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.

The outer membrane of Haemophilus influenzae type b: cell envelope associations of major proteins

1983 ◽  
Vol 29 (2) ◽  
pp. 280-287 ◽  
Author(s):  
J. W. Coulton ◽  
D. T. F. Wan
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Haemophilus Influenzae Type ◽  
Major Protein ◽  
Haemophilus Influenzae Type B ◽  
Type B ◽  
Molecular Weights ◽  
Triton X

Membrane proteins fom the cell envelope of Haemophilus influenzae type b ATCC 9795 were examined by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. When envelopes were extracted with a phosphate-based buffer containing 2% Triton X-100, a major protein of molecular weight 43 000 was detected in fractions containing cytoplasmic membrane proteins. The cell wall material which was Triton X-100 insoluble contained six major proteins of molecular weights 46 000, 40 000, 36 000, 30 000, 27 000, and 16 000. One of these proteins showed a shift in molecular weight from 27 000 to 36 000 when it was heated over a temperature range from 50 °C to 100 °C in buffer containing 2% sodium dodecyl sulphate, 5% 2-mercaptoethanol. This alteration in mobility could be demonstrated either by the membrane-bound form of the protein or by a detergent-soluble form of the protein. Enriched preparations of the 36 000 molecular weight form were obtained by a series of purification steps. Extraction of the Triton X-100 insoluble material with buffer containing 2% Triton X-100, 5.0 mM EDTA yielded chiefly one major protein molecular weight 30 000 and many minor protein species. Pretreatment of the Triton X-100 insoluble fraction with lysozyme followed by extraction with buffer containing 2% Triton X-100, 5.0 mM EDTA released two proteins of molecular weights 16 000 and 27 000 and few minor proteins. By these operational manipulations, the proteins of molecular weights 16 000 and 27 000 may be considered as peptidoglycan-associated proteins.

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 ISOLATION AND REACTIVATION OF THE AXOSTYLE

1973 ◽  
Vol 56 (1) ◽  
pp. 13-26 ◽  
Author(s):  
Mark S. Mooseker ◽  
Lewis G. Tilney
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Atpase Activity ◽  
Adenosine Triphosphate ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gels ◽  
Molecular Weights ◽  
Dodecyl Sulfate ◽  
Cilia And Flagella ◽  
Triton X

The contractile axostyle is a ribbon-shaped organelle present in certain species of flagellates found in the hindgut of wood eating insects. This organelle propagates an undulatory wave whose motion, like flagella and cilia, is related to microtubules. Unlike the axoneme of cilia and flagella, however, the axostyle is composed of singlet microtubules linked together in parallel rows. Axostyles were isolated from Cryptocercus gut protozoa with Triton X-100. Normal motility of the isolated axostyle could be restored with adenosine triphosphate (ATP); the specific conditions necessary for this reactivation were essentially identical with those reported for the reactivation of isolated flagella or whole sperm. ATPase activity of the isolated axostyle was comparable to the values reported for ciliary or flagellar axonemes. The axostyle was reasonably specific for ATP. Most of the proteins of the isolated axostyle comigrated with proteins of the ciliary axoneme on sodium dodecyl sulfate (SDS) polyacrylamide gels (i e. equivalent molecular weights). These included the following: the higher molecular weight component of dynein, tubulin, linkage protein (nexin), and various secondary proteins. Evidence for dynein in the axostyle is presented and a model proposed to explain how repeated propagated waves can be generated.

scholarly journals Effect of molecular size of 125I-labelled poly(vinylpyrrolidone) on its pinocytosis by rat visceral yolk sacs and rat peritoneal macrophages

1981 ◽  
Vol 196 (1) ◽  
pp. 49-55 ◽  
Author(s):  
R Duncan ◽  
M K Pratten ◽  
H C Cable ◽  
H Ringsdorf ◽  
J B Lloyd
Keyword(s):  
Molecular Weight ◽  
Molecular Size ◽  
Weight Fraction ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Yolk Sac ◽  
High Molecular Weight Fraction ◽  
Molecular Weights ◽  
Molecular Weight Distributions

Rates of pinocytosis of different molecular-weight distributions of 125I-labelled poly(vinylpyrrolidone) by rat visceral yolk sacs and rat peritoneal macrophages were measured in vitro. Four preparations of mean molecular weights 50 000, 84 000, 700 000 and 7 000 000, were used. Macrophages captured the highest-molecular-weight preparation more rapidly than the other preparations. In contrast, rate of capture by the yolk sac decreased with increasing molecular weight. Incubations with a very-high-molecular-weight fraction derived from the 7 000 000-average-mol. wt. preparation clearly demonstrated that very large polymer molecules are not accumulated by the yolk sac, but are preferentially captured by macrophages. Analysis of the 125I-labelled poly(vinylpyrrolidone) internalized by the two cell types confirmed that low-molecular-weight material is preferred by the yolk sac, whereas the macrophage is less discriminating.

scholarly journals Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions.

1987 ◽  
Vol 104 (3) ◽  
pp. 537-546 ◽  
Author(s):  
N Carlson ◽  
M Rechsteiner
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Size Distribution ◽  
High Molecular Weight ◽  
Hela Cells ◽  
Insoluble Fraction ◽  
Nuclear Fraction ◽  
Molecular Weights ◽  
Differential Sedimentation ◽  
Triton X

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.

Is High-Molecular-Weight-Renin Binding of Renin to the Protease Inhibitors and Lipoproteins?

1978 ◽  
Vol 55 (s4) ◽  
pp. 125s-128s
Author(s):  
K. Poulsen ◽  
A. H. Nielsen ◽  
S. Lykkegaard ◽  
C. Malling ◽  
J. Krøll ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Protease Inhibitors ◽  
Plasma Proteins ◽  
Tertiary Structure ◽  
Limited Proteolysis ◽  
Submaxillary Gland ◽  
Enzymic Activity ◽  
Molecular Weights ◽  
Α2 Macroglobulin

1. Two high-molecular-weight forms of renin (molecular weights 800 000 and 70 000) are present in mouse plasma. 2. The 800 000 form could be activated and converted into the fully active 40 000 form, by acid or limited proteolysis. The 70 000 form was activated without change in molecular weight. 3. In addition to its enzymic activity, renin was measured by a direct radioimmunoassay, which revealed that the current acid treatment of plasma did not activate all the renin present. 4. Renin is stored as fully active 40 000 renin, with a specific enzymic reactivity of 0·4 × 10−3 GU ng−1, in the submaxillary gland of mice. 5. Pure 125I-labelled 40 000 submaxillary renin did not bind to plasma proteins. However, by changing the tertiary structure of renin, it was bound to some of the plasma protease inhibitors; α2-macroglobulin, inter-α-trypsin inhibitor and α2-antithrombin. It was also bound to α1- and β1-lipoprotein, albumin and an unidentified plasma protein. No binding was seen to more than 50 other studied plasma proteins.

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 RABBIT MACROPHAGE INTERFERONS

1967 ◽  
Vol 125 (4) ◽  
pp. 579-593 ◽  
Author(s):  
Thomas J. Smith ◽  
Robert R. Wagner
Keyword(s):  
Molecular Weight ◽  
Peritoneal Macrophages ◽  
Biologically Active ◽  
Rabbit Kidney ◽  
Rabbit Serum ◽  
Molecular Component ◽  
Molecular Weights ◽  
Marker Proteins ◽  
Approximate Molecular Weight ◽  
Interferon Activity

Antiviral factors present in cultures of rabbit peritoneal macrophages or rabbit kidney (RK) cells infected with Newcastle disease virus (NDV) and those in cultures of uninfected macrophages all fulfilled the biological and physicochemical criteria for classification as interferons. Virus-induced macrophage and RK interferons were slightly more stable to heat or acid than "spontaneously produced" or endotoxin-induced macrophage interferon. Interferon activity in serum of NDV-infected rabbits was decidedly more labile than NDV-induced macrophage interferon. However, these differences in lability were too slight to serve as a useful basis for distinguishing one rabbit interferon from another. Rabbit interferons from various sources could be differentiated by filtration through Sephadex G-100 and their molecular weights estimated by comparison with elution profiles of a series of marker proteins of known molecular weight. Each of four different preparations of rabbit interferons was found to contain more than one molecular component. Elution peaks for three NDV-induced interferons were equivalent to the following molecular weights: RK ≃44,000–45,000 and > 134,000 (variable and < 1% when present); macrophage ≃37,000, 44,000–45,000, and > 134,000 (variable and <1% when present); and serum ≃50,000–52,000 and > 134,000 (∼10% and heat labile). NDV-induced serum interferon may also contain another molecular component of mol wt ≃45,000 represented by a trailing shoulder from the major 51,000 mol wt peak. Endotoxin-induced macrophage interferon proved to be polydisperse. Sephadex filtration of this interferon did not reveal clear and consistent elution patterns, partially owing to its low initial titer and lability. However, variable peaks of biological activity could be detected in Sephadex fractions equivalent to approximate molecular weight values of > 134,000, 72,000–78,000, 33,000–38,000, 28,000–30,000, and possibly a component of 42,000–45,000. A major component of mol wt ≃37,000 was present in all samples of endotoxin-induced macrophage interferon. The other constituents may be biologically active subunits or polymers. These data indicate that rabbit macrophages produce two primary kinds of interferon: (a) an RK-like component of mol wt ≃45,000 that is synthesized in greatest amount after viral induction, and (b) a different species of mol wt ≃ 37,000 that can also be synthesized in the absence of viral induction. The presence of major interferon constituents of mol wt ≃51,000 and > 134,000 in rabbit serum after viral induction suggests that macrophages are not the principal interferon-producing cells that respond to intravenous injection of NDV.

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