Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Alpha-actinin can be proteolytically cleaved into major fragments of 27 and 53 kD using the enzyme thermolysin. The 27-kD fragment contains an actin-binding site and we have recently shown that the 53-kD fragment binds to the cytoplasmic domain of beta 1 integrin in vitro (Otey, C. A., F. M. Pavalko, and K. Burridge. 1990. J. Cell Biol. 111:721-729). We have explored the behavior of the isolated 27- and 53-kD fragments of alpha-actinin after their microinjection into living cells. Consistent with its containing a binding site for actin, the 27-kD fragment was detected along stress fibers within 10-20 min after injection into rat embryo fibroblasts (REF-52). The 53-kD fragment of alpha-actinin, however, concentrated in focal adhesions of REF-52 cells 10-20 min after injection. The association of this fragment with focal adhesions in vivo is consistent with its interaction in vitro with the cytoplasmic domain of the beta 1 subunit of integrin, which was also localized at these sites. When cells were injected with greater than 5 microM final concentration of either alpha-actinin fragment and cultured for 30-60 min, most stress fibers were disassembled. At this time, however, many of the focal adhesions, particularly those around the cell periphery, remained after most stress fibers had gone. By 2 h after injection only a few small focal adhesions persisted, yet the cells remained spread. Identical results were obtained with other cell types including primary chick fibroblasts, BSC-1, MDCK, and gerbil fibroma cells. Stress fibers and focal adhesions reformed if cells were allowed to recover for 18 h after injection. These data suggest that introduction of the monomeric 27-kD fragment of alpha-actinin into cells may disrupt the actin cytoskeleton by interfering with the function of endogenous, intact alpha-actinin molecules along stress fibers. The 53-kD fragment may interfere with endogenous alpha-actinin function at focal adhesions or by displacing some other component that binds to the rod domain of alpha-actinin and that is needed to maintain stress fiber organization.
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PMID:Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin. 190 87

A novel form of free human chorionic gonadotrophin beta-subunit (hCG beta) was found in serum from ElBre, a woman with epidermoid carcinoma of unknown origin. ElBre hCG beta was larger than standard (pregnancy urine) hCG beta when analysed by gel chromatography (apparent molecular weight 54 000 vs 44 000). This size difference appeared to be due to a larger carboxyterminal extension (CTE) of ElBre hCG beta since thermolysin cleavage of the CTE from standard hCG beta and Elbre hCG beta yielded core products of the same size. Oligosaccharides, O-linked to serine or threonine, were present in ElBre hCG beta, presumably on its CTE as judged by the complete binding of desialylated ElBre hCG beta to immobilized peanut agglutinin (this lectin is specific for terminal galactose linked beta 1----3 to N-acetylgalactosamine, a disaccharide exposed after desialylation of the O-linked oligosaccharides of standard hCG beta). ElBre hCG beta, however, was incompletely recognized by antisera specific for the CTE of standard hCG beta, especially the carbohydrate-sensitive antiserum R141. The O-linked oligosaccharides of standard hCG beta are heterogeneous in size; 13% are of the largest (hexasaccharide) form. In contrast, over 50% of the O-linked oligosaccharides in hCG beta from the JAr choriocarcinoma cell line are hexasaccharides. Like desialylated ElBre hCG beta, desialylated JAr hCG beta bound completely to peanut agglutinin, but was incompletely recognized by antisera to the hCG beta-CTE. Furthermore, JAr hCG beta was intermediate in size between standard hCG beta and ElBre hCG beta when analysed by gel chromatography (apparent molecular weight 49 000).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A novel form of ectopic human chorionic gonadotrophin beta-subunit in the serum of a woman with epidermoid cancer. 241 52

Selected fragments of the central rod of chicken gizzard alpha-actinin were expressed as fusion proteins in Escherichia coli, with the aim of determining the positions in the sequence of the four successive spectrin-like repeats that make up this domain. The criteria for an independently folding unit were resistance to proteolysis and the high alpha helicity characteristic of the native protein. Sequences containing repeats 1-4, 2-4, 3-4 and 4 all generated stable fragments on digestion with trypsin and/or thermolysin and N-terminal sequencing gave the most probable starting position of each repeat. The sequences of all four inferred repeats and the sequences of the entire rod, were separately expressed and were shown to assume a stable, protease-resistant fold in solution. The repeat boundaries established in this way differed from those originally deduced from sequence alignments; the N-terminal boundaries of the repeats were 14-24 residues nearer the C-terminus than predicted. The ability to express individual repeats should facilitate identification of the binding sites for the cytoplasmic domains of beta 1 integrins and intercellular cell adhesion molecule-1 which have been localised to the rod domain of alpha-actinin.
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PMID:Analysis of the phasing of four spectrin-like repeats in alpha-actinin. 792 43

Cells interact with type IV collagen mainly via the integrins alpha 1 beta 1 and alpha 2 beta 1. A triple helical CNBr derived fragment CB3[IV], which contains the recognition sites for both integrins, was isolated from type IV collagen. Trypsin treatment of CB3[IV] gave rise to four smaller fragments, F1-F4, of which the smallest one, F4, contained the recognition site for alpha 1 beta 1. Further fragmentation of F4 by thermolysin treatment at 50 degrees C led to fragment TL1, which represents the C-terminal half of F4, and which was no longer able to interact with alpha 1 beta 1. Therefore the recognition site of alpha 1 beta 1 had to be located within the N-terminal half of F4, a position which was verified by electron micrographs of a crosslinked F2-alpha 1 beta 1 complex. Modification of the Arg and Asp residues, which abolished the binding activity of F4, led to the identification of Arg (461) within the alpha 2(IV) and Asp (461) within the alpha 1 (IV) chain as essential residues for the alpha 1 beta 1. The array of these two residues on the surface of the triple helix is discussed.
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PMID:The alpha 1 beta 1 integrin recognition site of the basement membrane collagen molecule [alpha 1(IV)]2 alpha 2(IV). 822 88

Divalent cation-dependent platelet adhesion to fibronectin (FN) is mediated by the integrin receptors alpha 5 beta 1 (GP Ic-IIa) and alpha IIb beta 3 (GP IIb-IIIa), which recognize the RGD (Arg-Gly-Asp) sequence in the cell-binding domain. However, FN can also support divalent cation-independent platelet adhesion. To determine which domain of FN mediates divalent cation-independent adhesion, proteolysis with thermolysin and affinity chromatography were used to isolate the cell-binding, gelatin-binding, and heparin-binding domains of FN. Unactivated and thrombin-activated platelets adhered to intact FN and the 45-Kd gelatin-binding domain in the presence of either Ca2+ or EDTA. Platelet spreading was mediated only by the 105-Kd cell-binding domain and required divalent cations. The heparin-binding domains did not support platelet adhesion. Reduction of intrachain disulfide bonds or removal of carbohydrate side chains on the gelatin-binding domain did not alter the ability to support platelet adhesion. Divalent cation-independent adhesion to the 45-Kd gelatin-binding domain was not inhibited by RGDS (Arg-Gly-Asp-Ser) synthetic peptides or monoclonal antibodies (MoAbs) directed against known platelet receptors. We conclude that platelets can adhere but not spread on the gelatin-binding domain of FN by a novel divalent cation-independent mechanism.
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PMID:Platelet interactions with fibronectin: divalent cation-independent platelet adhesion to the gelatin-binding domain of fibronectin. 846 64

Differentiation of ST-13 preadipocytes into adipocytes was inhibited almost completely by addition of rat plasma fibronectin (FN) (approximately 100 micrograms/mL), but was reversed by GRGDSP cell recognition peptide (1.5 mM) and anti-alpha 5 beta 1. On the contrary, the thermolysin digest of FN stimulated adipocyte differentiation in a dose-dependent manner, in which remarkable increases in the values of the differentiation indexes, the number of adipocytes (8-fold above the control), glycerophosphate dehydrogenase (GPD) activity (12-fold), and triacylglycerol content (5-fold), were observed by inclusion of the thermolysin digest (100 micrograms/mL). The increase in GPD activity by the thermolysin digest was inhibited remarkably (about 70% inhibition) by an antibody directed to the amino-terminal fibrin-binding (Fib 1) domain of FN and slightly (about 15%) by an antibody directed to the central cell-binding (Cell) domain, but not by anti-gelatin-binding domain and anti-carboxy-terminal fibrin-binding domain. Treatment of ST-13 cells by a purified 24K fragment (100 micrograms/mL) derived from the Fib 1 domain caused an over 20-fold augmentation of the GPD activity, accounting for a major part of the differentiation stimulatory activity of the thermolysin digest. The differentiation stimulatory effect of the 24K Fib 1 fragment was not affected by either GRGDSP peptide or anti-alpha 5 beta 1. Thus, FN can regulate adipose development of ST-13 cells by its two antipodal, inhibitory and stimulatory, activities, the latter of which is expressed only upon fragmentation. Proteolytic cleavage of FN may play an important role in controlling the action of FN on adipocyte differentiation.
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PMID:An amino-terminal fibronectin fragment stimulates the differentiation of ST-13 preadipocytes. 850 92