Endocytosis of hyaluronidase-1 by the liver

2010 ◽  
Vol 430 (2) ◽  
pp. 305-313 ◽  
Author(s):  
Marie-Christine Gasingirwa ◽  
Jacqueline Thirion ◽  
Jeannine Mertens-Strijthagen ◽  
Simone Wattiaux-De Coninck ◽  
Bruno Flamion ◽  
...  
Keyword(s):  
Lysosomal Enzyme ◽  
Mouse Liver ◽  
Sucrose Gradient ◽  
Intracellular Distribution ◽  
Wild Type ◽  
Serum Enzyme ◽  
Specific Density ◽  
Sinusoidal Cells ◽  
Intracellular Pathway ◽  
Isopycnic Centrifugation

It has been suggested that intracellular Hyal-1 (hyaluronidase-1), which is considered a lysosomal enzyme, originates via endocytosis of the serum enzyme. To test this proposal we have investigated the uptake and intracellular distribution of rhHyal-1 (recombinant human Hyal-1) by mouse liver, making use of centrifugation methods. Experiments were performed on wild-type mice injected with 125I-labelled rhHyal-1 and on Hyal-1−/− mice injected with the unlabelled enzyme, which were killed at various times after injection. Activity of the unlabelled enzyme was determined by zymography. Intracellular distribution of Hyal-1 was investigated by differential and isopycnic centrifugation. The results of the study indicated that rhHyal-1 is endocytosed by the liver, mainly by sinusoidal cells, and follows the intracellular pathway described for many endocytosed proteins that are eventually located in lysosomes. However, Hyal-1 endocytosis has some particular features. First, endocytosed rhHyal-1 is quickly degraded. Secondly, its distribution, as analysed by differential centrifugation, differs from the distribution of β-galactosidase, taken as the reference lysosomal enzyme. Further analysis by isopycnic centrifugation in a sucrose gradient shows endocytosed rhHyal-1 behaves like β-galactosidase shortly after injection. However the Hyal-1 distribution is markedly less affected than β-galactosidase, following a prior injection of Triton WR-1339, which is a specific density perturbant of lysosomes. The behaviour in centrifugation of endogenous liver Hyal-1, identified by hyaluronan zymography, exhibits some similarity with the behaviour of the endocytosed enzyme, suggesting that it could originate from endocytosis of the serum enzyme. Overall, these results can be explained by supposing that active endocytosed Hyal-1 is mainly present in early lysosomes. Although its degradation half-time is short, Hyal-1 could exert its activity due to a constant supply of active molecules from the blood.

scholarly journals Analytical study of microsomes and isolated subcellular membranes from rat liver. VI. Electron microscope examination of microsomes for cytochrome b5 by means of a ferritin-labeled antibody.

1976 ◽  
Vol 71 (2) ◽  
pp. 551-564 ◽  
Author(s):  
J Remacle ◽  
S Fowler ◽  
H Beaufay ◽  
A Amarcostesec ◽  
J Berthet
Keyword(s):  
Electron Microscope ◽  
Rat Liver ◽  
Sucrose Gradient ◽  
Analytical Study ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Microscope Examination ◽  
Group B ◽  
Isopycnic Centrifugation

The distribution of cytochrome b5 in rat liver microsomes, and in two microsomal subfractions isolated by density equilibration in a linear sucrose gradient, was studied under the electron microscope by means of a ferritin-labeled hybrid anti-cytochrome b5/anti-ferritin antibody. Results of this study show that cytochrome b5 is present in essentially all microsomal vesicles derived from endoplasmic reticulum (ER), whether rough or smooth. Thus, the dissociation of ER constituents into two groups (b and c), achieved by subfractionating microsomes by isopycnic centrifugation (Beaufay, H., A. Amar-Costesec, D. Thines-Sempoux, M. Wibo, M. Robbi, and J. Berthet. 1974. J. Cell Biol. 61:213-231), does not reflect the association of each group with distinct microsomal particles but reflects rather an enzymatic heterogeneity of the ER: the ratio of group c to group b enzymes increasing with the density and ribosome load of the particles.

Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis

2002 ◽  
Vol 205 (9) ◽  
pp. 1209-1219 ◽  
Author(s):  
Natalie Perzov ◽  
Vered Padler-Karavani ◽  
Hannah Nelson ◽  
Nathan Nelson
Keyword(s):  
Sucrose Gradient ◽  
Ph Sensor ◽  
Wild Type ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vacuolar Acidification ◽  
Atpase Subunits ◽  
Immunological Assays ◽  
Gradient Fractionation

SUMMARYSubunit a of V-ATPase in the yeast Saccharomyces cerevisiae, in contrast to its other subunits, is encoded by two genes VPH1 and STV1. While disruption of any other gene encoding the V-ATPase subunits results in growth arrest at pH 7.5, null mutants of Vph1p or Stv1p can grow at this pH. We used a polyclonal antibody to yeast Stv1p and a commercially available monoclonal antibody to Vph1p for analysis of yeast membranes by sucrose gradient fractionation, and two different vital dyes to characterize the phenotype of vph1 ▵ and stv1 ▵mutants as compared to the double mutant and the wild-type cells. Immunological assays of sucrose gradient fractions revealed that the amount of Stv1p was elevated in the vph1 ▵ strain, and that vacuoles purified by this method with no detectable endosomal contamination contain an assembled V-ATPase complex, but with much lower activity than the wild type. These results suggest that Stv1p compensates for the loss of Vph1p in the vph1 ▵ strain. LysoSensor Green DND-189 was used as a pH sensor to demonstrate unexpected changes in vacuolar acidification in stv1▵ as the Vph1p-containing V-ATPase complex is commonly considered to acidify the vacuoles. In the vph1 ▵ strain, the dye revealed slight but definite acidification of the vacuole as well. The lipophilic dye FM4-64 was used as an endocytic marker. We show that the null V-ATPase mutants, as well as the vph1 ▵ one, markedly slow down endocytosis of the dye.

Differences in renal metabolism of insulin and cytochrome c

1988 ◽  
Vol 254 (4) ◽  
pp. E419-E428 ◽  
Author(s):  
J. Herrman ◽  
R. E. Simmons ◽  
B. H. Frank ◽  
R. Rabkin
Keyword(s):  
Cytochrome C ◽  
Lysosomal Enzyme ◽  
Subcellular Distribution ◽  
Sucrose Gradient ◽  
Insulin Degradation ◽  
Renal Metabolism ◽  
Small Proteins ◽  
Cyt C ◽  
Alternate Routes

Kidneys degrade small proteins such as cytochrome c (CYT c) by the classic lysosomal pathway. However, because alternate routes for the transport and degradation of protein hormones have been identified in other tissues, we set out to determine whether extralysosomal sites might participate in the renal degradation of insulin. First, we compared the effect of the lysosomal inhibitor NH4Cl on insulin and CYT c degradation by isolated perfused rat kidneys. After kidneys were loaded with radiolabeled proteins to allow for absorption and transport to lysosomes, degradation was measured in the presence or absence of inhibitors. Control kidneys degraded 45 +/- 1.5% of the trapped CYT c per hour, and this was inhibited 62 +/- 1.3% by NH4Cl. In contrast, 86 +/- 2.4% of the trapped insulin was degraded per hour, and this was inhibited 26 +/- 4% by NH4Cl. Next we followed the subcellular distribution of 125I-labeled insulin in kidneys exposed to 125I-labeled insulin in vivo or when isolated and perfused. Under both circumstances the distribution of insulin on a linear sucrose gradient differed from that of the lysosomal enzyme N-acetyl-beta-glucosaminidase. In contrast, [14CH3]CYT c, injected in vivo, distributed over a density similar to the lysosomal marker. Thus important differences exist between the renal metabolism of CYT c, which proceeds in lysosomes, and the renal metabolism of insulin. These include rate of degradation, sensitivity to NH4Cl, and subcellular sites of localization. Accordingly, we suggest that insulin degradation may occur, at least in part, in a different compartment from the classic lysosomal site of protein degradation.

scholarly journals Glucagon receptors in endothelial and Kupffer cells of mouse liver.

1988 ◽  
Vol 36 (9) ◽  
pp. 1081-1089 ◽  
Author(s):  
J Watanabe ◽  
K Kanai ◽  
S Kanamura
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Kupffer Cells ◽  
Mouse Liver ◽  
Intracellular Transport ◽  
Coated Pits ◽  
Sinusoidal Cells ◽  
Nonparenchymal Cells ◽  
Glucagon Receptors

To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.

scholarly journals THE INFLUENCE OF PRECURSOR POOL SIZE ON MITOCHONDRIAL COMPOSITION IN NEUROSPORA CRASSA

1965 ◽  
Vol 24 (3) ◽  
pp. 445-460 ◽  
Author(s):  
David J. L. Luck
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Wild Type ◽  
Difference Spectra ◽  
Leucine Supplementation ◽  
Protein Ratio ◽  
Kinetic Measurements ◽  
Precursor Pool ◽  
Mutant Strains ◽  
Isopycnic Centrifugation

The chemical composition of mitochondria obtained from exponentially growing Neurospora can be varied by addition of choline or amino acids to the culture medium. The variation affects the phospholipid to protein ratio, and the density of mitochondria as determined by isopycnic centrifugation in sucrose gradients. These variations have been observed in biochemical mutant strains as well as wild type cultures. In a choline-requiring strain, two levels of choline supplementation to the medium have been defined: a low choline concentration just adequate to support maximal logarithmic growth, and a high choline concentration which permits maximal incorporation of radioactive choline into cellular lipids. Mitochondria isolated from cultures growing at the low choline concentration have one-half the phospholipid to protein ratio of those from high choline cultures, and their density is significantly higher. Artificial mixtures of the two types of mitochondria can be resolved into two populations by isopycnic centrifugation. The concentration of cytochromes (measured by mitochondrial difference spectra) and of malate and succinate dehydrogenases (measured by enzyme activity) were the same in both types of mitochondria, on a protein basis. The results suggest that during growth of the mitochondrial mass, the incorporation of phospholipid and protein components can vary independently. Direct kinetic measurements did indeed show that choline, added to a culture growing at low choline concentration, was incorporated into mitochondrial lipids at a rate faster than the incorporation of protein. The mitochondrial phospholipid to protein ratio can also be influenced by the level of leucine supplementation to a leucine-requiring mutant, so that with leucine concentrations above those required for maximal exponential growth, mitochondria of increasing density and decreasing phospholipid to protein ratio are produced. Additions of choline or amino acids to the minimal medium of wild type cultures influence mitochondrial composition in a manner directly comparable to that observed in biochemical mutant strains. The results suggest that mitochondrial composition, in general, is determined by rates of incorporation of the two major components, phospholipid and protein; that these rates can vary independently in response to precursor concentration in the culture medium; and that they normally operate at a precursor (substrate) concentration below saturation level.

scholarly journals Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.

1994 ◽  
Vol 126 (2) ◽  
pp. 343-352 ◽  
Author(s):  
T Ruscetti ◽  
J A Cardelli ◽  
M L Niswonger ◽  
T J O'Halloran
Keyword(s):  
Dictyostelium Discoideum ◽  
Heavy Chain ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Mutant Cells

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

scholarly journals Prolonged expression of a lysosomal enzyme in mouse liver afterSleeping Beauty transposon-mediated gene delivery: implications for non-viral gene therapy of mucopolysaccharidoses

2007 ◽  
Vol 9 (5) ◽  
pp. 403-415 ◽  
Author(s):  
Elena L. Aronovich ◽  
Jason B. Bell ◽  
Lalitha R. Belur ◽  
Roland Gunther ◽  
Brenda Koniar ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Lysosomal Enzyme ◽  
Mouse Liver ◽  
Viral Gene ◽  
Viral Gene Therapy

scholarly journals COLD-SENSITIVE MUTANTS OF DROSOPHILA MELANOGASTER DEFECTIVE IN RIBOSOME ASSEMBLY

Genetics ◽  
1975 ◽  
Vol 81 (4) ◽  
pp. 655-682
Author(s):  
Ernest V Falke ◽  
Theodore R F Wright
Keyword(s):  
Drosophila Melanogaster ◽  
Sucrose Gradient ◽  
Gradient Analysis ◽  
Gradient Centrifugation ◽  
Ribosome Assembly ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Ribosomal Subunits ◽  
Cold Sensitive ◽  
Lower Ratio

ABSTRACT Thirteen X-linked, cold-sensitive lethal, female-sterile mutants of Drosophila melanogaster located at eight separate loci were screened for their ability to assemble ribosomes at the restrictive temperature of 17°. Females were labelled with 3H-uridine for either 2 or 20 hours at 17°. A mitochondria-free extract was prepared and analyzed by means of sucrose gradient centrifugation. Four of the mutants, l(1)TW-2 cs, l(1)HM16cs, l(1)HM23cs, and l(1)HM20cs, had a lower ratio of cpm in the 40S subunit to cpm in the 60S subunit (40S:60S ratio) than wild type with a 2-hour label. The same was true of a 20-hour label of l(1)TW-2cs, l(1)HM16cs, and l(1)HM23cs, which are allelic, resulted in a 40S:60S ratio higher than wild type. Four other cs mutants were found to have less drastic effects on ribosome assembly. The ribosomal subunits of mutants l(1)HM16sc and l(1)HM20cs sediment at the same rate as their wild-type counterparts. The same is true for the RNA in their ribosomal particles. Sucrose gradient analysis of ribosomes from cold-sensitive lethal, female-sterile mutants appears to be an effective method for finding mutants that affect ribosome assembly.

Studies On The Structure And Cellular Location Of Various Ribosome And Ribosomal Rna Species In The Green Alga Chlamydomonas Reinhardi

1971 ◽  
Vol 8 (1) ◽  
pp. 153-183 ◽  
Author(s):  
D. P. BOURQUE ◽  
J. E. BOYNTON ◽  
N. W. GILLHAM
Keyword(s):  
Ribosomal Rna ◽  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Chlorophyll Synthesis ◽  
Sedimentation Velocity ◽  
Wild Type ◽  
Progressive Reduction ◽  
Lamellar System ◽  
Subunit Dissociation ◽  
Type C

Under ionic conditions effecting little or no subunit dissociation, Chlamydomonas reinhardi contains 2 major classes of ribosomes with generic sedimentation velocities of 83 and 70s and 3 minor classes with sedimentation velocities of 66, 54, and 41s. Ribosomal RNAs with sedimentation velocities of 25, 23, 18, 16 and 5s have been identified. The 70-s ribosomes are in the chloroplast and contain 23-, 16- and 5-s ribosomal RNA whereas the 83-s ribosomes are in the cytoplasm and contain 25-, 18- and 5-s ribosomal RNA. Numbers of chloroplast ribosome particles counted in electron micrographs of wild type C. reinhardi and the ac-20 and y mutants have been compared with relative amounts of 70-s ribosomes determined by sucrose gradient sedimentation and amounts of 23-, 16- and 5-s ribosomal RNA determined by gel electrophoresis. In response to reduced concentrations of magnesium the 70-s ribosomes of wild type are susceptible to a progressive reduction in sedimentation velocity whereas the 66-s ribosomes of the mutant ac-20 are not. Chlorophyll synthesis and the formation of the chloroplast lamellar system do not appear to be correlated with the relative amounts of chloroplast ribosomes.

Sign in / Sign up

Export Citation Format

Share Document