Heterogeneity and microtubule interaction of the CHO1 antigen, a mitosis-specific kinesin-like protein. Analysis of subdomains expressed in insect sf9 cells

1994 ◽  
Vol 107 (12) ◽  
pp. 3485-3499 ◽  
Author(s):  
R. Kuriyama ◽  
S. Dragas-Granoic ◽  
T. Maekawa ◽  
A. Vassilev ◽  
A. Khodjakov ◽  
...  
Keyword(s):  
Cho Cells ◽  
Sequence Similarity ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sf9 Cells ◽  
Coding Region ◽  
Expression Library ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Calculated Molecular Mass

The CHO1 antigen is a mitosis-specific kinesin-like motor located at the interzonal region of the spindle. The human cDNA coding for the antigen contains a domain with sequence similarity to the motor domain of kinesin-like protein (Nislow et al., Nature 359, 543, 1992). Here we cloned cDNAs encoding the CHO1 antigen by immunoscreening of a CHO Uni-Zap expression library, the same species in which the original monoclonal antibody was raised. cDNAs of CHO cells encode a 953 amino acid polypeptide with a calculated molecular mass of 109 kDa. The N-terminal 73% of the antigen was 87% identical to the human clone, whereas the remaining 27% of the coding region showed only 48% homology. Insect Sf9 cells infected with baculovirus containing the full-length insert produced 105 and 95 kDa polypeptides, the same doublet identified as the original antigen in CHO cells. Truncated polypeptides corresponding to the N-terminal motor and C-terminal tail produced a 56 and 54 kDa polypeptide in Sf9 cells, respectively. Full and N-terminal proteins co-sedimented with, and caused bundling of, brain microtubules in vitro, whereas the C-terminal polypeptide did not. Cells expressing the N terminus formed one or more cytoplasmic processes. Immunofluorescence as well as electron microscopic observations revealed the presence of thick bundles of microtubules, which were closely packed, forming a marginal ring just beneath the cell membrane and a core in the processes. The diffusion coefficient and sedimentation coefficient were determined for the native CHO1 antigen by gel filtration and sucrose density gradient centrifugation, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of intrinsic dimer and overexpressed monomeric forms of gamma-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas gamma-tubulin sequence

1995 ◽  
Vol 108 (3) ◽  
pp. 1083-1092 ◽  
Author(s):  
A. Vassilev ◽  
M. Kimble ◽  
C.D. Silflow ◽  
M. LaVoie ◽  
R. Kuriyama
Keyword(s):  
Molecular Mass ◽  
Gradient Centrifugation ◽  
Expression System ◽  
Sedimentation Coefficient ◽  
Apparent Molecular Mass ◽  
Size Exclusion ◽  
Sf9 Cells ◽  
Dependent Manner ◽  
Monomeric Form ◽  
Gamma Tubulin

A new member of the tubulin superfamily, gamma-tubulin, is localized at microtubule-organizing centers (MTOCs) in a variety of organisms. Chlamydomonas cDNA coding for the full-length sequence of gamma-tubulin was expressed in insect ovarian Sf9 cells using the baculovirus expression system. Approximately half of the induced 52 kDa gamma-tubulin was recovered in the supernatant after centrifugation of Sf9 cell lysates at 18,000 g for 15 minutes. When the cell supernatant was analyzed by FPLC on a Superdex 200 sizing column, Chlamydomonas gamma-tubulin separated into two major peaks. The lagging peak contained a monomeric form of gamma-tubulin with a sedimentation coefficient of 2.5 S, which interacted with the Superdex column in a salt-dependent manner. The leading peak, with an apparent molecular mass of 900 kDa, corresponded to a molecular chaperonin complex, and TCP1 chaperonin released folded gamma-tubulin polypeptide from the complex in the presence of MgATP. The released gamma-tubulin monomers were capable of binding to microtubules in vitro and biochemical quantities of active monomers were further purified using a combination of size-exclusion and ion-exchange column chromatography. The endogenous Sf9 cell gamma-tubulin migrated faster than Chlamydomonas gamma-tubulin with an apparent molecular mass of 49 kDa on gels. Analyses on gel filtration and sucrose density gradient centrifugation showed that, while overexpressed Chlamydomonas gamma-tubulin was present in a monomeric form, endogenous gamma-tubulin from Sf9 and HeLa cells exists as a dimer. These results may suggest the possibility that gamma-tubulin could form a heterodimer with hitherto unknown molecule(s).

DEMONSTRATION OF A CYTOPLASMIC RECEPTOR PROTEIN FOR OESTROGEN IN THE CANINE PROSTATE GLAND

1978 ◽  
Vol 78 (1) ◽  
pp. 131-139 ◽  
Author(s):  
N. CHAISIRI ◽  
Y. VALOTAIRE ◽  
BRONWEN A. J. EVANS ◽  
C. G. PIERREPOINT
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Prostate Gland ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Receptor Protein ◽  
Receptor Complex ◽  
Binding Properties ◽  
Sucrose Density Gradient Centrifugation ◽  
A Value

A receptor protein that selectively binds oestrogens has been demonstrated in the cytosol of the canine prostate gland. The steroid–receptor complex was found to have a sedimentation coefficient of 4–5 S with respect to bovine serum albumin after sucrose density-gradient centrifugation. The high affinity and low capacity of the protein for oestrogens was indicated by displacement studies, which gave a value of 3·8 ± 1·53 (s.d.) × 10−10 mol/l for the dissociation constant. A metastasizing prostatic tumour was also shown to possess this receptor, with binding properties similar to those exhibited by the receptor in normal prostatic cytosol. The implications of these findings are discussed with regard to normal prostatic function in the dog and the virtually inevitable advent of prostatic hyperplasia with age in this species.

Über die Spaltung physikalisch intakter Poliovirus-Teilchen in Nucleinsäure und leere Proteinhüllen durch Wärmebehandlung

1965 ◽  
Vol 20 (9) ◽  
pp. 870-878 ◽  
Author(s):  
O. Drees ◽  
Ch. Borna
Keyword(s):  
Protein Concentration ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Virus Protein ◽  
Analytical Ultracentrifuge ◽  
Sucrose Density Gradient Centrifugation ◽  
Virus Particles ◽  
Phosphate Buffered Saline

Purified preparations of poliovirus devoid of contaminating nucleic acid and so-called “C antigen” released their particle-bound ribonucleic acid (RNA) almost quantitatively on heating at 40°C for 48 hours in phosphate-buffered saline (pH 7.6). This process occurred without disruption of the virus protein and left, in addition to the free RNA and traces of undegraded virus particles, empty protein shells in the reaction mixture.The liberated RNA sedimented in the analytical ultracentrifuge as a very diffuse boundary with sedimentation coefficients (s20, w) in the range from about 8 S to less than 1 S. The shell material which could be isolated by means of sucrose density-gradient centrifugation proved to be homogeneous in the analytical ultracentrifuge. Its sedimentation coefficient (s20,w) extrapolated to zero protein concentration was found to be 78 S.

Purification and some properties of the glutamate dehydrogenase of Salmonella typhimurium

1973 ◽  
Vol 19 (4) ◽  
pp. 427-438 ◽  
Author(s):  
J. W. Coulton ◽  
M. Kapoor
Keyword(s):  
Salmonella Typhimurium ◽  
Glutamate Dehydrogenase ◽  
Ammonium Sulfate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ammonium Sulfate Fractionation ◽  
Sulfate Fractionation

NADP-specific glutamate dehydrogenase (GDH) from Salmonella typhimurium was purified 190-fold by heat treatment, ammonium sulfate fractionation, DEAE-Sephadex chromatography, reverse ammonium sulfate fractionation, and gel filtration. The enzyme proved to be stable to 55 °C, and displayed a pH optimum at 8.6 in the amination reaction. The sedimentation coefficient of GDH, as determined by sucrose density gradient centrifugation, was about 10.3 S. From gel filtration chromatography, the molecular weight and Stokes' radius for the enzyme were estimated at 280 000 daltons and 54 × 10−8 cm, respectively. Unusual resistance was displayed by the enzyme to high concentrations of the protein denaturants, urea, SDS, and guanidine hydrochloride.

Isolation and characterization of a novel dynein that contains C and A heavy chains from sea urchin sperm flagellar axonemes

1994 ◽  
Vol 107 (2) ◽  
pp. 345-351 ◽  
Author(s):  
E. Yokota ◽  
I. Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Heavy Chain ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Heavy Chains ◽  
Isolation And Characterization ◽  
Sea Urchin Sperm

A novel dynein (C/A dynein), which is composed of C and A heavy chains, two intermediate chains and several light chains, was isolated from sea urchin sperm flagella. The C/A dynein was released by the treatment with 0.7 M NaCl plus 5 mM ATP from the axonemes depleted of outer arm 21 S dynein. Sedimentation coefficient of this dynein was estimated by sucrose density gradient centrifugation to be 22–23 S. The C/A dynein particle appeared to be composed of three distinct domains; two globular head domains and one rod domain as seen by negative staining electron microscopy. The mobility of ‘A’ heavy chain of C/A dynein on SDS-gel electrophoresis was similar to that of A heavy chains (A alpha and A beta) of 21 S dynein. However, UV-cleavage patterns of C and A heavy chains of C/A dynein were different from those of A heavy chains of 21 S dynein. Furthermore, an antiserum raised against A heavy chain of C/A dynein did not crossreact with A heavy chains of 21 S dynein. Under the conditions in which the C/A dynein was released, some of inner arms were removed concomitantly from axonemes as observed by electron microscopy. These results suggested that C/A dynein is a component of the inner arms.

Biochemical and Structural Changes in Mitochondria and Other Cellular Components of Pea Cotyledons During Germination

1972 ◽  
Vol 50 (7) ◽  
pp. 725-737 ◽  
Author(s):  
T. Solomos ◽  
S. S. Malhotra ◽  
S. Prasad ◽  
S. K. Malhotra ◽  
Mary Spencer
Keyword(s):  
Electron Microscopy ◽  
Structural Changes ◽  
Gradient Centrifugation ◽  
Microscopic Analysis ◽  
Sucrose Density Gradient ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Cellular Components ◽  
Further Development

Integrated studies comprising biochemical and electron microscopic analysis suggested that the increase in respiratory activity of pea cotyledon mitochondria during germination results from further development of the original mitochondria present in dormant seeds. Electron microscopy of isolated mitochondria as well as mitochondria in situ has revealed that membranes are scarce in the mitochondria present in dormant seeds. Mitochondrial cristae become well developed during the initial stages of germination. Crude mitochondrial preparations from pea cotyledons were fractionated by sucrose density gradient centrifugation and analyzed through electron microscopy. These studies showed that, at all stages of germination, "peroxisome"-like structures were present in the fractions of higher sucrose densities than that containing mitochondria. Biochemical studies revealed that the activities of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) and peroxidase (guaicol:H2O2 oxidoreductase, EC 1.11.1.7) were associated mainly with these fractions and their activities increased during germination.

Molecular characteristics of cytostatic factors in amphibian egg cytosols

Development ◽  
1989 ◽  
Vol 106 (4) ◽  
pp. 799-808
Author(s):  
E.K. Shibuya ◽  
Y. Masui
Keyword(s):  
Density Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Molecular Characteristics ◽  
Small Peptides ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytostatic Factor

In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated ‘cytostatic factor, (CSF)’. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca(2+)-containing cytosols, a stable CSF activity develops. Therefore, the first Ca(2+)-sensitive CSF and the second Ca(2+)-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2 h at 25 degrees C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20–30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2 × 10(6), and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11.5 or by 4M-NaCl and LiCl and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, alpha-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.

INTERMICROSOMAL DISTRIBUTION OF AROMATIZING ENZYME SYSTEM IN EQUINE TESTICULAR TISSUE

1973 ◽  
Vol 72 (2) ◽  
pp. 366-375 ◽  
Author(s):  
Ran Oh ◽  
Bun-ichi Tamaoki
Keyword(s):  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Enzyme System ◽  
Gradient Centrifugation ◽  
Electron Microscopic Examination ◽  
Sucrose Density Gradient ◽  
Testicular Tissue ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytochrome P 450

ABSTRACT The microsomal fraction (10 000–105 000 × g precipitate) of equine testes was fractionated into the smooth- and the rough-surfaced microsomal subfractions by a sucrose density-gradient centrifugation in the presence of CsCl. The validity of this fractionating procedure was confirmed by electron microscopic examination and also by chemical analysis of the RNA contents in these subfractions. The aromatizing enzyme system (19-hydroxylase and aromatase) which was concentrated in the microsomal fractions among the organellae was found to be localized in the smoothsurfaced microsomal fraction. The cytochrome P-450 which was also involved in the process of enzymatic aromatization was detected exclusively in the smooth-surfaced microsomal fraction. The distribution of the aromatizing system between the two microsomal subfractions of equine testes was discussed in comparison with that in human full term placentae.

scholarly journals On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin.

1981 ◽  
Vol 91 (1) ◽  
pp. 83-94 ◽  
Author(s):  
R Füssle ◽  
S Bhakdi ◽  
A Sziegoleit ◽  
J Tranum-Jensen ◽  
T Kranz ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gradient Centrifugation ◽  
Membrane Damage ◽  
Sedimentation Coefficient ◽  
Water Soluble ◽  
Effective Diameter ◽  
Gel Chromatography ◽  
Alpha Toxin ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X

Rabbit or human erythrocytes lysed with Staphylococcus aureus alpha-toxin were solubilized with Triton X-100, and the toxin was subsequently isolated by gel chromatography, sucrose density gradient centrifugation, and reincorporation into liposomes. In the presence of Triton X-100, the toxin exhibited a sedimentation coefficient of 11S and eluted at a position between those of IgG and alpha 2-macroglobulin in gel chromatography. A single polypeptide subunit of 34,000 mol wt was found in SDS PAGE. In the electron microscope, ring-shaped or cylindrical structures were observed, 8.5-10 nm in diameter, harboring central pits or channels 2-3 nm in diameter. An amphiphilic nature of these structures was evident from their capacity to bind lipid and detergent, aggregation in the absence of detergents, and low elutability from biological and artificial membranes through ionic manipulations. In contrast to the membrane-derived form of alpha-toxin, native toxin was a water-soluble, 34,000 mol wt, 3S molecule, devoid of an annular structure. Because studies on the release of radioactive markers from resealed erythrocyte ghosts indicated the presence of circumscribed lesions of approximately 3-nm effective diameter in toxin-treated membranes, the possibility is raised that native alpha-toxin oligomerizes on and in the membrane to form an amphiphilic annular complex that, through its partial embedment within the lipid bilayer, generates a discrete transmembrane channel.

scholarly journals Claudin-1 and -2: Novel Integral Membrane Proteins Localizing at Tight Junctions with No Sequence Similarity to Occludin

1998 ◽  
Vol 141 (7) ◽  
pp. 1539-1550 ◽  
Author(s):  
Mikio Furuse ◽  
Kohji Fujita ◽  
Takashi Hiiragi ◽  
Kazushi Fujimoto ◽  
Shoichiro Tsukita
Keyword(s):  
Membrane Proteins ◽  
Tight Junctions ◽  
Sequence Similarity ◽  
Gradient Centrifugation ◽  
Broad Band ◽  
Multiple Integral ◽  
Sucrose Density Gradient ◽  
Transmembrane Domains ◽  
Integral Membrane Proteins ◽  
Sucrose Density Gradient Centrifugation

Occludin is the only known integral membrane protein localizing at tight junctions (TJ), but recent targeted disruption analysis of the occludin gene indicated the existence of as yet unidentified integral membrane proteins in TJ. We therefore re-examined the isolated junction fraction from chicken liver, from which occludin was first identified. Among numerous components of this fraction, only a broad silver-stained band ∼22 kD was detected with the occludin band through 4 M guanidine-HCl extraction as well as sonication followed by stepwise sucrose density gradient centrifugation. Two distinct peptide sequences were obtained from the lower and upper halves of the broad band, and similarity searches of databases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 amino acids, respectively. Hydrophilicity analysis suggested that both bore four transmembrane domains, although they did not show any sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy revealed that both proteins tagged with FLAG or GFP were targeted to and incorporated into the TJ strand itself. We designated them as “claudin-1” and “claudin-2”, respectively. Although the precise structure/function relationship of the claudins to TJ still remains elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands.

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