scholarly journals Association of methionyl-tRNA synthetase with detergent-insoluble components of the rough endoplasmic reticulum.

1983 ◽  
Vol 96 (4) ◽  
pp. 1138-1147 ◽  
Author(s):  
C V Dang ◽  
D C Yang ◽  
T D Pollard
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Antibody ◽  
Insoluble Fraction ◽  
Trna Synthetase ◽  
Insoluble Residue ◽  
Synthetase Activity ◽  
Fluorochrome Staining ◽  
Antibody Staining ◽  
Triton X ◽  
Methionyl Trna Synthetase

Using fluorescent antibody staining, we have established the association of methionyl-tRNA synthetase with the endoplasmic reticulum in PtK2 cells. After Triton X-100 extraction, 70% of the recovered aminoacyl-tRNA synthetase activity was found in the detergent-insoluble fraction. This fraction of the enzyme remained localized with insoluble endoplasmic reticulum antigens and with ribosomes, which were stained with acridine orange. By both fluorescence microscopy and electron microscopy the organization of the detergent-insoluble residue was found to depend on the composition of the extracting solution. After extraction with a microtubule-stabilizing buffer containing EGTA, Triton X-100, and polyethylene glycol (Osburn, M., and K. Weber, 1977, Cell, 12:561-571) the ribosomes were aggregated in large clusters with remnants of membranes. After extraction with a buffer containing Triton X-100, sucrose, and CaCl2 (Fulton, A. B., K. M. Wang, and S. Penman, 1980, Cell, 20:849-857), the ribosomes were in small clusters and there were few morphologically recognizable membranes. In both cases the methionyl-tRNA synthetase and some endoplasmic reticulum antigens retained approximately their normal distribution in the cell. Double fluorochrome staining showed no morphological association of methionyl-tRNA synthetase with the microtubule, actin, or cytokeratin fiber systems of PtK2 cells. These observations demonstrate that detergent-insoluble cellular components, sometimes referred to as "cytoskeletal" preparations, contain significant amounts of nonfilamentous material including ribosomes, and membrane residue. Caution is required in speculating about intermolecular associations in such a complex cell fraction.

scholarly journals Mutations in MARS identified in a specific type of pulmonary alveolar proteinosis alter methionyl‐tRNA synthetase activity

FEBS Journal ◽  
2018 ◽  
Vol 285 (14) ◽  
pp. 2654-2661 ◽  
Author(s):  
Martine Comisso ◽  
Alice Hadchouel ◽  
Jacques Blic ◽  
Marc Mirande
Keyword(s):  
Pulmonary Alveolar Proteinosis ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Alveolar Proteinosis ◽  
Methionyl Trna Synthetase

Increased transglutaminase in the aortas of cholesterol-fed rabbits: occurrence of buffer soluble and insoluble forms and an inhibitor

1991 ◽  
Vol 69 (12) ◽  
pp. 821-827 ◽  
Author(s):  
Rick I. Wiebe ◽  
Alan H. Tarr ◽  
J. Michael Bowness
Keyword(s):  
Soluble Fraction ◽  
Tissue Transglutaminase ◽  
Insoluble Fraction ◽  
Peanut Oil ◽  
Insoluble Residue ◽  
Transglutaminase Activity ◽  
Atherosclerotic Lesions ◽  
Triton X ◽  
Two Peaks

Rabbits were fed for 10–12 weeks on a normal pellet diet or on the same diet containing 1% cholesterol and 6% peanut oil. The animals were killed and the aortas divided into three layers which were homogenized and extracted. The extracts and the insoluble residues were assayed for transglutaminase activity and tissue transglutaminase antigen. When compared with normal aortas, the inner and middle layers of aortas with atherosclerotic lesions from cholesterol-fed rabbits showed higher transglutaminase activities in the buffer-soluble fraction without a corresponding increase in antigen. The buffer extracts showed two peaks (I and II) of activity and antigen on DE 52 chromatography; peak I was also found, together with lipid, in Triton X-100 extracts of the buffer-insoluble residue. The Triton X-100 insoluble fraction showed higher concentrations of both activity and antigen in the inner and middle layers of atherosclerotic aortas than in normal aortas, but the activity per nanogram of antigen was lower than in the buffer-soluble fraction. The activity in this insoluble residue was largely extracted, together with an inhibitor, by an NaCl – sucrose – dithiothreitol – Triton X-100 solution. DE 52 chromatography of this extract showed a third peak of activity and antigen (peak III) and an inhibitor peak that was distinct from the activity peaks.Key words: aorta, transglutaminase, inhibitor, cholesterol, atherosclerosis.

Calcium-induced desmosome formation in cultured kidney epithelial cells

1986 ◽  
Vol 85 (1) ◽  
pp. 95-111
Author(s):  
D.L. Mattey ◽  
D.R. Garrod
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Fluorescent Antibody ◽  
Cell Types ◽  
Cell Periphery ◽  
Cell Type ◽  
Antibody Staining ◽  
Mdbk Cells ◽  
Triton X ◽  
Do So

Previous work has shown that cultured keratinocytes do not form desmosomes at low [Ca2+] (less than 0.1 mM) but may be induced to do so by raising [Ca2+] to physiological levels (1.8-2 mM). Here, fluorescent antibody staining with specific anti-desmosomal antibodies and electron microscopy have been used to determine whether Ca2+-induced desmosome formation also occurs in simple epithelial cells. Both Madin-Darby canine and bovine kidney cells (MDCK and MDBK) exhibit Ca2+-induced desmosome formation, but there are significant differences between them. MDCK cells resemble keratinocytes in showing rapid desmosome formation characterized by the simultaneous appearance of four desmosomal antigens at the cell periphery within 15–20 min of raising the [Ca2+]. In contrast MDBK cells take between 7 and 8 h to form desmosomes after Ca2+ switching, and this is characterized by slow appearance of two desmosomal antigens, the 175–164(X 10(3)) Mr glycoprotein and desmoplakin, at the cell periphery. Differences in the pattern of staining for desmosomal antigens between the two cell types in low and high [Ca2+] are described and discussed in relation to desmosome formation and internalization. Triton X-100 extractability of desmosomal antigen staining is also considered. While most is non-extractable, staining for the glycoproteins known as desmocollins is completely extractable from MDCK cells in low [Ca2+], but that which reaches the cell periphery after Ca2+ switching becomes non-extractable. Although neither cell type forms desmosomes in low [Ca2+], both possess zonulae adhaerentes, suggesting a difference in Ca2+ requirement for formation of these two junctions.

scholarly journals Messenger RNA in the cytoskeletal framework: analysis by in situ hybridization.

1982 ◽  
Vol 95 (1) ◽  
pp. 1-7 ◽  
Author(s):  
W R Jeffery
Keyword(s):  
In Situ Hybridization ◽  
Messenger Rna ◽  
Significant Proportion ◽  
Insoluble Fraction ◽  
Radioactive Isotopes ◽  
Follicle Cells ◽  
Insoluble Residue ◽  
Framework Analysis ◽  
Triton X

The possibility of an association of mRNA with the cytoskeletal framework (CF) of ascidian (Styela plicata) follicle cells was examined in this study. The approach was to extract the follicle cells with Triton X-100 and determine whether mRNA persisted in the insoluble residue by two methods, in situ hybridization with poly(U) and actin DNA probes and the incorporation of radioactive isotopes into RNA. Triton X-100 extraction of follicle cells yielded a filamentous CF containing approximately 70% of the total poly (A) but only 9% of the total lipid, 23% of the total protein, and 28% of the total RNA. In situ hybridization with a poly (U) probe indicated that approximately 70% of the poly (A) was associated with the CF. In situ hybridization with a cloned actin DNA probe indicated that approximately 60% of the actin mRNA was associated with the CF. Autoradiography of detergent-extracted follicle cells, which had been labeled with [3H]uridine or [3H]adenosine, indicated that greater than 90% of the newly synthesized poly (A)+RNA was preserved in the CF. Thus more newly synthesized mRNA than steady-state mRNA may be present in the Triton X-100 insoluble fraction. It is concluded that a significant proportion of the mRNA complement of ascidian follicle cells is associated with the CF.

A Most Probable Number Method for the Enumeration of Legionella Bacteria in Water

1991 ◽  
Vol 24 (2) ◽  
pp. 143-147 ◽  
Author(s):  
N. A. Grabow ◽  
R. Kfir ◽  
W. O. K. Grabow
Keyword(s):  
Water Samples ◽  
Membrane Filtration ◽  
Quantitative Method ◽  
Fluorescent Antibody ◽  
Most Probable Number ◽  
Probable Number ◽  
Comparative Tests ◽  
Antibody Staining ◽  
Direct Fluorescent Antibody ◽  
Yeast Extract Agar

A new quantitative method for the enumeration of Legionella bacteria in water is described. Appropriate tenfold serial dilutions of water samples concentrated by membrane filtration are plated in triplicate on buffered charcoal yeast extract agar. After incubation for 3 days representative smears from individual plates are tested for the presence of Legionella by direct fluorescent antibody staining. The number of positive plates in each dilution is used to calculate the Legionella count by means of conventional most probable number statistics. In comparative tests on a variety of water samples this method yielded significantly higher counts than previously used procedures.

scholarly journals A MUTANT OF YEAST WITH A DEFECTIVE METHIONYL-tRNA SYNTHETASE

Genetics ◽  
1969 ◽  
Vol 61 (3) ◽  
pp. 557-566
Author(s):  
Calvin S McLaughlin ◽  
Leland H Hartwell
Keyword(s):  
Trna Synthetase ◽  
Methionyl Trna Synthetase
Sign in / Sign up

Export Citation Format

Share Document