scholarly journals Use of immobilized lactoperoxidase to label L cell proteins involved in adhesion to polystyrene.

1980 ◽  
Vol 85 (2) ◽  
pp. 402-413 ◽  
Author(s):  
N W Chin ◽  
K W Lanks
Keyword(s):  
Molecular Weight ◽  
Tissue Culture ◽  
Broad Spectrum ◽  
Peptide Mapping ◽  
Limited Proteolysis ◽  
Functional Association ◽  
One Dimensional ◽  
L Cells ◽  
L Cell ◽  
10 Nm Filaments

Proteins involved in the attachment of murine L cells to polystyrene have been identified by a technique designed to iodinate only those macromolecules coming into closet apposition to the substratum. Whereas soluble lactoperoxidase (LPO) catalyzes the radioiodination of a broad spectrum of polypeptides, the same enzyme immobilized on polystyrene tissue culture flasks discriminately labels 55,000 and 42,000 mol wt polypeptides that adhere tightly to the substratum after the cells are removed. One-dimensional peptide mapping following limited proteolysis showed that the labeled 55,000 mol wt polypeptide is similar to a component of comparable molecular weight present in the detergent-extracted cytoskeleton. The functional association of two cytoskeletal structures, presumably 10-nm filaments and actin, is discussed, and alternative explanations for their susceptibility to iodination by immobilized LPO are presented.

scholarly journals Identification of cytoskeletal components involved in attachment of L929 cells and macrophages to polystyrene.

1981 ◽  
Vol 89 (3) ◽  
pp. 691-694 ◽  
Author(s):  
K W Lanks ◽  
N W Chin
Keyword(s):  
Molecular Weight ◽  
Peptide Mapping ◽  
Limited Proteolysis ◽  
Detailed Examination ◽  
Peritoneal Macrophages ◽  
Intimate Contact ◽  
L929 Cells ◽  
Molecular Weights ◽  
Murine Peritoneal Macrophages ◽  
Triton X

We have previously shown that lactoperoxidase (LPO) covalently coupled to polystyrene tissue culture flasks can be used to radioiodinate monolayer cell proteins that come into intimate contact with the LPO-polystyrene surface. These studies have now been extended to include a detailed examination of the class of iodinated polypeptides migrating with apparent molecular weights of 50,000 and 55,000 in SDS polyacrylamide gels. Whereas in cultured L929 cells the 55,000 band is predominantly iodinated, in thioglycollate-activated murine peritoneal macrophages the 55,000 and 50,000 bands are of equal intensity. It is possible that the marked degree of exposure of the 50,000 mol wt polypeptide to immobilized LPO is related to the unique strength of macrophages attachment. After labeling of both L929 cells and macrophages with immobilized LPO, all polypeptides in this molecular weight region were subjected to peptide mapping by simultaneous limited proteolysis and electrophoresis in a second SDS polyacrylamide slab gel. The results clearly show that the two major polypeptides in this region are identical within the limits of resolution of this technique. The 55,000 mol wt polypeptide can also be identified in Triton X-100 cytoskeletons from L929 cells after labeling with soluble LPO either before or after detergent lysis. We conclude that this cell surface polypeptide is in continuity with the cytoskeleton and is preferentially exposed to the substratum during attachment to polystyrene.

Limited Proteolysis of the Purified Aα Chain of Human Fibrinosen by Plasmin

1975 ◽  
Author(s):  
L. Williams ◽  
G. Murano
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Peptide Mapping ◽  
Proteolytic Enzymes ◽  
Degradation Products ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Terminal Region ◽  
Low Molecular Weight ◽  
Human Fibrinogen

Based on evidence that a portion of circulating fibrinogen consists of a family of catabolic intermediates formed by proteolytic degradation of the COOH terminal region of Aα chains, we attempted to obtain early degradation products using the purified alkylated Aα chain derivative of human fibrinogen as the substrate and plasmin as the enzyme. Having established optimal conditions, a preparative quantity of material was digested in 0.1 M tris buffer pH = 9.5; time = 4 min; E/S ratio = 1/75 (mole/mole); temp = 37° C. Low molecular weight fragments were separated from the larger species, and further purified by gel filtration on Sephadex G-100. Selected early fragments were analyzed by polycrylamide gel electrophoresis, amino acid composition, peptide mapping and partial N-terminal amino acid sequence. Two of the earliest low molecular weight fragments released by plasmin were derived from the N-terminal region of the Aα chain. Their molecular size was estimated at about 10,000 daltons. One fragment contains fibrinopeptide A; both fragments extend beyond Met-51. Our data indicate that: a) the specificity of plasmin on the purified Aα chain differs from that on intact fibrinogen; or b) proteolytic enzymes other than or in addition to plasmin are responsible for the formation of early catabolic fibrinogen intermediates having a degraded Aα chain.(Supported by USPHS N. I. H. Grant HL 14142.)

scholarly journals Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells.

1978 ◽  
Vol 78 (1) ◽  
pp. 93-109 ◽  
Author(s):  
J M Starger ◽  
W E Brown ◽  
A E Goldman ◽  
R D Goldman
Keyword(s):  
Amino Acid ◽  
Peptide Mapping ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Potassium Phosphate ◽  
Baby Hamster Kidney ◽  
10 Nm Filaments ◽  
Filament Protein ◽  
Triton X ◽  
Phosphate Buffered Saline

Juxtanuclear birefringent caps (FC) containing 10-nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KCl, 1% Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10-nm filaments that is seen in situ. Up to 90% of the FC protein is resolved as two polypeptides of approximately 54,000 and 55,000 molecular weight on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels, and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10-nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10-nm filaments has been achieved by treatment of FC with 6 mM sodium-potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10-nm filaments upon the addition of 0.171 M sodium chloride.

scholarly journals Characterization of calcium-dependent membrane binding proteins of brain cortex

1985 ◽  
Vol 229 (3) ◽  
pp. 587-593 ◽  
Author(s):  
A R Rhoads ◽  
M Lulla ◽  
P B Moore ◽  
C E Jackson
Keyword(s):  
Brain Cortex ◽  
Peptide Mapping ◽  
Bovine Brain ◽  
Particulate Fraction ◽  
Structural Homology ◽  
One Dimensional ◽  
Calcium Dependent ◽  
Stokes Radius ◽  
The Brain

Proteins of Mr 68 000, 34 000 and 32 000 were selectively extracted by EGTA from brain cortex. The three proteins that were extracted along with calmodulin were acidic, monomeric, and did not exhibit structural homology, as demonstrated by one-dimensional peptide mapping. The Mr-68 000 protein was purified to homogeneity and had a Stokes radius of 3.54 nm and S20,W value of 5.1S. Purified calmodulin, Mr-68 000 protein and two proteins of Mr 34 000 and Mr 32 000, interacted with the brain particulate fraction, with half-maximal binding occurring at 3.5 microM, 8.3 microM and 150 microM-Ca2+ respectively. Proteins were bound independently of each other and calmodulin. Pretreatment of the particulate fraction with trypsin prevented the Ca2+-dependent binding of calmodulin; however, the binding of the Mr-68 000 protein or the Mr−32 000 and −34 000 proteins was unaffected. The Mr-68 000 protein of bovine brain did not cross-react immunologically with Mr-67 000 calcimedin from chicken gizzard.

scholarly journals Identification of two intermediates during processing of profilaggrin to filaggrin in neonatal mouse epidermis.

1984 ◽  
Vol 99 (4) ◽  
pp. 1372-1378 ◽  
Author(s):  
K A Resing ◽  
K A Walsh ◽  
B A Dale
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Tandem Repeats ◽  
Peptide Mapping ◽  
Two Dimensional ◽  
Major Event ◽  
Mouse Epidermis ◽  
Cell Stage ◽  
Processing Steps

A major event in the keratinization of epidermis is the production of the histidine-rich protein filaggrin (26,000 mol wt) from its high molecular weight (greater than 350,000) phosphorylated precursor (profilaggrin). We have identified two nonphosphorylated intermediates (60,000 and 90,000 mol wt) in NaSCN extracts of epidermis from C57/Bl6 mice by in vivo pulse-chase studies. Results of peptide mapping using a two-dimensional technique suggest that these intermediates consist of either two or three copies of filaggrin domains. Each of the intermediates has been purified. The ratios of amino acids in the purified components are unusual and essentially identical. The data are discussed in terms of a precursor containing tandem repeats of similar domains. In vivo pulse-chase experiments demonstrate that the processing of the high molecular weight phosphorylated precursor involves dephosphorylation and proteolytic steps through three-domain and two-domain intermediates to filaggrin. These processing steps appear to occur as the cell goes through the transition cell stage to form a cornified cell.

Generation and Maintenance of Immune Interferon-Producing Cells Induced by Allogeneic Stimulation in Mice

1980 ◽  
Vol 29 (2) ◽  
pp. 383-389
Author(s):  
Yasuhiko Ito ◽  
Hiizu Aoki ◽  
Yoshinobu Kimura ◽  
Michiko Takano ◽  
Koichiro Maeno ◽  
...  
Keyword(s):  
Tissue Distribution ◽  
Culture Fluid ◽  
Suppressive Effect ◽  
Interferon Production ◽  
Spleen Cells ◽  
Immune Interferon ◽  
L Cells ◽  
L Cell ◽  
Interferon Activity ◽  
Allogeneic Stimulation

When spleen cells derived from C57BL/6 mice immunized with L cells 7 days previously were cocultured with antigenic cells, immune interferon appeared in the culture fluid. We analyzed the tissue distribution of the immune interferon-producing cells (IIPC) which appeared in various lymphoid organs after allogeneic stimulation. Although fluid from cocultures of L-cell-sensitized thymocytes and L-cells could not detect interferon activity consistently, small numbers of IIPC could be detected by using the enumeration method of IIPC. The generation, maintenance, and nature of IIPC emerging in the spleen were different depending on how the host mice were immunized. Multiple antigenic stimulations were more effective and induced longer-lasting immune interferon production than a single stimulation. IIPC induced by a single stimulation appeared to be sensitive to cortisone, vinblastine, and cyclophosphamide and were relatively short lived. In contrast, IIPC induced by multiple stimulations seemed to be partially resistant to these drugs and long lived. When mice were immunized with intact L-cells, carrageenan, a known antimacrophage agent, had no effect on immune interferon production. However, when mice were immunized with solubilized L-cell antigen, this drug displayed a suppressive effect on immune interferon production.

scholarly journals The photocarcinogenicity of anthracence: photochemical binding to deoxyribonucleic acid in tissue culture

1975 ◽  
Vol 149 (1) ◽  
pp. 289-291 ◽  
Author(s):  
G M Blackburn ◽  
P E Taussig
Keyword(s):  
Molecular Weight ◽  
Tissue Culture ◽  
High Molecular Weight ◽  
Deoxyribonucleic Acid ◽  
Radiation Doses ◽  
Mammalian Tissue ◽  
Hydrocarbon Molecule ◽  
High Radiation ◽  
High Molecular Weight Dna ◽  
Low Radiation Doses

Anthracene becomes covalently bound to high-molecular-weight DNA in mammalian tissue culture as a result of irradiation at 365 nm after the incubation of cells with the hydrocarbon. At high radiation doses, the extent of binding exceeds one hydrocarbon molecule per 103 bases, and is lethal. At low radiation doses, much decreased binding is observed, but a majority of cells remain viable and can be recultured.

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