Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.6.4 (chondroitinase)
2,039 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 29-kDa monomeric dispase-digestive fragment of human plasma fibronectin has been purified by heparin affinity chromatography. The NH2-terminal sequence was determined as Ala1687-Val-Thr-Thr-Ile-Pro-Ala-Pro. By mass spectrometry the molecular weight was determined to be 30,241.9 with standard deviation of 3.9 amu. Therefore, we defined the C-terminal sequence of the 29-kDa fragment as Arg1957-Lys-Lys-Thr-Gly-Gln-Glu. This indicates that the fragment is composed of 277 amino acids. 125I-fibronectin and the 125I-labeled 29-kDa fragment bound to HL-60 (human acute promyelocytic leukemia) cells in a time-dependent, saturable, and reversible manner. Approximately 120 min was required to reach maximal binding. There were no differences in quantity or rate of binding of labeled fibronectin and 29-kDa fragment at temperatures of 4 degrees, 22 degrees, and 37 degrees C. The number of binding sites per HL-60 cell of fibronectin and the 29-kDa fragment were 140,000 with a Kd of 133 nM and 108,000 with a Kd of 250 nM, respectively. The binding of fibronectin to HL-60 cells was completely inhibited by this fragment, and by the peptides of RGDS and CS1 with IC50s of 3.6, 840, and 670 microM, respectively. Native fibronectin inhibited the direct binding of the 29-kDa fragment to HL-60 cells; however, RGDS peptide, peptide CS1, or two melanoma cell adhesion-promoting domain peptides in this 29-kDa fragment (peptide I; Tyr1906-Val1924, peptide II; Asp1946-Thr1960) did not block this binding. Neither heparitinase nor chondroitinase treatment of cells had any effect on these bindings. These results indicate that the C-terminal cell- and heparin-binding domain of fibronectin mediates HL-60 cell binding by direct interaction independently of RGD, CS1, and melanoma cell adhesion domains in this fragment.
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PMID:Binding site in human plasma fibronectin to HL-60 cells localizes in the C-terminal heparin-binding region independently of RGD and CS1. 769 49

Cell surface anionic charge is known to be related to various cellular functions. Therefore, we ultrastructurally localized anionic sites in normal and psoriatic human epidermis, using poly-L-lysine-gold complex (cationic gold), to assess their possible participation in the differentiation of keratinocytes and the pathogenesis of psoriasis. In normal and psoriatic epidermis, the cell membrane of keratinocytes showed positive staining at pH 2.0. At pH 7.4 the cytoplasm and nucleus were diffusely stained, in addition to the cell membrane. In normal epidermis, the intensity of labelling on the cell membrane at pH 2.0 was strong in the basal layer and lower stratum spinosum, and decreased in parallel with differentiation of keratinocytes. In psoriatic epidermis, the intensity of labelling on the cell membrane at pH 2.0 was stronger than in normal epidermis. In normal epidermis, heparitinase digested 63% and chondroitinase ABC digested 80% of cationic labelling. This suggests that heparan sulphate and chondroitin sulphate (and/or dermatan sulphate) constitute anionic sites in normal epidermis. In psoriatic epidermis, chondroitinase ABC-sensitive anionic sites were greatly increased, whereas heparitinase-sensitive anionic sites were the same, when compared with normal epidermis. This suggests that chondroitin sulphate and/or dermatan sulphate constitute anionic sites which are increased in psoriatic epidermis.
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PMID:Localization of anionic sites in normal and psoriatic epidermis: the effect of enzyme digestion on these anionic sites. 777 75

Rat tail tendons from 54-day-old and 900-day-old animals were incubated with different concentrations of the dibasic amino acids, lysine and arginine. We observed a significant incorporation of these amino acids into the tendons. Uniaxial tension tests and relaxation experiments were performed at strain levels within the linear portion of the stress-strain relationship. The incorporation of the amino acids resulted in a decrease of ultimate stress and maximum Young's modulus and, after separation of the elastic and viscous stress components, in a decrease of the elastic fraction. The incorporation of amino acids and the resulting mechanical alterations were more pronounced in the young animals. The reversibility of the effects induced by the amino acids was tested. After the glycosaminoglycan chains were digested with chondroitinase ABC, we showed that the dibasic amino acids bind predominantly to the proteoglycan matrix. A possible analogy to the effects of amino acid incorporation on biomechanics and swelling with a monovalent cation such as Na+ is discussed.
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PMID:Mechanical changes in rat tail tendons induced by dibasic amino acids as a function of age. 817 43

We localized anionic sites ultrastructurally in human eccrine and apocrine sweat glands with a poly-L-lysine-gold complex (cationic colloidal gold). Anionic sites were labeled by incubating Lowicryl K4M-embedded sections on droplets of cationic colloidal gold. In eccrine sweat glands, colloidal gold particles were restricted to the basolateral membrane of the secretory cells at low pH, whereas the luminal membrane did not react with the gold particles. Chondroitinase ABC digested these anionic sites. This indicates that chondroitin sulfate and/or dermatan sulfate constitutes anionic sites in the basal labyrinth of eccrine sweat glands. In apocrine sweat glands, the luminal membrane of the secretory cells showed strong reaction at low pH, whereas the contraluminal membrane did not show any reaction. Neuraminidase completely digested these anionic sites, which indicated that the anionic charge of the apocrine lumen was due to sialic acid. Differences in distribution and susceptibility to enzymes of anionic sites in cell membranes between eccrine and apocrine sweat glands may reflect functional differences between these glands. Dark cell granules in eccrine secretory cells were negative for the anionic sites when sections were labeled without any pre-treatment. However, pre-incubation of the grids on EGTA or deionized water unmasked the anionic sites on the dark cell granules. The positive staining after EGTA treatment was greatly decreased by reincubation with CaCl2. These results suggested that Ca blocked anionic sites in dark cell granules. Exposed anionic sites were digested with chondroitinase ABC. This indicated that chondroitinase ABC and/or dermatan sulfate composed the anionic sites in dark cell granules.
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PMID:Demonstration of anionic sites in human eccrine and apocrine sweat glands in post-embedded ultra-thin sections with cationic colloidal gold: effect of enzyme digestion on these anionic sites. 833 Dec 83

We have studied the binding, uptake, and degradation of a recombinant form of apolipoprotein[a] (r-apo[a]) using a cultured cell model. In HepG2 cells and in human fibroblasts, r-apo[a] complexed with low density lipoprotein(LDL) is bound and internalized via high affinity (Kd = 10 nM) receptors; in both cell types, low affinity (Kd = 200-300 nM) sites also mediate free apo[a] uptake. Using competition studies, we found that the high affinity binding component corresponds to the LDL receptor. Involvement of the LDL receptor in r-apo[a] uptake by fibroblasts was confirmed using fibroblasts derived from an individual homozygous for familial hypercholesterolemia; in contrast to normal fibroblasts, these cells lacked the high affinity r-apo[a] binding component. Cell association of 125I-labeled r-apo[a] was increased and decreased concomitantly with the up- and down-regulation of the LDL receptor in response to a number of compounds. The addition of alpha 2-macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease total specific binding of r-apo[a], suggesting that neither the low density lipoprotein receptor-related protein nor cell surface proteoglycans are involved in r-apo[a] clearance. The low affinity binding component present in both fibroblasts and HepG2 cells likely corresponds to the plasminogen receptor, as binding of r-apo[a] to these sites was specifically decreased by the addition of plasminogen or the lysine analogue epsilon-aminocaproic acid, but not by the addition of tissue-type plasminogen activator. Heparin abolished uptake of r-apo[a] by the LDL receptor component only; this indicates that apo[a] must be associated with LDL to be cleared by this receptor. In contrast, free apo[a] can be effectively cleared by the plasminogen receptor which may represent a significant route of clearance for free apo[a] in vivo.
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PMID:Interaction of a recombinant form of apolipoprotein[a] with human fibroblasts and with the human hepatoma cell line HepG2. 872 15

Decorin is a small dermatan sulfate-rich proteoglycan which binds to collagen type I in vitro and in vivo. In atherosclerotic lesions the contents of low density lipoprotein (LDL), decorin, and collagen type I are increased, and ultrastructural studies have suggested an association between LDL and collagen in the lesions. To study interactions between LDL, decorin, and collagen type I, we used solid phase systems in which LDL was coupled to a Sepharose column, or in which LDL, decorin, or collagen type I was attached to microtiter wells. The interaction between LDL and decorin in the fluid phase was evaluated using a gel mobility shift assay. We found that LDL binds to decorin by ionic interactions. After treatment with chondroitinase ABC, decorin did not bind to LDL, showing that the glycosaminoglycan side chain of decorin is essential for LDL binding. Acetylated and cyclohexanedione-treated LDL did not bind to decorin, demonstrating that both lysine and arginine residues of apoB-100 are necessary for the interaction. When collagen type I was attached to the microtiter plates, only insignificant amounts of LDL bound to the collagen. However, if decorin was first allowed to bind to the collagen, binding of LDL to the decorin-collagen complexes was over 10-fold higher than to collagen alone. Thus, decorin can link LDL with collagen type I in vitro, which suggests a novel mechanism for retention of LDL in collagen-rich areas of atherosclerotic lesions.
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PMID:The proteoglycan decorin links low density lipoproteins with collagen type I. 906 18

When cells dissociated from the neonatal rat brains are plated on a poly-lysine-coated surface in a serum-free medium, they display a strange morphology: a dark and extended cell body. Preincubation of the surface with fetal bovine serum was found to inhibit the appearance of this strange contraction of the basal cell sheets in a dose-dependent manner. This finding indicated the presence of a factor(s) in the serum, which might be an appropriate substratum for prolonged survival of brain neurons. In the current study, this factor was highly purified through DEAE ion-exchange chromatography followed by gel filtration. The factor was eluted from a Superose column at fractions corresponding to a molecular weight greater than 1000 kDa. By SDS-PAGE analysis, these fractions were found to contain a major band (>/=1000 kDa) positive for alcian blue and few minor bands faintly stainable with Coomassie blue. The activity of the purified sample, inducing the morphological change in cells, was diminished by incubation with chondroitinase ABC. Neither heparitinase II, hyaluronidase, nor trypsin modified the activity. An authentic chondroitin sulfate (type B) mimicked the serum action on the morphology of brain cells in early stages of culture. Taking these findings together, it is suggested that the factor in serum beneficial for the attachment of brain cells is composed of a chondroitin sulfate with a Mr greater than 1000 kDa. Cortical cells dissociated from the neonatal rat brain attached well to the purified factor-coated surface and displayed a healthy morphology: an optically-reflective cell body with thick neurites for at least 3 days in the absence of serum.
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PMID:A culture substratum appropriate for brain cells is a chondroitin sulfate glycosaminoglycan in serum. 947 1

The internalization of a basic peptide, 001-C8 [H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH2], into enterocyte-like Caco-2 cells was evaluated. Internalization of 125I-labeled 001-C8 (125I-001-C8) increased time dependently and reached steady state at 60 min. The steady-state internalization of 125I-001-C8 (7.24 +/- 0. 41 microl/mg protein) was temperature and concentration dependent and was significantly decreased by dansylcadaverine (500 microM), protamine (1 mM), poly-L-lysine (1 mM), E-2078 (1 mM), and ebiratide (1 mM), whereas poly-L-glutamic acid (1 mM), tyrosine (1 mM), and glycylglycine (25 mM) were not inhibitory. Predigestion of acid mucopolysaccharides by heparinase I, heparitinase, and chondroitinase ABC also decreased the internalization. The maximal internalization, the half-saturation constant, and the nonsaturable internalization of 125I-001-C8 were 1.13 +/- 0.23 pmol/mg protein, 0. 47 +/- 0.43 microM, and 3.13 +/- 0.19 microl/mg protein, respectively. Confocal microscopy also indicated the internalization of fluorescence-derived 001-C8 [001-C8-4-nitrobenz-2-oxa-1,3-diazole (001-C8-NBD)]. Granular staining seen within the cell, excluding nuclei, indicated the sequestration of 001-C8-NBD within endocytotic vesicles. Dansylcadaverine and protamine strongly decreased the granular distribution of 001-C8-NBD within the cell. These results demonstrate that 001-C8 is taken up by Caco-2 cells via adsorptive-mediated endocytosis.
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PMID:Adsorptive-mediated endocytosis of a basic peptide in enterocyte-like Caco-2 cells. 972 63

Lp(a) is a major inherited risk factor for premature atherosclerosis. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to interfere with plasminogen activation and its atherogenic potential as a lipoprotein particle after receptor-mediated uptake. We demonstrate that Lp(a) stimulates production of vascular cell adhesion molecule 1 (VCAM-1) and E-selectin in cultured human coronary artery endothelial cells (HCAEC). This effect resulted from a rise in intracellular free calcium induced by Lp(a) and could be inhibited by the intracellular calcium chelator, BAPTA/AM. The involvement of the LDL and VLDL receptors in Lp(a) activation of HCAEC were ruled out since Lp(a) induction of adhesion molecules was not prevented by an antibody (IgGC7) to the LDL receptor or by receptor-activating protein, an antagonist of ligand binding to the VLDL receptor. Addition of alpha2-macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease levels of VCAM-1 and E-selectin stimulated by Lp(a), suggesting that neither the low density lipoprotein receptor-related protein nor cell-surface proteoglycans are involved in Lp(a)-induced adhesion molecule production. Neither does the binding site on HCAEC responsible for adhesion molecule production by Lp(a) appear to involve plasminogen receptors, as levels of VCAM-1 and E-selectin were not significantly decreased by the addition of glu-plasminogen, the lysine analog epsilon-aminocaproic acid, or by trans-4-(aminomethyl)-cyclohexanecarboxymethylic acid (tranexamic acid), which acts by binding to the lysine binding sites carried on the kringle structures in plasminogen. In contrast, recombinant apolipoprotein (a) [r-apo(a)] competed with Lp(a) and attenuated the expression of VCAM-1 and E-selectin. In summary, we have identified a calcium-dependent interaction of Lp(a) with HCAEC capable of inducing potent surface expression of VCAM-1 and E-selectin that does not appear to involve any of the known potential Lp(a) binding sites. Because leukocyte recruitment to the vessel wall appears to represent one of the important early events in atherogenesis, this newly described endothelial cell-activating effect of Lp(a) places it at a crucial juncture in the initiation of atherogenic disease and may lead to a better understanding of the role of Lp(a) in the vascular biology of atherosclerosis.
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PMID:Expression of adhesion molecules by lp(a): a potential novel mechanism for its atherogenicity. 983 67

Using bone marrow embedded in hydrophilic resin Lowicryl K4M and cationic colloidal gold pH 1.0 labelling, we studied sites of sulphation and sulphated glycosaminoglycans ultrastructurally in various maturational stages of both eosinophil granulocytes and eosinophil granules of guinea pig. Eosinophil granules reacted positively to cationic gold, the pattern of labelling varying according to the degree of cell maturation. The formation of eosinophil granules takes place throughout the myelocyte stage. Early eosinophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late ones contain a small and less active Golgi apparatus. All the immature granules were labelled positively. However, mature granules with a central crystal bar lost their affinity towards colloidal gold. Interestingly, strong colloidal gold labelling was also observed in the trans to transmost Golgi apparatus, especially in immature eosinophil granulocytes. This indicates that sulphation of glycosaminoglycans occurs in the trans to transmost Golgi apparatus of eosinophil granulocytes. Prior absorption with poly-L-lysine prevented colloidal gold labelling of tissue sections. Methylation of sections at 37 degrees C did not alter the gold labelling, whereas the labelling disappeared after methylation at 60 degrees C. Prior treatment with chondroitinase ABC or heparinase I abolished the majority of colloidal gold labelling in immature eosinophil granules. Taking these results together, we conclude that immature eosinophil granules contain sulphated glycosaminoglycans including chondroitin sulphate or heparan sulphate or both.
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PMID:Sulphated glycosaminoglycans in guinea pig eosinophils studied by means of cationic colloidal gold. 987 Jul 69


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