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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the vitamin D-depleted rat, all nucleated tissues examined (brain, lung, heart, pancreas, liver, cartilage, muscle, bone, kidney, and intestine) contained a soluble substance which bound 25-hydroxy[3H]cholecalciferol in vitro specifically and sedimented at 6.3 S in linear sucrose gradients. The serum-steroid complex sedimented a 4.1 S, and erythrocyte lysates were apparently devoid of specific binding activity. The ability of these cytosols to specifically bind the steroid was destroyed by treatment with trypsin, but not by RNase, DNase, or 1 mM p-hydroxymercuribenzoate. The sedimentation pattern was not altered in sucrose gradients containing 0.5 M KCl or following cytosol preparation and ultracentrifugation in gradients containing 0.012 M dithiothreitol. The apparent avidity for 25-hydroxycholecalciferol (KA similar to 2 times 10- M) was slightly higher in muscle and kidney cytosols than in serum, but serum contained a large number of specific binding sites. The presence of widespread, high affinity binding proteins for 25-hydroxycholecalciferol raises the possibility that tissues other than the intestine, bone, and kidney may respond directly to vitamin D metabolites.
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PMID:Widespread, specific binding of 25-hydroxycholecalciferol in rat tissues. 114 Dec 9

The amino acid sequence for vitamin D-dependent bovine intestinal calcium binding protein has been established. It contains 85 amino acids in a single chain and lacks cysteine, tryptophan, methionine, histidine, and arginine. The NH2-terminal lysine is blocked by an N-acetyl group. Enzymatic digestion with trypsin, chymotrypsin, and pepsin yielded a number of peptides which were purified by two-dimensional high voltage paper electrophoresis. These peptides were examined by end group analysis and sequenced by the dansyl procedure. The absence of tryptophan permitted by a single cleavage of the molecule by N-bromosuccinimide at the tyrosine residue at position 8 and the larger fragment was subjected to automated Edman degradation. By these means, the following sequence was established: N-Ac-Lys-Gln-Ser-Pro-Leu-Glu-Tyr-Ala-Ala-Glu-Lys-Ser-Ile-Gln-Lys-Glu-Ile-Glu-Lys-Gly-Phe-Phe-Lys-Gln-Leu-Leu-Val-Ser-Val-Gln-Lys-Ala-Gly-Asp-Lys-Glu-Ser-Leu-Gln-Pro-Leu-Phe-Thr-Leu-Leu-Lys-Ser-Gly-Pro-Glu-Glu-Asn-Leu-Lys-Glu-Ser-Gln-Asn-Gly-Pro-Asp-Leu-Ls7-Ser-Gly-Pro-Gly-Asn-Asp-Leu-Glu-Glu-Lys-Gly-Thr-Asp-Val-Phe-Ser-Leu-Lys-Gln. Microheterogeneity may exist in the molecule at residue 76 in which position threonine may be replaced by serine. Comparison of the sequence of calcium-binding protein to the "test" sequence of Tufty and Kretsinger ((1975) Science 187, 167-169) proposed to identify E-F hands in muscle proteins suggests that intestinal calcium-binding protein may likewise contain one or possibly two E-F hands which could account for calcium-binding property. Dayhoff alignment scores, however, calculated for calcium-binding protein against nine E-F hands in muscle proteins parvalbumin, troponin and alkali light chains do not indicate that intestinal calcium-binding protein is homologous to these muscle protein chains.
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PMID:Calcium-binding protein of bovine intestine. The complete amino acid sequence. 117 41

When assessed by 1,25-dihydroxyvitamin D3 (1,25(OH)2-D3)-receptor (VDR) binding analysis or 1,25(OH)2-D3-VDR-directed bioresponsiveness, cultured cells from some New World primates (platyrrhines) demonstrate a variable decrement in VDR when compared with Old World primate (catarrhine) cells. To study this difference in VDR expression among primates, we performed immunoblot analysis of the VDR in cultured dermal fibroblasts from platyrrhines in the genera Pithecia and Aotus and from catarrhines in the genus Presbytis; although a platyrrhine, the owl monkey (Aotus) expresses a VDR of the catarrhine (wild type) phenotype. Despite a 10-fold difference in the content of VDR by ligand binding analysis among cells from the three prototypic primate genera, there was a less than or equal to 10% difference in the steady-state level of 50-kD VDR detected by immunoblot analysis of cellular extracts. We investigated this apparent discrepancy in the content of VDR in immunoblots and ligand binding analyses by mixing VDR-containing nuclear extracts of equivalent protein concentration from the various primates. Coincubation of Pithecia and Aotus fibroblast extracts with Presbytis extract diminished specific 1,25(OH)2-D3 binding in the mix by 90% and 95% respectively. Similar results were obtained by mixing nuclear extracts of the owl monkey cell line, OMK, and the vitamin D resistant marmoset B-lymphoblast cell line B95-8. A wild type 1,25(OH)2-D3-binding profile was restored in mixtures after trypsin or heat treatment of the B95-8 extract. These data indicate that some New World primate cells contain a soluble protein that prevents intracellular 1,25(OH)2-D3-VDR binding. It is possible that the quantitative differences in the expression of this protein are responsible for 1,25(OH)2-D3 and other steroid hormone resistant states of variable severity in New World primates.
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PMID:Endogenous blockade of 1,25-dihydroxyvitamin D-receptor binding in New World primate cells. 184 42

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] has been found to exert its effects in a manner entirely analogous to that of other steroid hormones and is known to induce the synthesis of a calcium-binding protein (CaBP). The effects of 1,25-(OH)2D3 and dietary alteration on genomic expression in rat kidney were studied by in vitro translation of poly(A+)-containing RNA and by immunoprecipitation. Poly(A+)RNA from rat kidneys was translated in a rabbit reticulocyte lysate system in the presence of [35S]methionine, and the renal CaBP mRNA translation product was identified and quantitated by specific immunoprecipitation. Total translation products and specific immunoprecipitable products were visualized on sodium dodecyl sulfate-gels, followed by fluorography. After the addition of affinity purified rat renal CaBP antiserum to the 35S-labeled translation products, only one protein band, electrophoretically indistinguishable from that of purified renal CaBP (mol wt, 28,000), was observed. When 10 micrograms purified renal CaBP were added to the translation product before addition of the antiserum, immunoprecipitation of the 35S-labeled 28,000 mol wt protein was not observed. A comparison of the peptides produced after limited digestion with trypsin of 125I-labeled CaBP and [3H]tyrosine-labeled translation product indicated a good coincidence of peaks from purified 125I-labeled CaBP and the immunoprecipitated translation product, suggesting that the immunoprecipitated translation product is indeed vitamin D-dependent renal CaBP. When 100 ng 1,25-(OH)2D3 were injected for 7 days to 8-week-old vitamin D-deficient rats, there was a 4-fold increase in CaBP mRNA levels in the kidney (quantitated by densitometry of immunoprecipitates analyzed on sodium dodecyl sulfate-polyacrylamide gels). This increase in mRNA was accompanied by a corresponding increase in the concentration of renal CaBP, as measured by RIA, thus establishing a bridge between CaBP and the putative transcriptional effect of 1,25-(OH)2D3 in rat kidney. Similarly, both the concentration of renal CaBP and renal CaBP mRNA levels increased 4-fold in rats fed low phosphorus diets, increased 2-fold in rats fed low calcium diets, and decreased 67% in rats fed low sodium diets, providing evidence that the nutritional induction or decrease in renal CaBP is accompanied by a corresponding alteration in the concentration of its specific translatable mRNA. These results are consistent with a transcriptional control mechanism for the induction of renal CaBP.
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PMID:Cell-free translational analysis of messenger ribonucleic acid coding for vitamin D-dependent rat renal calcium-binding protein. 241 31

The author examined a group of 143 patients with osteomalacia of different origin before treatment and after adequate treatment with vitamin D, using laboratory tests, assessment of body weight and muscular strength (grip of the dominant hand). After treatment there was a significant rise of calcaemia, phosphataemia and calciuria and a drop of alkaline phosphatase activity. The body weight increased within the first month of treatment on average by 1.27 kg, during the second month by another 1.15 kg. The patients gained a total of 2.42 kg. The muscular strength increased during the first month on average by 3.23 kg and during the second month by another 2.16 kg, i.e. a total of 5.39 kg. From these results it may be concluded that vitamin D may have a certain anabolic effect if used in pharmacological does either due to an increased nutrient absorption from the gut because of hypertrophy of the intestinal wall or indirectly via hypercalcaemia which increases the hydrochloric acid secretion in the stomach as well as pepsin secretion, and promotes activation of trypsin and lipase in the duodenum and moreover causes retardation of the intestinal transit. The increased muscular strength in due to a rise of calcaemia, improved muscle contraction and probably also due to the mentioned nutritional factors. There may be also the factor of an improved lifestyle due to the immunomodulating action of vitamin D and disappearance of bone pain.
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PMID:[Anabolic effects of vitamin D in patients with osteomalacia]. 263 59

In vitro studies were performed to assess the ability of term human trophoblastic tissue to metabolize 25-hydroxyvitamin D3 (25OHD3) and to compare this metabolism to that occurring in porcine renal mitochondria and microsomes. Human trophoblastic homogenates, containing a NADPH-generating system, were able to produce 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] at a rate of 225 pg/mg protein.h, but did not produce detectable quantities (less than 20 pg/mg protein.h) of 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3]. Similarly, mitochondria and microsomes isolated from term human trophoblastic tissue produced 1,25-(OH)2D3 [249 +/- 156 and 199 +/- 82 (mean +/- SD) pg/mg protein.h, respectively] in the presence of an NADPH-generating system, but failed to produce detectable quantities (less than 200 pg/mg protein.h) of 24,25-(OH)2D3. The production of 1,25-(OH)2D3 from the trophoblastic mitochondria and microsomes could be increased by adding 140,000 x g trophoblastic cytosol to the subcellular incubation tubes. This treatment had no effect on the production of 24,25-(OH)2D3. The component(s) present in trophoblastic cytosol responsible for the increased 1,25-(OH)2D3 production by trophoblastic mitochondria and microsomes was shown to be heat labile, trypsin resistant, and less than 1000 mol wt in size. Comparing characteristics of the porcine renal 1 alpha- and 24R-hydroxylase systems with those of the human trophoblastic system revealed that 1) 1,2-dianilinoethane and EDTA totally blocked synthesis of 1,25-(OH)2D3 in trophoblastic mitochondria and microsomes, but had no effect on the synthesis of 1,25-(OH)2D3 by renal mitochondria; and 2) ketoconazole greatly inhibited the synthesis of 1,25-(OH)2D3 and 24,25-(OH)2D3 by renal mitochondria, but had no effect on the production of 1,25-(OH)2D3 by trophoblastic mitochondria or microsomes. Finally, production of 24,25-(OH)2D3 could not be demonstrated in trophoblastic homogenates, mitochondria, or microsomes, while the production of this compound was readily evident in renal mitochondria, but not microsomes. The results of this study question the existence of the 25-hydroxyvitamin D3-1 alpha- and 24R-hydroxylase systems in the trophoblastic portion of the human placenta. This study also suggests that 1,25-(OH)2D3 can be produced in vitro by a mechanism other than enzymatic 1 alpha-hydroxylation. The possibility exists that the mechanism involves the insertion of oxygen at the 1 position of 25-(OH)D3 by free radical chemistry.
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PMID:In vitro metabolism of 25-hydroxyvitamin D3 by human trophoblastic homogenates, mitochondria, and microsomes: lack of evidence for the presence of 25-hydroxyvitamin D3-1 alpha- and 24R-hydroxylases. 275 24

The presence of a specific receptor for 1,25-dihydroxy-vitamin D3 was investigated in myoblasts released from chick embryo skeletal muscle by trypsin and collagenase treatment. Density gradient analysis of the cytosol obtained from these muscle cell preparations showed that 1,25-dihydroxy-vitamin D3 binds specifically to a 3.7 S macromolecule. Scatchard analysis yielded an equilibrium dissociation constant of 2.46 x 10(-10) M and a Nmax of 74 fmol/mg of cytosol protein. The data is in agreement with previous evidence which indicates that the action of the vitamin D metabolite on muscle Ca uptake is mediated by de novo protein and RNA synthesis, and supports the concept that muscle is a target organ for 1,25-dihydroxy-vitamin D3.
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PMID:Presence of a 1,25-dihydroxy-vitamin D3 receptor in chick skeletal muscle myoblasts. 298 76

In the transition from proliferation to hypertrophic cell zones in the growth plate, there is an increase in chondrocyte volume and a corresponding decrease in collagen content to accommodate the enlarging cells. It is postulated that collagenase accounts for this collagen loss. To test this hypothesis, tibial growth plates were obtained from normal rats, rachitic rats deficient in vitamin D and phosphate, and rats after 48 and 72 h of healing from rickets. Collagenase was quantitated by a pellet assay based on the release of solubilized collagen from the endogenous insoluble collagen in the tissue homogenates. A fourfold greater collagen release and a concomitant sixfold greater hypertrophic cell volume were measured in rachitic growth plates compared with normal age-matched controls. During healing of rickets, collagenase activity and hypertrophic cell volume returned almost to control levels. Rachitic growth plates were dissected into the juxtaepiphyseal 1/3 and the juxtametaphyseal 2/3. The latter portion contained greater than 95% of the hypertrophic cells and 86% of the collagenase. The collagen-degrading activity was extracted from this region and was shown to be a true collagenase by its production of typical A fragments of tropocollagen produced by collagenase action. The enzyme was activated by aminophenylmercuric acetate and trypsin and was inhibited by EDTA, 1,10-phenanthroline, and a tissue inhibitor of metalloproteinases from human articular cartilage. Inhibitors of aspartic, cysteine, and serine proteases had no effect. Micropuncture fluids aspirated from rachitic cartilage contained latent collagenase activity, indicating an extracellular localization. Negative tests for hemoglobin in the rachitic cartilage samples indicated that there was no contamination by capillaries and that this was not a source of collagenase. It is concluded that extracellular collagenase accounts for the loss of cartilage matrix in the hypertrophic zone, and that this process may be distinct from that of capillary invasion.
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PMID:Localization of collagenase in the growth plate of rachitic rats. 299 64

A squamous cell carcinoma of 33-yr-old patient who developed marked leukocytosis and hypercalcemia was transplanted into nude mice in which more marked leukocytosis and hypercalcemia also developed. This tumor (LJC-1-JCK) produced a colony-stimulating factor (CSF) and formed a cyst in the tumor from which a CSF-producing cell line (T3M-1) was established. The CSF causes predominantly formation of granulocytic colonies in addition to macrophage colonies. Bone-resorbing activity (BRA) was detected in the cystic fluid and was eluted as two separate peaks with proteins of an apparent molecular weight of 30,000-50,000 and 10,000-20,000. Colony-stimulating activity (CSA) was eluted at an apparent 30,000 mol wt. The conditioned medium of the T3M-1 cells also contained a BRA with an apparent 14,000 mol wt, whereas CSA eluted at an apparent 30,000 mol wt. PTH, epidermal growth factor, transforming growth factor-alpha, prostaglandin Es, and vitamin D could not account for the powerful BRA. In contrast to CSA, BRA was not inactivated by trypsin and more stable at 70 degrees C. When T3M-1 cells were transplanted into nude mice, marked hypercalcemia developed in addition to granulocytosis. Our findings suggest that the tumor produces and secretes a powerful BRA in vivo and in vitro, which is different from CSA in terms of molecular weight, heat stability, and trypsin treatment. We speculate that the synergistic action of CSF that stimulates macrophage colony formation and recruits osteoclast precursors, and BRA, which stimulates mononuclear phagocytes and/or osteoclasts were responsible for a marked increase in osteoclastic bone resorption and humoral hypercalcemia in the patient.
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PMID:Production of bone-resorbing activity and colony-stimulating activity in vivo and in vitro by a human squamous cell carcinoma associated with hypercalcemia and leukocytosis. 348 54

Osteoclast-activating factor (OAF) is a soluble mediator found in supernates of human peripheral leukocytes which have been cultured with antigens or phytomitogens. OAF is a potent stimulator of osteoclastic resorption of fetal bone in organ culture. The present studies were designed to characterize OAF chemically. Bone resorbing activity from supernates of leukocytes cultured without added plasma was not lost on dialysis using a membrane with a molecular weight cutoff of 3,500, but was lost when heated to 60 degrees C for 30 min. The activity was lost after treatment with trypsin or pronase but not after treatment with ribonuclease or neuraminidase. Papain, which inactivated parathyroid hormone at a concentration of 25 mug/ml, did not inactivate OAF at 250 mug/ml. OAF did not react with an antibody to bovine parathyroid hormone which cross-reacts with human parathyroid hormone. OAF was also distinguished from active metabolites of vitamin D and from prostaglandin by extraction procedures and immunoassay for prostaglandin E(2). When the medium from activated leukocytes cultured with autologous plasma was fractionated by gel filtration on Sephadex, bone resorbing activity eluated both with plasma proteins and in lower molecular weight fractions. However, when medium from leukocytes cultured without added plasma was chromatographed, all the OAF activity was eluted in a sharp low molecular weight peak located between chymotrypsinogen (25,000 molecular weight) and ribonuclease A (13,700 molecular weight). This peak contained about 4% of the total protein originally present in the supernate. Its activity was destroyed by overnight incubation at 37 degrees C at pH 6 or 8, but not at pH 7.2. After incubation at 4 degrees C, the activity was lost at pH 3 or 10, but not at pH 4-9. The active fraction from Sephadex G-100 was therefore chromatographed at pH 7.2 on DEAE cellulose and carboxymethyl cellulose. The active material was not adsorbed; however, about sevenfold further purification was achieved by removal of contaminants. The material obtained after sequential Sephadex, DEAE and, carboxymethyl cellulose chromatography stimulated resorption of fetal rat bone in culture at concentrations of 0.75-3 mug protein/ml, indicating that this preparation of OAF was nearly as potent as bovine parathyroid hormone in this system.
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PMID:Partial purification of osteoclast-activating factor from phytohemagglutinin-stimulated human leukocytes. 482 37


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