The mechanism of the vitamin K-dependent post-translational carboxylation of the y-carbon atom of... more The mechanism of the vitamin K-dependent post-translational carboxylation of the y-carbon atom of glutamic acid residues in proteins remains obscure. Experiments were performed in vivo and in vitro in an attempt to establish a role for biotin in the transfer of the carboxyl group. Weanling male rats were fed on a biotin-deficient diet until severe biotin deficiency was induced. Their degree of biotin deficiency was documented by assaying for liver acetyl-CoA carboxylase activity, which was about 15 % of normal. However, onestage and two-stage prothrombin times measured on the plasmas were normal. In addition, the liver microsomal fraction did not contain any more prothrombin precursor than did that of normal rat liver. Experiments were done in vitro in which vitamin K-dependent fixing of 14C02 was measured in the liver microsomal fraction from vitamin K-deficient male rats in the presence or absence of avidin. No evidence for an avidin-sensitive critical biotin-containing site was obtained. Thus neither series of experiments suggests a role for biotin; the data are compatible with carboxyl transfer occurring either through a carboxylated vitamin K intermediate, or via a yet to be identified intermediate, or perhaps via CO2 itself.
The pathogenic role of antineutrophil cytoplasmic autoantibodies (ANCA) remains controversial bec... more The pathogenic role of antineutrophil cytoplasmic autoantibodies (ANCA) remains controversial because of the difficulty in explaining how extracellular ANCA can interact with intracellular primary granule constituents. It has been postulated that cytokine priming of neutrophils (PMN), as may occur during a prodromal infection, is an important trigger for mobilization of granules to the cell surface, where they may interact with ANCA. We show by electron microscopy that apoptosis of unprimed PMN is also associated with the translocation of cytoplasmic granules to the cell surface and alignment just beneath an intact cell membrane. Immunofluorescent microscopy and FACS ® analysis demonstrate reactivity of ANCA-positive sera and antimyeloperoxidase antibodies with apoptotic PMN, but not with viable PMN. Moreover, we show that apoptotic PMN may be divided into two subsets, based on the presence or absence of granular translocation, and that surface immunogold labeling of myeloperoxidase occurs only in the subset of PMN showing translocation. These results provide a novel mechanism that is independent of priming, by which ANCA may gain access to PMN granule components during ANCA-associated vasculitis. A ntineutrophil cytoplasmic autoantibodies (ANCA) 1 are associated with systemic vasculitides, especially Wegener's granulomatosis and microscopic polyarteritis (1-4). ANCA are also seen with idiopathic crescentic glomerulonephritis without immune deposits (2), and several other inflammatory or rheumatic diseases (3, 4). These autoAb are mainly directed against proteins in PMN primary granules and monocyte lysosomes (5). When detected by indirect immunofluorescence (IF) of ethanol-fixed PMN, there are two major patterns of ANCA staining-cytoplasmic (C-ANCA) and perinuclear (P-ANCA) (2). The major C-ANCA Ag is proteinase 3 (PR3) (6), a 29 kD serine proteinase. The major P-ANCA Ag is myeloperoxidase (MPO) (2). Although PR3 and MPO are located in the primary granules of PMN, ethanol fixation leads to solubilization and nuclear redistribution of MPO, leading to an artifactual perinuclear staining pattern (2, 7). Other minor ANCA Ag have been described, leading to both C-and P-ANCA patterns, but these account for Ͻ 5% of positive ANCA (5). The pathogenic role of ANCA remains controversial, in part because it is difficult to explain how extracellular ANCA interact with intracellular primary granule components. Al
The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein ... more The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein receptor (ASGP-R), were investigated by immunofluorescence and lateral mobility measurements combined with antibody-mediated crosslinking and immobilization. Immunofluorescence microscopy revealed two ASGP-R populations on the cell surface, one homogeneously distributed and the other in micropatches. This was observed both in stably transfected NIH 3T3 lines expressing H1 and/or H2, and in the human hepatoma cell line HepG2. In transfected cells expressing both polypeptides (the 1-7-1 line), H1 and H2 were colocalized in the same micro aggregates. Moreover, enhancement of the patching of, e.g., H1 by IgG-mediated crosslinking was accompanied by copatching of H2. To quantify H1-H2 complex formation, the lateral diffusion of H1 and H2 was measured at 12°C (to
Journal of the American Society of Nephrology, 1998
Although the inner medullary collecting duct (IMCD) plays a major role in urinary acidification, ... more Although the inner medullary collecting duct (IMCD) plays a major role in urinary acidification, the molecular identification of many of the specific components of the transport system in this nephron segment are lacking. A cultured line of rat IMCD cells was used to characterize the mediators of cellular HCO3 exit. This cell line functionally resembles alpha-intercalated cells. Physiologic experiments document that HCO3- transport is a reversible, electroneutral, Cl dependent, Na+-independent process. It can be driven by Cl-gradients and inhibited by stilbenes such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid. Immunohistochemical analysis, using a rabbit polyclonal antibody against the carboxy-terminal 12 amino acids of anion exchanger 1 (AE1), revealed a distribution of immunoreactive protein that is consistent with a basolateral localization of AE in cultured cells and in alpha-intercalated cells identified in sections of rat kidney cortex. Immunoblot rev...
the binding of vitamin B,2 to cell membrane prepara-tions from human placenta. The transcobalamin... more the binding of vitamin B,2 to cell membrane prepara-tions from human placenta. The transcobalamin II-vitamin B12 complex (TCII-B12), which has a much greater affinity for the membranes than vitamin Bl2 alone, binds to a single saturable binding site with an approximate K = 7.2 mM-1. The binding requires a divalent cation and is temperature-dependent. Free TCII can compete with TCII-B12 for the binding site but has somewhat less affinity than does TCII-B12. Rat TCII-B12 has an affinity constant that is less than one-fifth that of human TCII-B12; human TCI-B12, bovine TCII-B12, hog intrinsic factor-B12 (IF-B12), and human IF-B12 do not bind to the membranes. Pre-treating the membranes with trypsin causes a marked decrease in subsequent binding; this suggests the bind-ing site includes a relativdly exposed membrane pro-tein. These data suggest that a specific cell surface receptor for the TCII-B12 complex exists in placenta. This TCII-B12 receptor can be solubilized with Triton x-100.
Anti-phospholipid autoantibodies (aPL) are associated with a clinical syndrome of hypercoagulabil... more Anti-phospholipid autoantibodies (aPL) are associated with a clinical syndrome of hypercoagulability, thrombocytopenia, and fetal loss. Several groups have shown that the in vitro target of many aPL is not a pure phospholipid Ag, but is either a complex between anionic phospholipid and the plasma protein beta2-glycoprotein I (beta 2GPI) or the protein beta 2GPI alone. Anionic phospholipids are normally absent from the extracellular surface of cell membranes but redistribute from the inner to the outer leaflet during apoptosis. We show that aPL bind specifically to apoptotic, but not viable, thymocytes, and that binding is dependent upon the presence of beta 2GPI. Moreover, we show that beta 2GPI binds selectively to the surface of apoptotic thymocytes to generate an epitope for antiphospholipid autoantibodies. These findings suggest that apoptotic cells may be the natural immunogen and/or target for aPL. Moreover, we propose that the interaction of circulating beta 2GPI with redistributed anionic phospholipid may itself generate a novel ligand by which apoptotic cells are recognized directly for phagocytic clearance.
The human asialoglycoprotein receptor is composed of two homologous subunits, H1 and H2. By expre... more The human asialoglycoprotein receptor is composed of two homologous subunits, H1 and H2. By expressing the two subunits in transfected fibroblast cell lines, it has been shown previously that the formation of a hetero-oligomeric complex is necessary for the transport of H2 to the plasma membrane and for high-affinity ligand binding. Here we show that subunit H1, when expressed in the absence of H2, is capable of internalization through coated pits and recycling. The kinetics of these processes are very similar to those of the H1-H2 complex. To study endocytosis in the absence of ligand binding, the cell surface was labeled at 4 degrees C with the 125I-iodinated impermeant reagent sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate, the cells were incubated at 37 degrees C for different times and the amount of internalized receptor was determined by protease digestion of surface proteins and immunoprecipitation. Similarly, recycling of surface-labeled and then internalized receptor prot...
Monoclonal antibody (MAb) 5-1-6 identifies a 51-kDa protein (p51) on rat podocyte foot processes ... more Monoclonal antibody (MAb) 5-1-6 identifies a 51-kDa protein (p51) on rat podocyte foot processes and causes severe complement- and leukocyte-independent proteinuria when injected into rats. In the studies reported here, we used various immunohistological techniques to define the precise location of p51 and its relationship to ZO-1, a known component of the podocyte slit diaphragm in adult rat glomeruli. Our results demonstrate that p51 and ZO-1 lie close to each other on opposite sides of the podocyte plasma membrane at the point of insertion of the slit diaphragm: ZO-1 on the cytoplasmic face and p51 on the slit diaphragm and adjoining outer leaflet of the plasma membrane bordering the filtration slits. In addition to their geographic proximity, there appears to be a relationship between p51 and ZO-1. After MAb 5-1-6 injection, there was a progressive decline in stainable ZO-1 in the podocytes of heavily proteinuric rats. In addition, Western blot analysis of glomerular lysates sho...
An acetone powder, prepared from the liver microsomes of vitamin K-deficient rats, retains an act... more An acetone powder, prepared from the liver microsomes of vitamin K-deficient rats, retains an active vitamin K-dependent gamma-glutamyl carboxylase. While the basic requirements of the enzyme are similar to those of the carboxylase of either resuspended microsomes or detergent-solubilized microsomes, the acetone powder preparation reveals some additional properties of the carboxylase. Carboxylation of the synthetic pentapeptide substrate phenylalanylleucyl-glutamyl-glutamyl-valine can occur in the absence of nonionic detergent; however, when vitamin K hydroquinone drives the acetone powder carboxylation nonionic detergent is require for maximal activity. Experiments are described in which the acetone powder is incubated with the pentapeptide, pelleted by centrifugation, resuspended with fresh reactants, and incubated again. They suggest that the low V for the carboxylase, observed by all investigators, is, at least in part, not the result of irreversible enzyme inactivation nor depl...
We determined that the rate of insulin-stimulated autophosphorylation of the insulin receptor is ... more We determined that the rate of insulin-stimulated autophosphorylation of the insulin receptor is independent of receptor concentration and thus proceeds via an intramolecular process. This result is consistent with the possibility that ligand-dependent autophosphorylation may be a means by which cells can distinguish occupied from unoccupied receptors. We employed dithiothreitol to dissociate tetrameric receptor into alpha beta halves in order to further elucidate the structural requirements for the receptor-mediated kinase activity. Dithiothreitol had a complex biphasic effect on insulin-stimulated receptor kinase activity. Marked stimulation of kinase activity was observed at 1-2 mM dithiothreitol when the receptor was predominantly tetrameric and kinase activity diminished when dimeric alpha beta receptor halves predominate (greater than 2 mM dithiothreitol). N-Ethylmaleimide inhibits insulin-stimulated receptor kinase activity. We suggest that the tetrameric holoreceptor is the ...
A 2.5-kilobase cDNA clone (AF7), encoding 785 amino acids, was isolated from a rat liver cDNA lib... more A 2.5-kilobase cDNA clone (AF7), encoding 785 amino acids, was isolated from a rat liver cDNA library constructed in the expression vector lambda gt11. M13 vector sequence analysis yielded a deduced protein primary structure that was 89% homologous to the prototype alpha 1-inhibitor III (alpha 1I3) sequence presented in the preceding paper by Braciak et al. (Braciak, T. A., Northemann, W., Hudson, G. O., Shields, B. R., Gehring, M. R., and Fey, G. H. (1988) J. Biol. Chem. 263, 3999-4012) with regard to exact matches and 92% homologous when considering chemically conserved residues. The clone also possessed 100% homology to the putative bait region of a variant clone (pRLA1I3/27J) of alpha 1I3. Such sequence data demonstrates that the AF7 clone corresponds to a member of the family of variant alpha 1I3 mRNAs. Furthermore, this report presents the entire mRNA sequence corresponding to the 3'-half of alpha 1I3 variant 27J. We have utilized AF7 cDNA to study the expression of alpha ...
The biogenesis of p51, the target of nephritogenic monoclonal antibody 5-1-6, was studied in the ... more The biogenesis of p51, the target of nephritogenic monoclonal antibody 5-1-6, was studied in the developing glomerulus by immunolocalization and metabolic labeling. The localization of p51 was compared with that of ZO-1, a component of the cytoplasmic face of the epithelial slit diaphragm, and with that of podocalyxin, and apical marker of the podocyte. p51 first became faintly, but clearly, detectable on the basal and lateral sides of the developing podocytes at the S-shaped body stage. Staining intensity increased with further maturation and was restricted to the visceral epithelial cells. On immunoelectron microscopy, the antigen was seen along the basal and lateral surfaces below occluding junction at the early capillary loop stage and later, with the interdigitation of foot processes, became concentrated in the slit pores. At no stage was p51 seen on the apical surface. p51 and ZO-1 were closely localized in the mature glomerulus but arrived at their final positions from opposi...
The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein ... more The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein receptor (ASGP-R), were investigated by immunofluorescence and lateral mobility measurements combined with antibody-mediated cross-linking and immobilization. Immunofluorescence microscopy revealed two ASGP-R populations on the cell surface, one homogeneously distributed and the other in micropatches. This was observed both in stably transfected NIH 3T3 lines expressing H1 and/or H2, and in the human hepatoma cell line HepG2. In transfected cells expressing both polypeptides (the 1-7-1 line), H1 and H2 were colocalized in the same micro aggregates. Moreover, enhancement of the patching of, e.g., H1 by IgG-mediated crosslinking was accompanied by copatching of H2. To quantify H1-H2 complex formation, the lateral diffusion of H1 and H2 was measured at 12 degrees C (to avoid internalization) by fluorescence photobleaching recovery. H1 (or H2) was immobilized by crosslinking with specific Ig...
We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron mic... more We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the hi...
The mechanism of the vitamin K-dependent post-translational carboxylation of the y-carbon atom of... more The mechanism of the vitamin K-dependent post-translational carboxylation of the y-carbon atom of glutamic acid residues in proteins remains obscure. Experiments were performed in vivo and in vitro in an attempt to establish a role for biotin in the transfer of the carboxyl group. Weanling male rats were fed on a biotin-deficient diet until severe biotin deficiency was induced. Their degree of biotin deficiency was documented by assaying for liver acetyl-CoA carboxylase activity, which was about 15 % of normal. However, onestage and two-stage prothrombin times measured on the plasmas were normal. In addition, the liver microsomal fraction did not contain any more prothrombin precursor than did that of normal rat liver. Experiments were done in vitro in which vitamin K-dependent fixing of 14C02 was measured in the liver microsomal fraction from vitamin K-deficient male rats in the presence or absence of avidin. No evidence for an avidin-sensitive critical biotin-containing site was obtained. Thus neither series of experiments suggests a role for biotin; the data are compatible with carboxyl transfer occurring either through a carboxylated vitamin K intermediate, or via a yet to be identified intermediate, or perhaps via CO2 itself.
The pathogenic role of antineutrophil cytoplasmic autoantibodies (ANCA) remains controversial bec... more The pathogenic role of antineutrophil cytoplasmic autoantibodies (ANCA) remains controversial because of the difficulty in explaining how extracellular ANCA can interact with intracellular primary granule constituents. It has been postulated that cytokine priming of neutrophils (PMN), as may occur during a prodromal infection, is an important trigger for mobilization of granules to the cell surface, where they may interact with ANCA. We show by electron microscopy that apoptosis of unprimed PMN is also associated with the translocation of cytoplasmic granules to the cell surface and alignment just beneath an intact cell membrane. Immunofluorescent microscopy and FACS ® analysis demonstrate reactivity of ANCA-positive sera and antimyeloperoxidase antibodies with apoptotic PMN, but not with viable PMN. Moreover, we show that apoptotic PMN may be divided into two subsets, based on the presence or absence of granular translocation, and that surface immunogold labeling of myeloperoxidase occurs only in the subset of PMN showing translocation. These results provide a novel mechanism that is independent of priming, by which ANCA may gain access to PMN granule components during ANCA-associated vasculitis. A ntineutrophil cytoplasmic autoantibodies (ANCA) 1 are associated with systemic vasculitides, especially Wegener's granulomatosis and microscopic polyarteritis (1-4). ANCA are also seen with idiopathic crescentic glomerulonephritis without immune deposits (2), and several other inflammatory or rheumatic diseases (3, 4). These autoAb are mainly directed against proteins in PMN primary granules and monocyte lysosomes (5). When detected by indirect immunofluorescence (IF) of ethanol-fixed PMN, there are two major patterns of ANCA staining-cytoplasmic (C-ANCA) and perinuclear (P-ANCA) (2). The major C-ANCA Ag is proteinase 3 (PR3) (6), a 29 kD serine proteinase. The major P-ANCA Ag is myeloperoxidase (MPO) (2). Although PR3 and MPO are located in the primary granules of PMN, ethanol fixation leads to solubilization and nuclear redistribution of MPO, leading to an artifactual perinuclear staining pattern (2, 7). Other minor ANCA Ag have been described, leading to both C-and P-ANCA patterns, but these account for Ͻ 5% of positive ANCA (5). The pathogenic role of ANCA remains controversial, in part because it is difficult to explain how extracellular ANCA interact with intracellular primary granule components. Al
The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein ... more The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein receptor (ASGP-R), were investigated by immunofluorescence and lateral mobility measurements combined with antibody-mediated crosslinking and immobilization. Immunofluorescence microscopy revealed two ASGP-R populations on the cell surface, one homogeneously distributed and the other in micropatches. This was observed both in stably transfected NIH 3T3 lines expressing H1 and/or H2, and in the human hepatoma cell line HepG2. In transfected cells expressing both polypeptides (the 1-7-1 line), H1 and H2 were colocalized in the same micro aggregates. Moreover, enhancement of the patching of, e.g., H1 by IgG-mediated crosslinking was accompanied by copatching of H2. To quantify H1-H2 complex formation, the lateral diffusion of H1 and H2 was measured at 12°C (to
Journal of the American Society of Nephrology, 1998
Although the inner medullary collecting duct (IMCD) plays a major role in urinary acidification, ... more Although the inner medullary collecting duct (IMCD) plays a major role in urinary acidification, the molecular identification of many of the specific components of the transport system in this nephron segment are lacking. A cultured line of rat IMCD cells was used to characterize the mediators of cellular HCO3 exit. This cell line functionally resembles alpha-intercalated cells. Physiologic experiments document that HCO3- transport is a reversible, electroneutral, Cl dependent, Na+-independent process. It can be driven by Cl-gradients and inhibited by stilbenes such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid. Immunohistochemical analysis, using a rabbit polyclonal antibody against the carboxy-terminal 12 amino acids of anion exchanger 1 (AE1), revealed a distribution of immunoreactive protein that is consistent with a basolateral localization of AE in cultured cells and in alpha-intercalated cells identified in sections of rat kidney cortex. Immunoblot rev...
the binding of vitamin B,2 to cell membrane prepara-tions from human placenta. The transcobalamin... more the binding of vitamin B,2 to cell membrane prepara-tions from human placenta. The transcobalamin II-vitamin B12 complex (TCII-B12), which has a much greater affinity for the membranes than vitamin Bl2 alone, binds to a single saturable binding site with an approximate K = 7.2 mM-1. The binding requires a divalent cation and is temperature-dependent. Free TCII can compete with TCII-B12 for the binding site but has somewhat less affinity than does TCII-B12. Rat TCII-B12 has an affinity constant that is less than one-fifth that of human TCII-B12; human TCI-B12, bovine TCII-B12, hog intrinsic factor-B12 (IF-B12), and human IF-B12 do not bind to the membranes. Pre-treating the membranes with trypsin causes a marked decrease in subsequent binding; this suggests the bind-ing site includes a relativdly exposed membrane pro-tein. These data suggest that a specific cell surface receptor for the TCII-B12 complex exists in placenta. This TCII-B12 receptor can be solubilized with Triton x-100.
Anti-phospholipid autoantibodies (aPL) are associated with a clinical syndrome of hypercoagulabil... more Anti-phospholipid autoantibodies (aPL) are associated with a clinical syndrome of hypercoagulability, thrombocytopenia, and fetal loss. Several groups have shown that the in vitro target of many aPL is not a pure phospholipid Ag, but is either a complex between anionic phospholipid and the plasma protein beta2-glycoprotein I (beta 2GPI) or the protein beta 2GPI alone. Anionic phospholipids are normally absent from the extracellular surface of cell membranes but redistribute from the inner to the outer leaflet during apoptosis. We show that aPL bind specifically to apoptotic, but not viable, thymocytes, and that binding is dependent upon the presence of beta 2GPI. Moreover, we show that beta 2GPI binds selectively to the surface of apoptotic thymocytes to generate an epitope for antiphospholipid autoantibodies. These findings suggest that apoptotic cells may be the natural immunogen and/or target for aPL. Moreover, we propose that the interaction of circulating beta 2GPI with redistributed anionic phospholipid may itself generate a novel ligand by which apoptotic cells are recognized directly for phagocytic clearance.
The human asialoglycoprotein receptor is composed of two homologous subunits, H1 and H2. By expre... more The human asialoglycoprotein receptor is composed of two homologous subunits, H1 and H2. By expressing the two subunits in transfected fibroblast cell lines, it has been shown previously that the formation of a hetero-oligomeric complex is necessary for the transport of H2 to the plasma membrane and for high-affinity ligand binding. Here we show that subunit H1, when expressed in the absence of H2, is capable of internalization through coated pits and recycling. The kinetics of these processes are very similar to those of the H1-H2 complex. To study endocytosis in the absence of ligand binding, the cell surface was labeled at 4 degrees C with the 125I-iodinated impermeant reagent sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate, the cells were incubated at 37 degrees C for different times and the amount of internalized receptor was determined by protease digestion of surface proteins and immunoprecipitation. Similarly, recycling of surface-labeled and then internalized receptor prot...
Monoclonal antibody (MAb) 5-1-6 identifies a 51-kDa protein (p51) on rat podocyte foot processes ... more Monoclonal antibody (MAb) 5-1-6 identifies a 51-kDa protein (p51) on rat podocyte foot processes and causes severe complement- and leukocyte-independent proteinuria when injected into rats. In the studies reported here, we used various immunohistological techniques to define the precise location of p51 and its relationship to ZO-1, a known component of the podocyte slit diaphragm in adult rat glomeruli. Our results demonstrate that p51 and ZO-1 lie close to each other on opposite sides of the podocyte plasma membrane at the point of insertion of the slit diaphragm: ZO-1 on the cytoplasmic face and p51 on the slit diaphragm and adjoining outer leaflet of the plasma membrane bordering the filtration slits. In addition to their geographic proximity, there appears to be a relationship between p51 and ZO-1. After MAb 5-1-6 injection, there was a progressive decline in stainable ZO-1 in the podocytes of heavily proteinuric rats. In addition, Western blot analysis of glomerular lysates sho...
An acetone powder, prepared from the liver microsomes of vitamin K-deficient rats, retains an act... more An acetone powder, prepared from the liver microsomes of vitamin K-deficient rats, retains an active vitamin K-dependent gamma-glutamyl carboxylase. While the basic requirements of the enzyme are similar to those of the carboxylase of either resuspended microsomes or detergent-solubilized microsomes, the acetone powder preparation reveals some additional properties of the carboxylase. Carboxylation of the synthetic pentapeptide substrate phenylalanylleucyl-glutamyl-glutamyl-valine can occur in the absence of nonionic detergent; however, when vitamin K hydroquinone drives the acetone powder carboxylation nonionic detergent is require for maximal activity. Experiments are described in which the acetone powder is incubated with the pentapeptide, pelleted by centrifugation, resuspended with fresh reactants, and incubated again. They suggest that the low V for the carboxylase, observed by all investigators, is, at least in part, not the result of irreversible enzyme inactivation nor depl...
We determined that the rate of insulin-stimulated autophosphorylation of the insulin receptor is ... more We determined that the rate of insulin-stimulated autophosphorylation of the insulin receptor is independent of receptor concentration and thus proceeds via an intramolecular process. This result is consistent with the possibility that ligand-dependent autophosphorylation may be a means by which cells can distinguish occupied from unoccupied receptors. We employed dithiothreitol to dissociate tetrameric receptor into alpha beta halves in order to further elucidate the structural requirements for the receptor-mediated kinase activity. Dithiothreitol had a complex biphasic effect on insulin-stimulated receptor kinase activity. Marked stimulation of kinase activity was observed at 1-2 mM dithiothreitol when the receptor was predominantly tetrameric and kinase activity diminished when dimeric alpha beta receptor halves predominate (greater than 2 mM dithiothreitol). N-Ethylmaleimide inhibits insulin-stimulated receptor kinase activity. We suggest that the tetrameric holoreceptor is the ...
A 2.5-kilobase cDNA clone (AF7), encoding 785 amino acids, was isolated from a rat liver cDNA lib... more A 2.5-kilobase cDNA clone (AF7), encoding 785 amino acids, was isolated from a rat liver cDNA library constructed in the expression vector lambda gt11. M13 vector sequence analysis yielded a deduced protein primary structure that was 89% homologous to the prototype alpha 1-inhibitor III (alpha 1I3) sequence presented in the preceding paper by Braciak et al. (Braciak, T. A., Northemann, W., Hudson, G. O., Shields, B. R., Gehring, M. R., and Fey, G. H. (1988) J. Biol. Chem. 263, 3999-4012) with regard to exact matches and 92% homologous when considering chemically conserved residues. The clone also possessed 100% homology to the putative bait region of a variant clone (pRLA1I3/27J) of alpha 1I3. Such sequence data demonstrates that the AF7 clone corresponds to a member of the family of variant alpha 1I3 mRNAs. Furthermore, this report presents the entire mRNA sequence corresponding to the 3'-half of alpha 1I3 variant 27J. We have utilized AF7 cDNA to study the expression of alpha ...
The biogenesis of p51, the target of nephritogenic monoclonal antibody 5-1-6, was studied in the ... more The biogenesis of p51, the target of nephritogenic monoclonal antibody 5-1-6, was studied in the developing glomerulus by immunolocalization and metabolic labeling. The localization of p51 was compared with that of ZO-1, a component of the cytoplasmic face of the epithelial slit diaphragm, and with that of podocalyxin, and apical marker of the podocyte. p51 first became faintly, but clearly, detectable on the basal and lateral sides of the developing podocytes at the S-shaped body stage. Staining intensity increased with further maturation and was restricted to the visceral epithelial cells. On immunoelectron microscopy, the antigen was seen along the basal and lateral surfaces below occluding junction at the early capillary loop stage and later, with the interdigitation of foot processes, became concentrated in the slit pores. At no stage was p51 seen on the apical surface. p51 and ZO-1 were closely localized in the mature glomerulus but arrived at their final positions from opposi...
The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein ... more The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein receptor (ASGP-R), were investigated by immunofluorescence and lateral mobility measurements combined with antibody-mediated cross-linking and immobilization. Immunofluorescence microscopy revealed two ASGP-R populations on the cell surface, one homogeneously distributed and the other in micropatches. This was observed both in stably transfected NIH 3T3 lines expressing H1 and/or H2, and in the human hepatoma cell line HepG2. In transfected cells expressing both polypeptides (the 1-7-1 line), H1 and H2 were colocalized in the same micro aggregates. Moreover, enhancement of the patching of, e.g., H1 by IgG-mediated crosslinking was accompanied by copatching of H2. To quantify H1-H2 complex formation, the lateral diffusion of H1 and H2 was measured at 12 degrees C (to avoid internalization) by fluorescence photobleaching recovery. H1 (or H2) was immobilized by crosslinking with specific Ig...
We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron mic... more We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the hi...
Uploads
Papers by Michael Shia