Search results for "Isozyme"
showing 10 items of 102 documents
Differential subcellular localization of endogenous and transfected soluble epoxide hydrolase in mammalian cells: evidence for isozyme variants
1999
AbstractEndogenous, constitutive soluble epoxide hydrolase in mice 3T3 cells was localized via immunofluorescence microscopy exclusively in peroxisomes, whereas transiently expressed mouse soluble epoxide hydrolase (from clofibrate-treated liver) accumulated only in the cytosol of 3T3 and HeLa cells. When the C-terminal Ile of mouse soluble epoxide hydrolase was mutated to generate a prototypic putative type 1 PTS (-SKI to -SKL), the enzyme targeted to peroxisomes. The possibility that soluble epoxide hydrolase-SKI was sorted slowly to peroxiosmes from the cytosol was examined by stably expressing rat soluble epoxide hydrolase-SKI appended to the green fluorescent protein. Green fluorescent…
[26] Isoforms of nitric-oxide synthase: Purification and regulation
1994
Publisher Summary Nitric-oxide synthase (NOS) catalyzes the five-electron oxidation of L-arginine to the nitric oxide radical (.NO) and L-citrulline. Molecular oxygen is the cosubstrate of the enzyme. NO synthase activity has been found in a large variety of cells and tissues. The enzyme exists in several isoforms, three of which have been purified, characterized, and cloned. The activities of all three isoforms are found distributed between the soluble and particulate fractions of cells. Isoform I (from brain) and isoform II (from cytokine-induced macrophages) are mostly soluble proteins. Isoform III from endothelial cells is myristoylated and found predominantly in the particulate fractio…
Molecular size and net charge of pathogenesis-related enzymes from barley (Hordeum vulgare L., v. Karat) infected withDrechslera teres f. teres (Sacc…
1998
Molecular size and net charge of isoforms of pathogenesis-related (PR) chitinase, beta-1,3-glucanase and peroxidase were studied in uninfected barley (Hordeum vulgare L., v. Karat) leaves and in barley leaves infected with the pathogenic fungus Drechslera teres f. teres (Sacch.) Shoem. Molecular characteristics were determined by time-dependent polyacrylamide gradient gel electrophoresis under native conditions and by applying an extended version of the computer program MOL-MASS (Rothe, G. M., Weidmann, H., Electrophoresis 1991, 12, 703-709). Uninfected barley leaves contained predominantly one peroxidase isozyme but also three very weak peroxidases. Activities of all of these three peroxid…
Determination of molecular weights and Stokes' radii of non-denatured proteins by polyacrylamide gradient gel electrophoresis. 2. Determination of th…
1982
Under certain conditions in polyacrylamide gradient gel electrophoresis (PAGGE), a linear correlation between the logarithm of the size of calibration proteins (log MW or log Rs) and the square root of their migration distance (√D) can be observed; slope and intercept of the calibration curve depend on the duration of electrophoresis; linearity, however, is maintained over a wide range (4-60 h, 200 V) (Rothe and Purkhanbaba, Electrophoresis 1982, 3, 33–42.) Using this method the reaction of plant isozyme systems penetrating a linear polyacrylamide (PAA) gradient gel was investigated: lactate dehydrogenase (LDH) from potato tubers behaves similarly to animal calibration proteins. The enzyme …
Determination of molecular weights and Stokes' radii of non-denatured proteins by polyacrylamide gradient gel electrophoresis 3. Estimation of the up…
1985
Studying the separation behavior of various native carbonic anhydrase isozymes from mammalian erythrocytes we found that the migration of these enzymes differs from that of the marker proteins commonly used in gradient gel electrophoresis. In alkaline buffer systems the enzymes from human, bovine, rabbit, and canine erythrocytes start to migrate with a size apparently 6 to 12 times larger than their monomeric size, then gradually lose in apparent size and finally end up in a size equivalent to their monomeric mol mass. We determined the monomeric mol mass of the various carbonic anhydrase forms to be 23 000 to 39 000 (g/mol). These values are in accordance with different data in the literat…
Homologies Between Different Forms of 2-5A Synthetases
1994
(2′-5′) Oligoadenylate synthetases (2-5A synthetases; EC 2.7.7.19) are present in mammalian cells and tissues and synthesize from ATP a series of oligomers termed 2-5A [general formula: ppp(A2′p)nA; with 1 ≤ n < 18 and usually 1 ≤ n < 6] (Hovanessian 1991). For full enzymic activity of the 2-5A synthetases, binding of double-stranded RNA is required (Sen 1982). Three principal 2-5A synthetase isoenzymes have been described with Mr’s of 40–46, 69, and 100 kDa (Chebath et al. 1987; Hovanessian et al. 1987, 1988). In the following they are classified as 2-5A synthetase I [Mr 40–46 000], II [Mr 69 000] and III [Mr 100 000]. All three isoforms are induced in cells by interferon (Cohen et al. 198…
2′,5′-oligoadenylate synthetase from a lower invertebrate, the marine sponge Geodia cydonium, does not need dsRNA for its enzymatic activity
2002
AbstractRecently, the presence of 2′,5′-linked oligoadenylates and a high 2′,5′-oligoadenylate synthetase activity were discovered in a lower invertebrate, the marine sponge Geodia cydonium. It has been demonstrated that mammalian 2–5A synthetase isozymes require a dsRNA cofactor for their enzymatic activity. Our results show that, unlike mammalian 2–5A synthetases, the 2–5A synthetase from the sponge acts in a dsRNA-independent manner in vitro. A prolonged incubation of the G. cydonium extract with a high concentration of a micrococcal nuclease had no effect on the activity of the 2–5A synthetase. At the same time, the micrococcal nuclease was effective within 30 min in degrading dsRNA nee…
Semi-automatic quantitative RT-PCR to measure CYP induction by drugs in human hepatocytes
2003
An assay has been developed for the quantitative measurement of CYP mRNA content of the major human isoforms (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) in human hepatocytes. The method is based on the conversion of mRNAs into their corresponding cDNAs, followed by PCR amplification using appropriate primers. Making use of appropriate internal and external standards it is possible to estimate changes in CYP mRNA content of hepatocytes. The technique has been standardised to run semi-automatically. This procedure can be used to assess the CYP induction potential of new pharmaceuticals at a pre-clinical stage of development. To this aim, human hepatocytes obtained from functional l…
Efficient Expression and Crystallization System of Cancer-Associated Carbonic Anhydrase Isoform IX.
2015
Human carbonic anhydrase IX (CA IX) is overexpressed in a number of solid tumors and is considered to be a marker for cellular hypoxia that it is not produced in most normal tissues. CA IX contributes to the acidification of the extracellular matrix, which, in turn, favors tumor growth and metastasis. Therefore, CA IX is considered to be a promising anti-cancer drug target. However, the ability to specifically target CA IX is challenging due to the fact that the human genome encodes 15 different carbonic anhydrase isoforms that have a high degree of homology. Furthermore, structure-based drug design of CA IX inhibitors so far has been largely unsuccessful due to technical difficulties regar…
Nitric Oxide: Biological Synthesis and Functions
2012
The pluripotent gaseous messenger molecule nitric oxide (NO) controls vital functions such as neurotransmission or vascular tone (via activation of soluble guanylyl cyclase), gene transcription, mRNA translation (via iron-responsive elements), and post-translational modifications of proteins (via ADP-ribosylation). In higher concentrations, NO is capable of destroying parasites and tumor cells by inhibiting iron-containing enzymes or directly interacting with the DNA of these cells. In view of this multitude of functions of NO, it is important to understand the mechanisms by which cells accomplish and regulate the production of this molecule. In mammals, three isozymes of NO synthase (NOS; …