摘要:

AbstractProtamine sulfate blocked125I‐PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced125I‐PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose‐dependent decrease in the PDGF‐dependent incorporation of [3H]‐thymidine into 3T3 cells and a decreased PDGF‐stimulated tyrosine‐specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked125I‐PDGF binding to simian sarcoma virus transformed cells (SSV‐NIH 3T3 and SSV‐NPl cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of125I‐EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in125I‐EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect125I‐EGF binding to membranes from 3T3 cells or the EGF‐stimulated 3T3 cell membrane tyrbsinc specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G‐50 gel filtration columns; the half maximum inhibition concentration of125I‐PDGF binding to 3T3 cells of protamines I and II (MW ∼ 11,000 daltons and 7,000 daltons, respectively) is ∼ 0.4 μM. Protamine II (MW ∼ 4,800 daltons) was equally active (half maximum inhibition concentration ∼ 0.4 μM); protamine IV (MW ∼ 3,300 daltons) was substantially less active (half maximum inhibition concentration ∼ 2.8 μM).These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely

ISSN:0730-2312    

DOI:10.1002/jcb.240260402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

摘要:

AbstractDimethyl sulfoxidc (DMSO) stimulates tyrosine phosphorylation of the hepatic EGF receptor in isolated membrane preparations. To determine whether DMSO affects EGF binding, primary cultures of rat hepatocytes were incubated with 1–10% DMSO for 30 min prior to the addition of125I‐EGF. DMSO (1–2%) reduced specific125I‐EGF binding; the effect was maximal (a 40–60% reduction) at 5–7.5% DMSO and was reversed by removing the DMSO. Scatchard analysis showed that the reduction in binding was due to a change in receptor affinity. The decrease in binding was not seen when other, slightly less polar, solvents (eg, acetone and ethanol) were tested. DMSO also reduced125I‐EGF binding to purified rat liver plasma membranes. This reduction was seen in the absence of added ATP and in membranes that had been pretreated with TLCK, a tyrosine kinase inhibitor. Thus, completion of the receptor autophosphorylation reaction was not necessary to effect the change. The data arc consistent with a DMSO‐induced alteration of receptor conformation that rcversibly reduces re

ISSN:0730-2312    

DOI:10.1002/jcb.240260403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

摘要:

AbstractSeveral hormones, serum proteins, toxins, and viruses are brought into the cell by receptor‐mediated endocytosis. Initially, many of these molecules and particles are internalized into a common endocytic compartment via the clathrin‐coated pit pathway. Subsequently, the ligands and receptors are routed to several destinations, including lysosomes, the cytosol, or the plasma membrane. We have examined the mechanism by which sorting of internalized molecules occurs. A key step in the process is the rapid acidification of endocytic vesicles to a pH of 5.0–5.5 This acidification allows dissociation of several ligands from their receptors, the release of iron from transferring, and the penetration of diptheria toxin and some viral nucleocapsids into the cytoplasm. Transferrin, a ligand that cycles through the cell with its receptor, has been used as a marker for the recycling receptor pathway. We have found that in Chinese hamster ovary (CHO) cells transferrin is rapidly segregated from other ligands and is routed to a complex‐of small vesicles and/or tubules near the Golgi apparatus. The pH of the transferrin‐containing compartment is approximately 6.4, indicating that it is not in continuity with the more acidic endocytic vesicles which contain ligands destined to be degraded in

ISSN:0730-2312    

DOI:10.1002/jcb.240260404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

摘要:

AbstractA ternary complex of hyaluronic acid‐binding region and link protein bound to hyaluronic acid was isolated from limit clostripain digests of proteoglycan aggregates isolated from the Swarm rat chondrosarcoma. Under these conditions, the hyaluronic acid‐binding region has a molecular weight of ≅ 65,000 (HA‐BR65). N‐terminal amino acids in the complex were selectivelyl4C‐carbamylated. The resulting derivatized HA‐BR65was isolated, and tryptic peptide maps were prepared and developed on two‐dimensional TLC sheets. A single, labeled peptide was obtained which gave a Mrby ≅ 8,000 by SDS‐PAGE. Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA‐BR65to a polypeptide of ≅ 55,000 (HA‐BR55) which still retains the same N‐terminal tryptic peptide. Partial digestion of proteoglycan aggregates with clostripain generated a series of larger intermediates with the hyaluronic acid‐binding region. Direct SDS‐PAGE analysis revealed one major intermediate with Mr≅ 109,000 (HA‐BR109) as well as HA‐BR65. After chondroitinase digestion, two additional prominent intermediates were observed on a SDS‐PAGE gel at Mr≅ 120,000 (HA‐BR120) and ≅ 140,000 (HA‐BR140). All the intermediates were recognized by a monoclonal antibody specific for the hyaluronic acid‐binding region, and all of them contained the same N‐terminal tryptic peptide. The results indicate that the N terminus of the core protein is at the hyaluronic acid‐binding end of the proteoglycan and that the chondroitin sulfate chains are first present on the core protein in a region between 109,000

ISSN:0730-2312    

DOI:10.1002/jcb.240260405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

摘要:

AbstractBiosynthesis of cartilage proteoglycan was examined in a model system of cultured chondrocytes from a transplantable rat chondrosarcoma. Extensive modification with the addition of chondroitin sulfate glycosaminoglycan, N‐linkcd oligosac‐charide, and O‐linked oliogosaccharide is required to convert a newly synthesized core protein precursor into a proteoglycan. Kinetic analyses revealed the presence of a large pool of core protein precursor (t1/2 ∼ 90 min) awaiting completion into proteoglycan. The large t1/2 of this pool allowed kinetic labeling experiments with a variety of radioactive precursors to distinguish between early biosynthetic events associated primarily with the rough endoplasmic reticulum from late events associated primarily with the Golgi apparatus. The results of a series of experiments indicated that the addition of N‐linked oligosaccharide chains occurs early in the biosynthetic process in association with the rough endoplasmic reticulum, whereas the initiation and completion of O‐linked oligosaccharides occurs much later, at about the same time as chondroitin sulfate synthesis. This also indicated that keratan sulfate chains, when present in the completed molecule, are added in the Golgi apparatus, as they are probably built on oligosaccharide primers closely related to the O‐oligosaccharide chains. Furthermore, when3H‐glucose was used as the precursor, the entry of label into xylose, the linkage sugar between the core protein and the chondroitin sulfate chain, was found to occur within 5 min of the entry of label into galactose and galactosamine in the remainder of the chondroitin sulfate chain. This indicated that the initiation and completion of the chondroitin sulfate chain occurs late in the pathway probably entirely in the Golgi apparatus. Thus, proteoglycan synthesis can be described as occurring in two stages in this system, translation and N‐glycosylation of a core protein precursor which has a long half‐life in the rough endoplasmic reticulum, followed by extensive rapid modification in the Golgi complex in which the majority of glycosaminoglycan and oligosaccharide chains are added to the core protein precursor with subsequent rapid secretion into the e

ISSN:0730-2312    

DOI:10.1002/jcb.240260406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

ISSN:0730-2312    

DOI:10.1002/jcb.240260401

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY