipids were detected in chemical assays. Major amounts of chymotryptic, tryptic, and elastase activities were not present, nor were peroxidase or lysozyme activities detectable in substantial amounts. These data show that NK tumor cell cytoplasmic granules contain a potent lytic activity and have biochemical properties that distinguish them from granules present in granulocytes and mast cells.     

Identification of two subpopulations of thyroid lysosomes: relation to the thyroglobulin proteolytic pathway.

Using a combination of differential centrifugation and isopycnic centrifugation in Percoll gradients, we obtained a highly purified preparation of thyroid lysosomes [Alquier, Guenin, Munari-Silem, Audebet & Rousset (1985) Biochem. J. 232, 529-537] in which we identified thyroglobulin. From this observation, we postulated that the isolated lysosome population could be composed of primary lysosomes and of secondary lysosomes resulting from the fusion of lysosomes with thyroglobulin-containing vesicles. In the present study, we have tried to characterize these lysosome populations by (a) subfractionation of purified lysosomes using iterative centrifugation on Percoll gradients and (b) by functional studies on cultured thyroid cells. Thyroglobulin analysed by soluble phase radioimmunoassay, Western blotting or immunoprecipitation was used as a marker of secondary lysosomes. The total lysosome population separated from other cell organelles on a first gradient was centrifuged on a second Percoll gradient. Resedimented lysosomes were recovered as a slightly asymmetrical peak under which the distribution patterns of acid hydrolase activities and immunoreactive thyroglobulin did not superimpose. This lysosomal material (L) was separated into two fractions: a light (thyroglobulin-enriched) fraction (L2) and a dense fraction (L1). L1 and L2 subfractions centrifuged on a third series of Percoll gradients were recovered as symmetrical peaks at buoyant densities of 1.12-1.13 and 1.08 g/ml, respectively. In each case, protein and acid hydrolase activities were superimposable. The specific activity of acid phosphatase was slightly lower in L2 than in L1. In contrast, the immunoassayable thyroglobulin content of L2 was about 4-fold higher than that of L1. The overall polypeptide composition of L, L1 and L2 analysed by polyacrylamide-gel electrophoresis was very similar, except for thyroglobulin which was more abundant in L2 than in either L or L1. The functional relationship between L1 and L2 lysosome subpopulations has been studied in cultured thyroid cells reassociated into follicles. Thyroid cells, prelabelled with 125I-iodide to generate 125I-thyroglobulin, were incubated in the absence of in the presence of inhibitors of intralysosomal proteolysis. The fate of 125I-thyroglobulin, and especially its appearance in the lysosomal compartment, was studied by Percoll gradient fractionation and immunoprecipitation. Treatment of prelabelled thyroid cells with chloroquine and leupeptin induced the accumulation of immunoprecipitable 125I-thyroglobulin into a lysosome fraction corresponding to the L2 subpopulation. In control cells, in which intralysosomal proteolysis was n     

Isolation of two lysosomal populations from iron-overloaded rat liver with different iron concentration and proteolytic activity

Iron overload results in an accumulation of electron-dense iron-containing particles (IPs) such as ferritin and hemosiderin within the lysosomes of rat liver cells. In order to evaluate the effect or iron overload on lysosomal function, efforts were made to isolate lysosomes with different iron contents by means of ultracentrifugation in Percoll and Metrizamide gradients. Lysosomes isolated on the Percoll gradient were characterized ultrastructurally by a uniform matrix consisting mainly of IPs and these lysosomes contained a high iron concentration and showed a very low proteolytic activity. They may, therefore, constitute, or be equated, with a special type of residual bodies. They were also fragile, as judged by their significant release of enzymes during incubation in vitro. Lysosomes isolated in the Metrizamide gradient contained remnants of sequestered organelles and some IPs. These organelles displayed a somewhat impeded proteolytic activity compared with control lysosomes, as well as preserved membrane stability during incubation in vitro. We suggest that these may be precursors of the heavily iron-laden lysosomes recovered in the Percoll gradient. Our findings demonstrate that different populations of lysosomes exist in iron-overloaded rat liver cells, which show specific characteristics with regard to ultrastructural appearance, iron content and proteolytic activity. Differing iron contents is the most likely reason for their diverging densities and membrane integrities, whereas the difference in proteolytic activity could be a result of varying amounts of degradable substrate.     

Atrial natriuretic peptide hormonal system in plants.

To determine if atrial natriuretic peptides are present in plants as well as animals, where they are important for water and sodium metabolism, the leaves and stems of the Florida Beauty (Dracena godseffiana) were examined. The N-terminus consisting of amino acids (a.a.) 1-98 (i.e., pro ANF 1-98), the mid portion of the N-terminus (a.a. 31-67; pro ANF 31-67), and C-terminus (a.a. 99-126; ANF) of the 126 a.a. atrial natriuretic factor (ANF) prohormone were all present in the leaves and stems of this plant. The concentrations of pro ANF 1-98, pro ANF 31-67 and ANF-like peptides of 120 +/- 20, 123 +/- 21, and 129 +/- 20 ng/g of plant tissue in leaves and 109 +/- 20, 96 +/- 21, and 124 +/- 18 ng/g of tissue, respectively, in the stems were lower (P less than 0.05) than their concentrations in rat (Rattus norvegicus) heart atria of 196 +/- 40, 192 +/- 28, and 189 +/- 15 ng/g of tissue respectively, but higher (P less than 0.001) than their respective concentrations of 4.3 +/- 1.4, 4.1 +/- 1.2, and 3.9 +/- 1 ng/g of rat heart ventricular tissue. We conclude that the atrial natriuretic peptide-like hormonal system is present in the plant kingdom as well as in the animal kingdom.     

Phagocytosis by catfish neutrophils

Channel catfish peripheral blood leucocytes were separated on a Percoll gradient to establish the phagocytic function of the neutrophils. Four fractions of leucocytes were formed on the Percoll gradient, including a fraction that contained 50–80% neutrophils at a density of 1.08–1.09 g ml⁻¹ and a fraction that contained 10% monocytes at a density of 1.071–1.074 g ml⁻¹. Phagocytic assays, using ³H-uridine, showed that the two fractions had similar phagocytic indices, although neutrophils were less phagocytic than monocytes. Neutrophils were confirmed to be phagocytic when examined with transmission electron microscopy. Staining with 3,3-diaminobenzidine-tetrahydrochloride demonstrated peroxidase-positive granules in the cytoplasm of actively phagocytic cells as well as peroxidase reaction products in a number of phagosomes containing bacteria. Phagocytosis of bacteria by channel catfish neutrophils was further confirmed by differential staining of external bacteria and cell surfaces with ruthenium red during the fixation process.     

Capsaicin-sensitive nerves influence the release of atrial natriuretic factor by atrial stretch in the rat.

Although many factors may modulate the release of atrial natriuretic factor (ANF), the primary mechanism has been demonstrated to be atrial stretch. Recent studies have led to the suggestion that the peptidergic innervation of the heart, through the release of peptides, may be involved in the control of ANF secretion. We have examined the influence of chronic capsaicin treatment on three models of atrial stretch that release ANF. This treatment inhibited ANF released through in vivo blood volume expansion and through balloon inflation in the right atrium of in vitro isolated perfused hearts. Immunohistochemical and electron microscopical analysis confirmed the absence of innervation of the heart by calcitonin gene related peptide and substance P immunoreactive nerve fibres and apparent lack of effect on atrial granules in capsaicin treated rats. We conclude that capsaicin-sensitive cardiac innervation is a component modulating the release of ANF, stimulated by atrial stretch in the rat.     

Isolation of peroxisomes from rat liver using sucrose and Percoll gradients

Peroxisomes were isolated from the livers of both control and clofibrate-treated rats. Two procedures, one with a sucrose gradient, and a second with Percoll gradients, were utilized. The Percoll procedure allowed contamination of the isolated peroxisome fraction on protein basis, by lysosomes (8%), by mitochondria (5%) and by microsomes (2%). The peroxisome fraction isolated by the sucrose gradient showed no significant contamination with mitochondria, but the fraction contained 13% microsomes. In addition to established peroxisomal enzymes, the isolated peroxisomes also contained cytochrome b5, NADH-cytochrome c reductase and NADPH-isocitrate dehydrogenase. The peroxisomal membranes were also separated from the content, and they were found to have a relatively high phospholipid/protein ratio (0.55).     

Proatrial natriuretic factor is phosphorylated by rat cardiocytes in culture

Proatrial natriuretic factor (proANF) is phosphorylated in primary cultures of neonatal rat cardiocytes. Rittenhouse et al. (Rittenhouse, J., Moberly, L., O'Donnell, M. E., Owen, N. E., and Marcus, F. (1986) J. Biol. Chem. 261, 7607-7610) observed that cyclic AMP-dependent protein kinase phosphorylated synthetic peptides related to atrial natriuretic factor (ANF) and that phosphorylated ANF peptides were more effective in stimulating Na/K/Cl cotransport in smooth muscle cells than nonphosphorylated forms. In our studies, rat cardiocytes in culture were incubated with [32P]orthophosphoric acid, and ANF-related peptides in cell extracts and culture media were isolated using antisera to ANF. Both atrial and ventricular cardiocytes contained and secreted phosphorylated proANF, a 126-amino acid precursor of ANF. Phosphorylated and nonphosphorylated isoforms of proANF were resolved by isoelectric focusing; approximately 35% of the proANF secreted by cardiocytes was phosphorylated. proANF is phosphorylated on a serine residue localized to a 42-amino acid tryptic fragment (proANF residues 26-67). We conclude that proANF is phosphorylated by rat cardiocytes but not within the portion of the molecule destined to become ANF (proANF residues 99-126). Phosphorylation may have a role in the cellular mechanisms of proANF storage and secretion or in the modulation of potential biological activities of the circulating amino-terminal portion of proANF.