Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:APRD00631 (Gel)
 14,881 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method for beta-D-galactosidase isolation from cattle gastric juice has been developed. Gastric juice mucus was removed by and addition of equimolar amounts of Na2HPO4 and CaCl2. The removal of proteases and other proteins was achieved by the treatment with resins KB-51X2 and AN-22. The resulting preparation had specific activity of 0.14 units per mg of protein. Gel filtration on Sepharose 4B led to an increase of specific activity of the preparation up to 0,4 units per mg of protein. Some properties of the beta-D-galactosidase obtained were compared to relation of lactose and o-nitrophenyl-beta-D-galactoside (pH optima, temperature optimym and thermostability).
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PMID:[Isolation and characterization of beta-D-galactosidase from gastric juice]. 2 Sep 88

Myosin was extracted from frozen squid brain and purified by a modification of the procedure of Pollard et al. (Pollard, T.D., Thomas, S.M., and Niederman, R. (1974) Anal. Biochem. 60, 258-266). Myosin was eluted from Bio-Gel A-15m column as a single peak of (K+-EDTA)-activated ATPase ((K+-EDTA)-ATPase) activity with an average partition coefficient (Kav) of 0.22. In sodium dodecyl sulfate-acrylamide gel electrophoresis, the purified myosin showed a predominant band with similar electrophoretic mobility as the heavy chain of rabbit skeletal muscle myosin, and two less intense bands near the bottom of the gel. No actin band was seen. The properties of the (K+-EDTA)-ATPase activity were: (a) the time course of the reaction was biphasic at 25 degrees but linear at 32 degrees; (b) the optimum rate of reaction was obtained between 0.3 and 0.8 M KCl; (c) the pH optimum was between 8.0 and 9.0; (d) the reaction was specific for ATP with an apparent Km of 0.19 mM. ATPase activity in 0.06 M KCl and 5 mM MgCl2 was increased about 1.5 times by a 10-fold excess of rabbit skeletal muscle F-actin and about 5 times by a 40-fold excess. The actin activation was inhibited slightly by the addition of 0.2 mM CaCl2 and completely by the addition of 10 mM CaCl2. Myosin formed arrowhead patterns with rabbit skeletal muscle F-actin as observed by electron microscopy of negatively stained samples. It also aggregated in bipolar filaments which attached to decorated actin filaments at different angles, as well as formed cross-connections and ladder-like patterns between actin filaments. These two forms of interactions between myosin and actin were abolished by treatment with MgATP.
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PMID:Purification and characterization of squid brain myosin. 13 40

Phosphodiesterase activities for adenosine and guanosine 3':5'-monophosphates (cyclic AMP and cyclic GMP) were demonstrated in particulate and soluble fractions of rat anterior pituitary gland. Both fractions contained higher activity for cyclic GMP hydrolysis than that for cyclic AMP hydrolysis when these activities were assayed at subsaturating substrate concentrations. Addition of protein activator and CaCl2 to either whole homogenate, particulate or supernatant fraction stimulated both cyclic AMP and cyclic GMP phosphadiesterase activities. Almost 80% of cyclic AMP and 90% of cyclic GMP hydrolyzing activities were localized in soluble fraction. Particulate-bound cyclic nucleotide phosphodiesterase activity was completely solubilized with 1% Triton X-100. Detergent-dispersed particulate and soluble enzymes were compared with respect to Ca2+ and activator requirements and gel filtration profiles. Particulate, soluble and partially purified phosphodiesterase activities were also characterized in relation to divalent cation requirements, kinetic behavior and effects of Ca2+, activator and ethyleneglycol-bis-(2-aminoethyl)-N,N'-tetraacetic acid. Gel filtration of either sonicated whole homogenate or the 10500 X g supernatant fraction showed a single peak of activity, which hydrolyzed both cyclic AMP and cyclic GMP and was dependent upon Ca2+ and activator for maximum activity. Partially purified enzyme was inhibited by 1-methyl-3-isobutylxanthine and papaverine with the concentration of inhibitor giving 50% inhibition at 0.4 muM substrate being 20 muM and 24 muM for cyclic AMP and 7 muM and 10 muM for cyclic GMP, respectively. Theophylline, caffeine and theobromine were less effective. The rat anterior pituitary also contained a protein activator which stimulated both pituitary cyclic nucleotide phosphodiesterase(s) as well as activator-deficient brain cyclic GMP and cyclic AMP phosphodiesterases. Chromatography of the sonicated pituitary extract on DEAE-cellulose column chromatography resolved the phosphodiesterase into two fractions. Both enzyme fractions hydrolyzed cyclic AMP and cyclic GMP and had comparable apparent Km values for the two nucleotides. Hydrolysis of cyclic GMP and cyclic AMP by fraction II enzyme was stimulated 6--7-fold by both pituitary and brain activator in the presence of micromolar concentrations of Ca2+.
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PMID:Cyclic nucleotide phosphodiesterases from rat anterior pituitary. Characterization of multiple forms and regulation by protein activator and Ca+. 19 11

Raw extract in 2 m CaCl2 of bovine nasal septum cartilage was eluted from 4 per cent agarose gel to give a "void volume" Fraction v-4, which was indistinguishable in composition and behavior on viscometric and sedimentation analysis from the densest fraction obtained by associative centrifugation in a cesium chloride density gradient. The sulfated proteoglycan was precipitated (Fraction A) by cetylpyridinium chloride from acidic solutions of Fraction v-4 or of dialyzed raw ectract. Neutralization under conditions of low ionic strength precipitated a further small fraction (B), which contained from 0.5 to 1 per cent of the uronic acid in the original extract. Analysis by associative and dissociative density gradient centrifugation demonstrated that Fraction B resembled in effective density known samples of hyaluronic acid from other sources. Gel chromatography of proteolytic digests of Fractions A and B on 6 per cent agarose indicated that cetylpyridinium chloride precipitation essentially separated sulfated proteoglycan (A) from hyaluronic acid (B). A viscosity-average molecular weight of about 5 x 10(5) was estimated for a sample of Fraction B purified in a dissociative (4 M guanidine hydrochloride + CsCl) density gradient. Sedimentation velocity data were consistent with this result. Analysis of hexosamines showed that the sample contained 96 per cent glucosamine, confirming the identification of hyaluronic acid. The proteoglycan fraction (A) resembled "subunits" in its sedimentation behavior.
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PMID:Isolation and physical characterization of hyaluronic acid prepared from bovine nasal septum by cetylpyridinium chloride precipitation. 83 35

Phospholipase A2 (EC 3.1.1.4) from the insoluble pulmonary secretions that accumulate in the lungs of patients with alveolar proteinosis has been purified. The pure enzyme gives a single sharp band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. Amino acid analysis of the protein shows high content of cystine, aspartic acid, glutamic acid, serine, glycine, leucine and lysine. Only one N-terminal residue, alanine, can be detected. Gel filtration as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis indicate an apparent molecular weight of 75 000 for the enzyme. The enzyme activity has a pH optimum between 7.5 and 8.5 and is stimulated by sodium deoxycholate and CaCl2.
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PMID:Characterization of phospholipase A from pulmonary secretions of patients with alveolar proteinosis. 92 32

Plasma, cryoprecipitate, Hemofil, and human factor VIII concentrate were dissolved in 1.0 M NaCl and chromatographed on Bio-Gel A-5m. With high concentrations of factor VIII the activity eluted as a symmetrical peak in the void volume; with a low factor VIII concentration the procoagulant activity was retarded. Dilution curves were performed for several human factor VIII concentrates. When the concentration of factor VIII was decreased, elution patterns showed a gradual transition from a peak in the void volume to a peak with a Ve/Vo of 1.7. Cryoprecipitate exhibited a similar behavior in 1.0 M NaCl, but the percent dissociation was greater than expected at high concentrations of factor VIII. When gel filtration was performed with 0.25 M CaCl2, significant dissociation occurred at all concentrations of factor VIII tested. The behavior of factor VIII in 1.0 M NaCl closely fit a theoretically derived curve for the dissociation of a protein from its binder. We conclude that the dissociation of factor VIII in 1 M NaCl is dependent on the concentration and purification of the procoagulant protein.
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PMID:Concentration-dependent dissociation of factor VIII in 1 M NaCl. 94 52

We have purified to homogeneity the 33-kDa phosphatidylinositol-specific phospholipase C (PI-PLC) from the culture fluid of Listeria monocytogenes, a facultative intracellular pathogen. The protein was overexpressed, and secretion of PI-PLC was further enhanced by the addition of divalent cations to the culture medium. The basic protein (pI, approximately 9.4) was complexed with anionic proteins in the crude culture fluid. It bound to DEAE-Sepharose and was eluted from Sephacryl S-200 near the void volume in low-ionic-strength buffer, suggesting aggregates of greater than or equal to 150 kDa. Gel filtration chromatography on Sephacryl S-200 in the presence of 1 M ammonium sulfate resulted in disaggregation and complete separation of PI-PLC, which interacted with the column matrix. Amino-terminal sequencing of the pure protein gave results consistent with the previously deduced sequence and showed that the signal cleavage site was between alanine 29 and tyrosine 30. The enzyme was specific for PI and showed no activity with phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It did not cleave PI-4-phosphate or PI-4,5-bisphosphate, but it was active on the membrane form of the variable surface glycoprotein from Trypanosoma brucei, a PI-glycan-anchored protein. When assayed with deoxycholate-mixed micelles of PI, activity was highly dependent on added salt. Activation by salt was also observed with Triton X-100-mixed micelles. The optimal concentration of CaCl2 or MgCl2 was lower than that of KCl or (NH4)2SO4, but activity was not specifically dependent on divalent cations and was not inhibited by addition of EDTA. With deoxycholate, the optimum pH was 7.0. A broader pH optimum ranging from 5.5 to 6.5 was observed with Triton X-100-mixed micelles. These results are consistent with a postulated role for secreted PI-PLC in the acidified primary phagocytic vesicle of infected cells.
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PMID:Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C. 139 18

Demineralized dentin matrix induces the ectopic formation of bone, in vivo, when implanted subcutaneously or in muscle pouches. In these situations the bone induction follows a chondrogenic pathway. As part of the strategy for the assay and isolation of the factors responsible for initiating induction, we have developed a cell culture system in which the addition of soluble factors extracted from the dentin matrix appears to initiate chondrogenesis. Indicators of chondrogenesis, relative to control cultures, were taken as an increase of 35S-sulfate incorporation into proteoglycan (PG), an altered size of the PG, production of type II collagen, and changes in cell morphology and matrix histochemistry. Our studies have taken two directions: the use of the cell culture system under standard conditions to select fractions inducing one or more of the above indica-tors; and, the purification and characterization of the in vitro chondrogenesis inducing factor(s). Here we report the identification of a peptide fraction which acts in culture to satisfy each of the above indicators of chondrogenesis. An EDTA extract of rat incisor dentin was fractionated by CaCl2 precipitation, Sephacryl S-100 chromatography, and reverse phase HPLC. A single peptide fraction from the HPLC, evidenced by the existence of a single spot on 2-D Gel Electrophoresis, was found to be a potent enhancer of 35S-sulfate incorporation during the standard assay, with maximal activity in the 1-10 ng/ml range. Further detailed studies showed that the heightened incorporation occurred without any increase in cell number. The neonatal rat muscle explant fibroblasts exposed to this fraction for 7 days in monolayer culture formed dense cell nodules which stained intensely with Alcian blue relative to controls.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A rat incisor dentin matrix protein can induce neonatal rat muscle fibroblasts, in culture, to express phenotypic products of chondroblastic cells. 186 59

Type X collagen was extracted from ends of canine growth plates by pepsin digestion after 4 M guanidine hydrochloride extraction, purified by stepwise salt precipitation (2.0 M NaCl in 0.5 M acetic acid), and chromatographed on a Bio-Gel A1.5 M column in 1.0 M CaCl2. Without reduction on sodium dodecyl sulfate (SDS) polyacrylamide gels, the preparation yielded a single, high-molecular-weight (mol wt) band; after reduction, a single band of relative mol wt 5.0 x 10(4) was found. Polyclonal sera were raised against the purified collagen and used in the immunolocalization of canine type X collagen. As expected, indirect immunoperoxidase (IP) or indirect immunofluorescent staining with the polyclonal sera demonstrated that most of the immunoreactivity was localized in the zone of provisional calcification of the growth plate and in cartilage remnants in the metaphyseal region of the physis. A progressive decrease in staining toward the diaphysis of the fetal canine long bone was apparent as the trabecular structures were remodeled to bone. Unexpectedly, type X collagen was also detected in the zone of calcified, mature articular cartilage. It was concentrated in the pericellular matrix of the chondrocytes, appeared at or just above the tidemark, and was expressed immediately before mineralization. Identification of type X collagen in both the canine growth plate and the zone of calcified articular cartilage suggests that cells in the deep layer of cartilage and in the zone of calcified cartilage in the adult animal retain some characteristics of a growth plate and may be involved in regulation of mineralization at this critical interface. The expression of growth plate-like properties would allow the deep chondrocytes of mature articular cartilage to play a role in remodeling of the joint with age and in the pathogenesis of osteoarthritis.
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PMID:Localization of type X collagen in canine growth plate and adult canine articular cartilage. 204 75

Solubilization of the microsomal fraction from bovine kidney by Triton X-100 or by 3-[(3-cholamidopropyl)-dimethylammonio] 1-propanesulfonate (CHAPS) increased 2-fold the thermodynamic association constant for hGH. While solubilization with CHAPS did not change the 13-fold preferential binding of human growth hormone (hGH) over ovine prolactin (oPRL), solubilization with Triton X-100 increased this preference to 47-fold. The binding was optimal at pH 7-7.5 in the presence of 10 mM of MgCl2 or CaCl2. The association rate with hGH was identical in the microsomal and Triton X-100 solubilized fractions but the dissociation was slower in the latter. Only partial dissociation was observed at neutral pH. Full dissociation was, however, achieved by lowering the pH to 4-5, indicating that the binding was not covalent. Gel filtration studies of the Triton X-100 solubilized fraction after preincubation in the presence of reducing agent revealed two sharp peaks of activity, one having Mr of greater than 700 kDa that represented the aggregated receptor, and the second, with Mr 110-115 kDa. The specificity of the partially purified receptors clearly shows that they are lactogenic and not somatogenic. They resemble lactogenic receptors found in other bovine organs, but differ from other species particularly in their differential affinities of PRL and hGH.
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PMID:Solubilization and characterization of lactogenic hormone receptor from kidney of lactating cow. 274 17


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