scholarly journals Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation

1987 ◽  
Vol 247 (1) ◽  
pp. 165-174 ◽  
Author(s):  
M Whitman ◽  
D Kaplan ◽  
T Roberts ◽  
L Cantley
Keyword(s):  
Kinase Activity ◽  
Energy Charge ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Type I ◽  
Type Ii ◽  
Ionic Detergents ◽  
Rous Sarcoma ◽  
3T3 Fibroblasts ◽  
Specific Association

Phosphatidylinositol (PtdIns) kinase activities from non-transformed and polyoma-middle-T-transformed murine fibroblasts were examined. Both normal and transformed 3T3 fibroblasts have two PtdIns kinases, which can be separated by anion-exchange chromatography. One of these activities (Type I) has a Km for ATP of 10 microM, is resistant to inhibition by adenosine, AMP or ADP, and is inhibited by non-ionic detergents. The other activity (Type II) has a somewhat higher Km for ATP (35 microM) and is inhibited competitively by ADP, AMP and adenosine at concentrations suggesting regulation of this activity by the energy charge of the cell. The Type II PtdIns kinase is activated by non-ionic detergents. We have previously reported the specific association of a PtdIns kinase activity with polyoma-middle-T immunoprecipitates [Whitman, Kaplan, Schaffhausen, Cantley & Roberts (1985) Nature (London) 315, 239-242; Kaplan, Whitman, Schaffhausen, Raptis, Garcea, Pallas, Roberts & Cantley (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3624-3628]. Comparison of the immunoprecipitated PtdIns kinase with the activities identified by ion-exchange chromatography indicates that it is the Type I enzyme which specifically associates with the middle-T/pp60c-src complex. This PtdIns kinase activity is separable from both middle T and pp60c-src. Type I PtdIns kinase also associates with pp60v-src immunoprecipitates from Rous-sarcoma-virus-transformed cells. Furthermore, this PtdIns kinase appears to co-precipitate with partially purified platelet derived growth factor (PDGF) receptor. The amount of this activity found in anti-phosphotyrosine immunoprecipitates or in wheat-germ-lectin-agarose precipitates is increased 50-fold by stimulation of quiescent Balb/C 3T3 fibroblasts with PDGF. These results suggest that the Type I PtdIns kinase is regulated by agents which affect cell growth and transformation, whereas the Type II PtdIns kinase may be regulated by the local [ATP]/[ADP] ratio.

scholarly journals Sorting out IF networks: consequences of domain swapping on IF recognition and assembly.

1991 ◽  
Vol 113 (5) ◽  
pp. 1111-1124 ◽  
Author(s):  
M B McCormick ◽  
P A Coulombe ◽  
E Fuchs
Keyword(s):  
Coiled Coil ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Genetically Engineered ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Part Type ◽  
Structural Aberrations ◽  
Cellular Organelles

Vimentin and keratin are coexpressed in many cells, but they segregate into two distinct intermediate filament (IF) networks. To understand the molecular basis for the sorting out of these IF subunits, we genetically engineered cDNAs encoding hybrid IF proteins composed of part vimentin and part type I keratin. When these cDNAs were transiently expressed in cells containing vimentin, keratin, or both IFs, the hybrid IF proteins all recognized one or the other or both networks. The ability to distinguish networks was dependent upon which segments of IF proteins were present in each construct. Constructs containing sequences encoding either helix 1B or helix 2B seemed to be the most critical in conferring IF recognition. At least for type I keratins, recognition was exerted at the level of dimer formation with wild-type type II keratin, as demonstrated by anion exchange chromatography. Interestingly, despite the fact that swapping of helical domains was not as deleterious to IF structure/function as deletion of helical domains, keratin/vimentin hybrids still caused structural aberrations in one or more of the cytoplasmic IF network. Thus, sequence diversity among IF proteins seems to influence not only coiled-coil but also higher ordered associations leading to 10-nm filament formation and/or IF interactions with other cellular organelles/proteins.

Purification and Characterization of Laccase fromChaetomium thermophilium and Its Role in Humification

1998 ◽  
Vol 64 (9) ◽  
pp. 3175-3179 ◽  
Author(s):  
Benny Chefetz ◽  
Yona Chen ◽  
Yitzhak Hadar
Keyword(s):  
Municipal Solid Waste ◽  
Solid Waste ◽  
Laccase Activity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Type I ◽  
Light Spectrum ◽  
Solid Media ◽  
Wide Range

ABSTRACT Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process.C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45°C both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70°C and had half-lives of 24 and 12 h at 40 and 50°C, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.

scholarly journals Control of types I and II collagen and fibronectin gene expression in chondrocytes delineated by viral transformation.

1985 ◽  
Vol 5 (5) ◽  
pp. 1002-1008 ◽  
Author(s):  
E S Allebach ◽  
D Boettiger ◽  
M Pacifici ◽  
S L Adams
Keyword(s):  
Rous Sarcoma Virus ◽  
Type I Collagen ◽  
Skin Fibroblasts ◽  
Transcriptional Level ◽  
Type I ◽  
Type Ii ◽  
Intact Cells ◽  
Rous Sarcoma ◽  
Synthetic Rate ◽  
Sarcoma Virus

We have analyzed the effects of transformation by Rous sarcoma virus on expression of types I and II collagen and fibronectin genes in vertebral chondrocytes and compared them with expression of these genes in skin fibroblasts. Transformed chondrocytes display a dramatically decreased amount of type II collagen RNA, which can account fully for the decreased synthetic rate of this protein. Paradoxically, these cells also display greatly increased amounts of type I collagen RNAs, which are translated efficiently in vitro, but not in the intact cells. We show here that the type I collagen RNAs in transformed chondrocytes are nearly indistinguishable from those found in skin fibroblasts, and that they clearly differ from the type I collagen RNAs found in normal chondrocytes. Transformed chondrocytes also display an increased amount of fibronectin RNAs, which can account fully for the increased synthetic rate of this protein. Thus, the effects of transformation by Rous sarcoma virus on type I collagen and fibronectin RNAs in chondrocytes are the opposite of those observed in fibroblasts, which display decreased amounts of these three RNAs. These data indicate that the effects of transformation on the genes encoding type I collagen and fibronectin must be modulated by host cell-specific factors. They also imply that the types I and II collagen genes may be regulated by different mechanisms, the type I genes being controlled at both transcriptional and posttranscriptional levels, and the type II gene being controlled primarily at the transcriptional level.

scholarly journals Posttranslational regulation of keratins: degradation of mouse and human keratins 18 and 8.

1989 ◽  
Vol 9 (4) ◽  
pp. 1553-1565 ◽  
Author(s):  
D A Kulesh ◽  
G Ceceña ◽  
Y M Darmon ◽  
M Vasseur ◽  
R G Oshima
Keyword(s):  
Cell Types ◽  
Immunofluorescent Staining ◽  
Type I ◽  
Type Ii ◽  
Tail Domain ◽  
Proteolytic System ◽  
L Cells ◽  
Adult Mice ◽  
3T3 Fibroblasts ◽  
Keratin Filament

Human keratin 18 (K18) and keratin 8 (K8) and their mouse homologs, Endo B and Endo A, respectively, are expressed in adult mice primarily in a variety of simple epithelial cell types in which they are normally found in equal amounts within the intermediate filament cytoskeleton. Expression of K18 alone in mouse L cells or NIH 3T3 fibroblasts from either the gene or a cDNA expression vector results in K18 protein which is degraded relatively rapidly without the formation of filaments. A K8 cDNA containing all coding sequences was isolated and expressed in mouse fibroblasts either singly or in combination with K18. Immunoprecipitation of stably transfected L cells revealed that when K8 was expressed alone, it was degraded in a fashion similar to that seen previously for K18. However, expression of K8 in fibroblasts that also expressed K18 resulted in stabilization of both K18 and K8. Immunofluorescent staining revealed typical keratin filament organization in such cells. Thus, expression of a type I and a type II keratin was found to be both necessary and sufficient for formation of keratin filaments within fibroblasts. To determine whether a similar proteolytic system responsible for the degradation of K18 in fibroblasts also exists in simple epithelial cells which normally express a type I and a type II keratin, a mutant, truncated K18 protein missing the carboxy-terminal tail domain and a conserved region of the central, alpha-helical rod domain was expressed in mouse parietal endodermal cells. This resulted in destabilization of endogenous Endo A and Endo B and inhibition of the formation of typical keratin filament structures. Therefore, cells that normally express keratins contain a proteolytic system similar to that found in experimentally manipulated fibroblasts which degrades keratin proteins not found in their normal polymerized state.

scholarly journals Isolation of two myosin light-chain kinases from bovine carotid artery and their regulation by phosphorylation mediated by cyclic AMP-dependent protein kinase

1982 ◽  
Vol 203 (3) ◽  
pp. 583-592 ◽  
Author(s):  
Ramesh C. Bhalla ◽  
Ram V. Sharma ◽  
Ramesh C. Gupta
Keyword(s):  
Protein Kinase ◽  
Carotid Artery ◽  
Cyclic Amp ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Type I ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Myosin light-chain kinase was purifed from bovine carotid artery. Approx. 90% of myosin kinase was extracted in the supernatant fraction with buffer containing EDTA during myofibril preparation. The soluble fraction yielded two distinct peaks on DEAE-Sephacel chromatography. Peak I was eluted at a conductance of 11–12mmho and was completely dependent on Ca2+–calmodulin for its activity. Peak II was eluted at a conductance of 13–14mmho and showed approx. 15% Ca2+-independent activity. The myosin kinases I and II were further purified by affinity chromatography by using calmodulin coupled to Sepharose 4B, which resulted in 960-and 650-fold purification of type I and type II kinases respectively. Myosin kinase II activity was completely Ca2+-dependent after affinity chromatography on the calmodulin–Sepharose column. Myosin kinases I and II were phosphorylated by cyclic AMP-dependent protein kinase. In the presence of bound calmodulin 0.5–0.7mol of phosphate was incorporated/mol of myosin kinases I and II. On the other hand, in the absence of bound calmodulin 1–1.4mol of phosphate was incorporated/mol of kinases I and II. Phosphorylation in the absence of calmodulin significantly decreased the myosin kinase activity of both enzymes, and the decrease in myosin kinase activity was due to a 3–5-fold increase in the amount of calmodulin required for half-maximal stimulation of both type I and type II kinases. The regulation of myosin kinase activity by cyclic AMP-dependent phosphorylation would suggest that β-adrenergic-mediated relaxation of vascular smooth muscle may be partly due to the direct interaction of cyclic AMP at the site of contractile proteins.

scholarly journals Protective Effects of Type I and Type II Interferons toward Rous Sarcoma Virus-Induced Tumors in Chickens

Virology ◽  
1999 ◽  
Vol 256 (1) ◽  
pp. 85-91 ◽  
Author(s):  
Jiřı́ Plachý ◽  
Kirsten C. Weining ◽  
Elisabeth Kremmer ◽  
Florian Puehler ◽  
Karel Hala ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Type I ◽  
Protective Effects ◽  
Type Ii ◽  
Rous Sarcoma ◽  
Induced Tumors ◽  
Sarcoma Virus

Plasma ß-Thromboglobulin and Glycosylated Hemoglobin(Hb A1) in Type I and Type II Diabetes Mellitus

1979 ◽  
Author(s):  
G. Schernthaner ◽  
K. Silberbauer ◽  
H. Sinzinger ◽  
M. Müller
Keyword(s):  
Diabetes Mellitus ◽  
Metabolic Control ◽  
Platelet Function ◽  
Glycosylated Hemoglobin ◽  
Type Ii Diabetes Mellitus ◽  
Exchange Chromatography ◽  
Type I ◽  
Type Ii ◽  
Type Ii Diabetics ◽  
Insulin Dependent

Platelet hyperactivity is a common feature in diabetes meliitus. ß-thrombo-globulrn (ß-TG) - which is released during platelet aggregation - seems to be a useful Index of platelet involvement in thrombotic disease. Howeverthere is no detained information regarding metabolic control on platelet function in diabetes me 1litus, Therefore we studied the plasma ß-TG concentrations and the hemoglobin A1(Hb Airparameter of diabetic longterm control)levels in 7o patients with type-I(insulin-dependent) diabetes(D), ibo patients with type II D (non-insulin-dependent)as well as in 70 healthy controls (C). Plasma-ß-TG levels were determined by radioimmunoassay, Hb Al concentrations by ion-exchange chromatography. ß-TG levels were significantly increased in type-I and in type-II D compared to C (p<0,01, resp.<0,0011. Hb Al concentrations were significantly higher in both groups of diabetics than in the C <p< 0,005, resp. <0,005). No correlation could be found between the ß-TG levels and the Hb A1 concentrations neither in the type I nor in the type II diabetics. These data suggest that the degree of metabolic control is not the main factor in disturbed platelet function in diabetes mellitus.

scholarly journals Keratin incorporation into intermediate filament networks is a rapid process.

1991 ◽  
Vol 113 (4) ◽  
pp. 843-855 ◽  
Author(s):  
R K Miller ◽  
K Vikstrom ◽  
R D Goldman
Keyword(s):  
Ion Exchange ◽  
Intermediate Filament ◽  
Immunofluorescence Microscopy ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Type I ◽  
Type Ii ◽  
Filament Networks ◽  
Cytoskeletal Elements

The properties of keratin-containing intermediate filament (IF) networks in vivo were studied following the microinjection of biotinylated keratin. Keratin-IFs were biotinylated, disassembled, and separated into type I and type II proteins by ion exchange chromatography. Recombination of these derivatized type I and type II keratins resulted in the formation of 10-nm diameter IF. The type I keratins were microinjected into epithelial cells and observed by immunofluorescence microscopy. Biotin-rich spots were found throughout the cytoplasm at 15-20 min after injection. Short biotinylated fibrous structures were seen at 30-45 min after injection, most of which colocalized with the endogenous bundles of IF (tono-filaments). By 1 1/2 to 2 h after microinjection, extensive biotinylated keratin IF-like networks were evident. These were highly coincident with the endogenous tonofilaments throughout the cell, including those at desmosomal junctions. These results suggest the existence of a relatively rapid subunit incorporation mechanism using numerous sites along the length of the endogenous tonofilament bundles. These observations support the idea that keratin-IFs are dynamic cytoskeletal elements.

scholarly journals Expression, purification, and bioactivity of a soluble recombinant ovine interferon-tau in Escherichia coli

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Hai-Yang Yu ◽  
Dong-Mei Gao ◽  
Wei Zhou ◽  
Bing-Bing Xia ◽  
Zhi-Yuan He ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Restriction Enzymes ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Soluble Form ◽  
Type I ◽  
Interferon Tau ◽  
E Coli ◽  
Nickel Affinity Chromatography

Abstract Introduction Ovine interferon-tau (oIFN-τ) is a newly discovered type I interferon. This study used biochemical techniques to transform the oIFN-τ gene into Escherichia coli to obtain the mass and soluble expression of the recombinant protein. Materials and Methods First, total RNA was extracted from fresh sheep embryonic tissues with TRIzol reagent and then used as a template to reverse transcribe and amplify the mature oIFN-τ gene with RT-PCR. The amplified product was next digested with the HindIII and XhoI restriction enzymes and inserted into the pET-32a(+) vector to construct the prokaryotic expression plasmid. The corrected in-frame recombinant plasmid, pET-32a(+)-oIFN-τ, was transformed into E. coli Rosetta (DE3) competent cells. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was detected in bacteria. Finally, the bacteria were lysed by sonication, and the recombinant protein was purified by nickel affinity chromatography and DEAE anion exchange chromatography. Results The protein was confirmed to be oIFN-τ, which mainly existed in the soluble lysate fraction, as proven by SDS-PAGE and Western blot assays. Conclusion Purified IFN-τ exists mostly in a soluble form, and its anti-vesicular stomatitis virus (VSV) activity reached 7.08×10(6)IU/mL.

Phosphatidylinositol kinase activity associates with viral p60src protein

1989 ◽  
Vol 9 (4) ◽  
pp. 1651-1658
Author(s):  
Y Fukui ◽  
H Hanafusa
Keyword(s):  
High Performance ◽  
Kinase Activity ◽  
Cell Transformation ◽  
Heat Stability ◽  
Temperature Sensitive ◽  
Type I ◽  
Temperature Sensitive Mutants ◽  
Rous Sarcoma ◽  
Gradient Fractionation ◽  
Pi Kinase

Immunoprecipitates of p60v-src proteins from chicken embryo fibroblasts infected with Rous sarcoma virus were assayed for phosphatidylinositol (PI) kinase activity in the absence of detergents. The product of the PI kinase reaction, phosphatidylinositol monophosphate (PIP), migrated slightly slower than did the authentic phosphatidylinositol-4-monophosphate marker in thin-layer chromatography and was indistinguishable from phosphatidylinositol-3-monophosphate produced by PI kinase type I. Furthermore, the deacylated product comigrated with glycerophosphoinositol-3-phosphate in high-performance liquid chromatography. Both sucrose gradient fractionation and the heat stability of PI kinase activity from cells infected with temperature-sensitive mutants suggest that the PI kinase activity is not intrinsic to p60v-src but is a property of another molecule complexed with p60v-src. All transforming variants of p60src were associated with PI kinase activity, whereas this enzyme activity was hardly detectable in immunoprecipitates from cells infected with nontransforming viruses encoding p60c-src or an enzymatically inactive variant. However, PI kinase activity was found in p60src immunoprecipitates from cells infected with nonmyristylated, nontransforming mutants as well as temperature-sensitive mutants at the nonpermissive temperature, which indicated that simple association of PI kinase activity with p60src is not sufficient for cell transformation.

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