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Biomedical subjects

K E Howell

Publications and source records attributed to K E Howell.

At least 55 records · Page 3Linked to original sources
PubMed1988

Isolation of a fraction enriched in the trans-Golgi network from baby hamster kidney cells.

Incubation of cultured cells at 20 degrees C blocks the transport of newly synthesized plasma membrane proteins, and the proteins accumulate intracellularly in a terminally glycosylated form. When baby hamster kidney cells are infected with the ts O45 mutant of vesicular stomatitis virus, and incubated at 20 degrees C, the terminally glycosylated spike glycoprotein G of the virus accumulates in the membranes of a tubular network localized on the trans side of the Golgi cisternae, the trans-Golgi network (TGN). We have used the G protein of ts O45 as a marker for the TGN and isolated a TGN fraction using a combination of conventional cell fractionation techniques and immunoisolation. The TGN was separated from the bulk of the endoplasmic reticulum, mitochondria, lysosomes, plasma membrane, and endosomes, while the activity of trans-Golgi marker galactosyltransferase copurified with the G protein. Using G protein as the TGN marker we have determined that the TGN was enriched 25-fold in the final fraction relative to the total homogenate. Several polypeptides (Mr 75,000, 87,000, 92,000, and 120,000) copurified with the G protein in the isolated TGN fraction and most likely represent resident markers of the compartment.

Animals↗
PubMed1988

Immunoselection of stably transformed MDCK cells expressing the vesicular stomatitis virus G-protein at the basolateral surface.

We have used immunoisolation on a magnetic solid support for the positive selection of stable MDCK transformants which express VSV-G protein via genomic integration of a cloned cDNA. This method is a simple, inexpensive alternative to selection with a fluorescence-activated cell sorter. The G-protein is synthesized in the absence of other viral proteins and is transported to the plasma membrane. The G-positive cells were enriched by immunoselection during normal passage of the transformed population. Using sterile conditions, antibodies to G were incubated with a suspension of transformed cells at 4 degrees C, unbound antibodies were then removed, and the cells were incubated with the immunoabsorbent (3 micron magnetic beads; J. Ugelstad et al. (1983) Nature (London) 303, 95) containing bound IgG molecules against the Fc portion of rabbit IgG. The magnetic properties of the beads were used to retrieve and further wash the immunoselected population. The cells are then removed from the beads by the same trypsinization conditions used for routine passaging and returned to culture. Using this selection scheme we have been able to increase the number of G-expressing cells five- to sevenfold per round; with repeated rounds enrichment from 2 to 74% was obtained. When grown on filters the immunoselected cells were shown to have the same morphology and electrical resistance (150-200 ohm.cm2) as untransformed MDCK II cells. Indirect immunofluorescence staining and [125I]protein A binding assays carried out on these cells demonstrated that G protein was localized exclusively to the basolateral surface as is observed with viral infection.

Animals↗
PubMed1987

Two threshold values of low pH block endocytosis at different stages.

The influence of low extracellular pH on endocytosis was studied in baby hamster kidney cells. When the extracellular medium was adjusted to pH 5.7, the intracellular pH decreased within 2 min to pH 6.2 and the endocytosis of horseradish peroxidase (HRP) in the fluid phase dropped to an undetectable level. With an external pH of 6.3, the internal pH dropped to pH 6.8 and HRP was internalized at a normal rate for 5 min but accumulation during longer incubation times did not occur. Morphologically, HRP was visualized in the lumen of a subpopulation of tubular and vesicular endosomes. These observations were confirmed by subcellular fractionation studies using free flow electrophoresis. Low extracellular pH also had an effect on the endocytosis of the membrane-spanning glycoprotein G of vesicular stomatitis virus which was implanted into the plasma membrane. The internalization of G-protein was quantitated by a surface fluoroimmunoassay. The endocytosis of G-protein was not affected when the external pH was dropped to 6.3, but was reduced at an external pH of 5.7. The intracellular ATP was not depleted and the reduction of endocytosis was reversible upon return to physiological pH. Clathrin coated pits were detected by electron microscopy at the plasma membrane of the low-pH-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1987

A recombinant apoA-1--protein A hybrid reproduces the binding parameters of HDL to its receptor.

We have constructed a plasmid, pLM8, containing the coding sequence of the mature human apoA-1 fused to the coding sequence of the IgG-binding domains of protein A (PA) from Staphylococcus aureus. The hybrid gene is transcribed in Escherichia coli under the control of a heat-sensitive repressor, leading to the synthesis of large amounts of hybrid protein (apoA-1--PA). The hybrid protein was purified by denaturation with urea and alkali, renaturation and affinity chromatography on an IgG Sepharose column. ApoA-1--PA is soluble and has an Mr of 316 kd, as determined by gel filtration. This is five times the monomer size of 62 kd, predicted from the sequence and found by SDS-PAGE analysis. Cell surface binding activity of the hybrid protein was tested using two different cell types (J774 macrophages and Fao hepatocytes) and compared to human high density lipoprotein (HDL). High-affinity binding was found for both ligands in both cell lines (Kd = 3.4 X 10(-8)M in Fao cells, 4.9 X 10(-8) M in J774 cells for apoA-1--PA and 3.0 X 10(-8) M in Fao cells, 2.8 X 10(-8) M in J774 cells for HDL), with approximately 2 X 10(5) high-affinity binding sites per cell. ApoA-1--PA and HDL effectively competed with each other for binding to the cell surface. Additionally, they both bound to a 110-kd polypeptide on a ligand blot, identifying an HDL receptor. The binding parameters of HDL were very similar to those of apoA-1--PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The Fao cell. A tissue culture model for lipoprotein synthesis and secretion. I. Characterization of the system.

In this study we found that a differentiated cultured rat hepatocyte cell line, Fao, synthesizes and secretes lipoproteins qualitatively similar to those synthesized by the rat hepatocyte in vivo, but quantitatively differing considerably in apoprotein composition and density distribution. Immunoprecipitation demonstrates that all the major apoproteins are synthesized, including both forms of apoB, apoE and apoA-I. Particles of all density classes are formed, apoB associating with the lighter particles and apoA-I with the heavier. ApoE is a major apoprotein in all but the lightest density classes. The general finding is that most particles formed have a density exceeding 1.10 g/ml, while very few of the lighter, apoB-containing particles form, probably because the normal growth medium of the cultured cells is lipid-poor as compared with rat serum. In ref. [20] we show that the composition of the lipoproteins synthesized can be effectively modulated by lipid depletion and lipid supplementation of the growth medium.

Animals↗
PubMed1987

The Fao cell. A tissue culture model for lipoprotein synthesis and secretion. II. Modulation by lipid depletion and supplementation.

We have shown that the Fao cell, a differentiated rat hepatoma line, is an excellent model for the study of the synthesis of lipoproteins (Scarino, M L & Howell, K E, Exp cell res 170 (1987) 1 [1]. Here we demonstrate that variation of the lipid composition of the growth medium significantly modulates the composition and quantity of particles formed. Three growth conditions were compared: normal, lipid-depleted, and lipid-supplemented. The synthesis of both the protein and lipid moieties of the lipoproteins was quantitated using the radioactive metabolic precursors [35S]methionine and [14C]acetate. The total secretion of the cells was collected and fractionated into four density classes equivalent to plasma lipoproteins and a bottom fraction equivalent to plasma proteins. Each density class was evaluated for the apoprotein distribution after separation by SDS-PAGE and for lipid distribution and composition after lipid extraction. ApoE accounts for approx. 15% of the total protein synthesized and is the major apoprotein. The amount synthesized remains relatively constant under all growth conditions. In contrast, the amount of apoB synthesis varies over 600-fold. In lipid-depleted conditions, only 0.01 times the normal amount was synthesized, while in lipid-supplemented conditions 6.2 times the normal amount was synthesized. ApoB was associated with the lighter fraction; therefore the modulation increased the quantity of low-density particles formed. A similar but far less pronounced variation of the heavier particles and the apoA-I concentration was obtained. Under lipid-depleted conditions, 0.75 times the normal amount was synthesized, while under lipid-supplemented conditions 2.6 times the normal quantity was synthesized.

Acetates↗
PubMed1987

An internalized transmembrane protein resides in a fusion-competent endosome for less than 5 minutes.

We have used our assay for the cell-free reconstitution of vesicle fusion occurring in endocytosis to investigate the fusion competence of defined endosomal fractions containing the G protein of vesicular stomatitis virus, G protein was first implanted into the plasma membrane, and endocytosis was then allowed to proceed for defined periods of time. Endosomal fractions were prepared by "immuno-isolation" on a solid support with a monoclonal antibody against the cytoplasmic domain of the G protein. Maximal internalization of the G protein occurred within 5 min at 37 degrees C. From this early endosome the G molecules follow a branched pathway: 50% recycles to the cell surface, while 50% is transported along the endocytic route to the lysosomal compartment. The proportion of G protein following each pathway can be modulated. When the amount of implanted G protein was increased, the fraction of G molecules recycling to the cell surface was reduced. When the G molecules were cross-linked with an antibody prior to the internalization step, recycling to the cell surface was abolished. The cell-free analysis of vesicle fusion was carried out with endosomal fractions immuno-isolated after 5, 10, 15, and 30 min of G-protein internalization at 37 degrees C. Fusion competence was at a maximum with the fraction isolated 5 min after internalization and then decreased with a half-life of approximately equal to 3 min with fractions isolated at later time points. The fusion-competent compartment is the early endosome where sorting of the transmembrane G protein to recycling or degradation occurs.

Animals↗
PubMed1987

Endosomal compartment of toad bladder epithelium.

Apical exocytosis and increased permeability are induced by antidiuretic hormone (ADH). After this, endocytosis is also induced by ADH and is associated with the decline in ADH-induced water permeability at the apical surface of the toad urinary bladder (9, 19, 20). During this process, horseradish peroxidase (HRP), a fluid phase marker, is taken up from the mucosal solution into endocytic tubules and multivesicular bodies. We now report that we can introduce from the apical (mucosal) side, a viral transmembrane protein (the G-protein of VSV) and that this protein can be retrieved as an integral membrane protein in endocytic membranes. This was demonstrated by immunoisolation of endosomal vesicles loaded with HRP using a monoclonal antibody against the cytoplasmic domain of the G-protein.

Animals↗
PubMed1987

Isolation of an endocytic compartment from A431 cells using a density modification procedure employing a receptor-specific monoclonal antibody complexed with colloidal gold.

Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endocytosis in human epidermoid carcinoma (A431) cells. The isolation protocol involves density modification of endosome elements in A431 cells, caused by the receptor-dependent binding and internalization at 20 degrees C of colloidal gold-transferrin receptor antibody (B3/25) particles. The use of 125I-labelled gold-B3/25 provides a radioactive marker for the endosome compartment, the major peak being recovered at the bottom of a continuous sucrose gradient at a density of 1.23g ml-1. Enzyme markers characteristic of other cytoplasmic compartments are present only in negligible amounts in this fraction and L-[35S]methionine-labelling of the cells indicates approximately a 200-fold enrichment of 125I-labelled gold-B3/25 versus protein. Electron microscopy of the endosome-rich fraction reveals that we have isolated a highly purified population of small gold-containing vesicles and tubules from which the transferrin receptor can be immunoprecipitated using the B3/25 antibody. Gel electrophoresis and fluorography of L-[35S]-methionine-labelled cells suggests that these elements contain a characteristic profile of approximately 10 major proteins of which three appear to be specifically enriched. In cells incubated with [125I]transferrin, 12% of the ligand sediments with the gold-labelled elements. We conclude, therefore, that the components we have isolated play a role in the intracellular processing of the transferrin-transferrin receptor complexes.

Antibodies, Monoclonal↗
PubMed1987

One antigen, one gold? A quantitative analysis of immunogold labeling of plasma membrane 5'-nucleotidase in frozen thin sections.

We present an evaluation of the efficiency of immunogold labeling for a low abundance plasma membrane protein. Several independent methods were used to determine the density of 5'-nucleotidase on the plasma membrane of the Fao cell. These methods include morphometry in combination with either enzymology or cell surface radiometric assay. Immunocytochemistry of frozen thin sections with either single or double layers of antibody and visualized with protein A complexed with 5 nm colloidal gold was used to estimate the same density. The application of a balance sheet to immunogold labeling demonstrates that the labeling is never quantitative. For example, labeling of the cell surface is always greater than labeling on the section. We show that departures from the "one antigen, one gold" ideal are systematic, so that an efficiency can be calculated and quantitative results can be obtained. The ability to obtain reliable quantitative results from immunogold labeling extends the utility of this already powerful technique.

5'-Nucleotidase↗
PubMed1986

Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system.

We have used defined subcellular fractions to reconstitute in a cell-free system vesicle fusions occurring in the endocytic pathway. The endosomal fractions were prepared by immuno-isolation using as antigen an epitope located on a foreign protein, the transmembrane glycoprotein G (G-protein) of vesicular stomatitis virus. The G-protein was first implanted in the cell plasma membrane and subsequently endocytosed for 15 to 30 min at 37 degrees C. The endosomal fractions were immuno-isolated on a solid support using as antigen the cytoplasmic domain of the G-protein in combination with a specific monoclonal antibody. For comparative studies the plasma membrane was immuno-isolated from cells in the absence of G internalization with a monoclonal antibody against the exoplasmic domain of the G-protein. The immuno-isolated endosomal vesicles contained 70% of horseradish peroxidase internalized in the endosome fluid phase, exhibited an acidic luminal pH as shown by acridine orange fluorescence and differed in their protein composition from the immuno-isolated plasma membrane fraction. The fusion of endocytic vesicles originating from different stages of the pathway was studied in a cell-free assay using both a bio-chemical and a morphological detection system. These well defined endosomal vesicles were immuno-isolated with the G-protein on the solid support and provided the recipient compartment of the fusion (acceptor). They were mixed with a post-nuclear supernatant containing endosomes loaded with exogenous lactoperoxidase (donor) at 37 degrees C. Fusion delivered the donor peroxidase to the lumen of acceptor vesicles permitting fusion-specific iodination of the G-protein itself. The fusion of vesicles required ATP and was detected only with an endosomal fraction prepared after internalization of the G-protein for 15 min at 37 degrees C but not with a plasma membrane or with an endosomal fraction prepared after 30 min G-protein internalization.

Animals↗
PubMed1985

Immuno-isolation of a plasma membrane fraction from the Fao cell.

A plasma membrane was immuno-isolated from a post-nuclear supernatant of a cultured rat hepatocyte, the Fao cell, using a cellulose immuno-adsorbent and antibodies raised against a variety of endogenous antigens of hepatocytes: 5'-nucleotidase, a plasma membrane fraction and the whole Fao cell. The antibodies which recognize antigens on the cell surface were selected from the total serum by first binding the antiserum to suspension cells. Alternatively, the plasma membrane and Fao antisera were affinity purified on a column prepared from a Triton X-114 extract of a plasma membrane fraction. The immuno-isolation was most efficient when carried out with either the plasma membrane or the Fao anti-serum. When alkaline phosphodiesterase I or 5'-nucleotidase was used as the plasma membrane marker, 40-60% of the plasma membrane of the post-nuclear supernatant was isolated representing a maximum 34-fold increase in the specific activity of the enzymes in the bound material. Using the NaB-[3H]4-labelled glycoproteins of the plasma membrane or the IgG bound to the plasma membrane as alternative markers, an 80% isolate of the plasma membrane of the post-nuclear supernatant was achieved, resulting in an estimated 40-fold purification. The non-specific binding was low despite the use of a post-nuclear supernatant as the input fraction. The characterization of the bound materials suggested that the whole plasma membrane was immuno-isolated and not a particular domain.

Animals↗
PubMed1985

Biogenesis of the polymeric IgA receptor in rat hepatocytes. I. Kinetic studies of its intracellular forms.

The polymeric IgA receptor (or secretory component [SC]) is a major biliary secretory protein in the rat. It was identified as an 80,000-mol-wt (80 K) glycoprotein by coprecipitation (with IgA) by anti-IgA antibodies (Sztul, E. S., K. E. Howell, and G. E. Palade, 1983, J. Cell Biol., 97:1582-1591) and was used as antigen to raise anti-SC antibodies in rabbits. Pulse labeling with [35S]cysteine in vivo, followed by the immunoprecipitation of solubilized total microsomal fractions with anti-SC sera, made possible the identification of three intracellular forms of SC (all apparently membrane proteins) and the definition of their kinetic and structural interrelations. At 5 min postinjection of [35S]cysteine, a major band of Mr 105,000 was maximally labeled. This peptide lost radioactivity concomitantly with the appearance of a radioactive doublet of Mr 116,000 and 120,000 at 15-30 min postinjection. Loss of radioactivity from 116K paralleled increased labeling of the 120K peptide which appears to be the mature form of the receptor. The 105K form was sensitive to endoglycosidase H which converted it to a 96K peptide. The 116K and 120K forms were resistant to endoglycosidase H but sensitive to endoglycosidase F which converts them to 96K and 100K forms, respectively. Taken together, these findings support the following conclusions: (a) All rat hepatic SC forms are the products of a single gene; (b) all SC forms are N-glycosylated; (c) the 116K form is the result of the terminal glycosylation of the 105K form; and (d) the 120K peptide is probably produced by modifications at other sites than its complex oligosaccharide chains.

Animals↗
PubMed1985

Biogenesis of the polymeric IgA receptor in rat hepatocytes. II. Localization of its intracellular forms by cell fractionation studies.

In the companion paper (Sztul, E. S., K. E. Howell, and G. E. Palade, J. Cell Biol., 100:1248-1254), we have shown that pulse labeling of hepatic proteins with [35S]cysteine can be obtained in vivo in intact rats. Soluble label clears the plasma in approximately 5 min, and incorporated label reaches peak values in the liver approximately 20 min after injection. In the present study, we show that the 105,000-mol-wt protein (105K), kinetically the earliest intracellular form of secretory component (SC), is the predominant form found, between 5 and 20 min postinjection, in homogeneous rough microsomal fractions. The second kinetically defined form, i.e., 116K, is the predominant species present in relatively homogeneous, light Golgi fractions in which it appears at approximately 15 min, and peaks at approximately 25 min, postinjection. The third kinetically defined form, 120K, is found 30 min after injection as the major SC species (albeit still accompanied by its immediate precursor, 116K), in a sinusoidal plasmalemmal fraction isolated by immunoadsorption to anti-SC-coated Sepharose beads. These findings lead to the following conclusions: (a) SC is synthesized on polysomes attached to the rough endoplasmic reticulum (ER) membrane; (b) it is partially translocated across the ER membrane and core glycosylated co-translationally to give a 105K peptide; (c) 105K moves from the ER to the Golgi complex where it is terminally glycosylated to give the 116K form; (d) the latter moves to the sinusoidal plasmalemma where it appears together with the final mature form, 120K. Kinetic evidence indicates that the vesicular carriers involved in the transport of SC from the Golgi complex to the sinusoidal plasmalemma, and from the latter to the biliary front of the hepatocytes, are present in a Golgi heavy fraction and a crude carrier vesicle fraction from which they remain to be isolated, purified, and characterized.

Animals↗
PubMed1985

Immuno-isolation of vesicles using antigenic sites either located on the cytoplasmic or the exoplasmic domain of an implanted viral protein. A quantitative analysis.

In this study, we present a new general approach for immuno-isolation: a foreign integral membrane protein, the G-protein of vesicular stomatitis virus (VSV), is implanted into the plasma membrane for subsequent immuno-isolation. A quantitative analysis was accomplished using the erythrocyte plasma membrane as a model system. The virus was artificially bound to the membrane via a lectin and subsequently fused at low pH. Vesicles of two opposite orientations were prepared from erythrocytes with fused G-protein. Right-side-out and inside-out vesicles expose the exoplasmic and the cytoplasmic domains of the G-protein on their surfaces respectively. In immuno-isolation experiments antibodies against each of the domains of the G-protein were used. Vesicles were presented to an immunoadsorbent (ImAd) consisting of a solid support with appropriate antibodies bound to its surface. Two commonly used immunoadsorbents prepared from either polyacrylamide beads or fixed Staphylococcus aureus cells were compared and found to have identical immuno-isolation efficiencies. It was possible to control and quantitate the amount of implanted antigen. Therefore, we were able to show that the critical antigen density required for immuno-isolation is 50 G molecules/micron2 plasma membrane surface area for both types of vesicle/antibody couples. This analysis showed that vesicles presenting either the cytoplasmic or the exoplasmic domain of the G-protein are immuno-isolated with the same efficiency.

Animals↗
PubMed1983

Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes.

The intra- and transcellular transports of hepatic secretory and membrane proteins were studied in rats in vivo using [3H]fucose and [35S]cysteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile, and plasma were separated by SDS PAGE and identified by fluorography. 3H-radioactivity in Golgi fractions peaked at 10 min postinjection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from Golgi occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 30 min later than the bulk of content proteins. A major 80,000-dalton form of secretory component (SC) was identified in the bile by co-precipitation with (IgA)2 by an anti-IgA antibody. An antibody (raised in rabbit) against the biliary 80,000-dalton peptide recognized two larger forms (116,000 and 94,000 dalton), presumably precursors, in Golgi membranes. A comparative study of kinetics of transport of 35S-SC and 35S-albumin showed that albumin peaked in bile at approximately 45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins that are delivered to the bile canaliculus.

Albumins↗