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)

Cytosolic factors in a 50--75% (NH4)2SO4 fraction of the 105 000 x g supernatant of the renal cortex modulated adenylate cyclase activity in membrane preparations enriched in renal tubular cell basal--lateral membranes. The crude factor preparation had no effect on basal activity but it contained components that augmented the stimulated of the enzyme by NaF, parathyroid hormone (PTH), prostaglandin E1 (PGE1), and inhibited the activation of the enzyme by GMP--PNP. The factor(s) potentiating the stimulation by the hormones was partially purified (13-fold) by DEAE-cellulose and Sephadex G-75 chromatography. During purification, the component(s) that increased hormone-stimulated adenylate cyclase was separated from those affecting the activity in the presence of NaF and GMP--PNP. The factor(s) enhanced the PTH- and PGE1-stimulated enzyme at all concentrations of hormone, suggesting that the affinity for the hormone was not affected. The factor(s) was heat-stable. Partial proteolysis with chymotrypsin greatly reduced the ability of the factor(s) to enhance hormonal responsive adenylate cyclase. However, the factor(s) was resistant to trypsin digestion. The effect of the factor was not due to GTP, nor was GTP necessary for its action. Ca2+ was not needed for the enhancing activity of the factor(s). These findings suggest the presence in the cytosol of the kidney cortex of a protein(s) that regulates the response of renal adenylate cyclase to hormones. The relationship between this kidney cytosolic factor and those reported in other tissues remains to be established.
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PMID:Regulation of hormone(PTH and PGE1)-stimulated adenylate cyclase by renal cytosolic factors. 721 2

Peptide maps of recombinant human parathyroid hormone (rhPTH) were determined by both trypsin and V-8 protease digestion with subsequent fast-atom bombardment mass spectrometry (FAB-MS). Coverage of the sequence was 85% when using trypsin and 90% when using V-8 protease. Five rhPTH variants that were recombinantly produced as models of Asn deamidated type degradation products were measured, and molecular weight differences between their respective deamidated peptide fragments were completely detected. In the V-8 protease digests of some variants, characteristic peptide ions caused by the deamidation were observed and this greatly facilitated the assignment and recognition of the deamidated position. Our data suggest that FAB-mapping of rhPTH via the protease digestion methods used, appears to have great potential for structural investigations of the peptide.
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PMID:Peptide mapping of recombinant human parathyroid hormone by enzymatic digestion and subsequent fast-atom bombardment mass spectrometry. 775 99

We established a useful assay system for evaluating osteoclast-mediated bone resorption based on the use of unfractionated bone cells obtained from 10- to 11-day-old mice. When cells from 10 to 11 mice were treated for 7 days with rat parathyroid hormone (rPTH, 10(-8) M), a total of 4 to 5 x 10(7) cells could be obtained from the culture by treatment with 0.05% trypsin and 0.02% EDTA in PBS. These harvested cells contained about 20% tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells. When the harvested cells were cultured on dentine slices without rPTH, after 1 day, they formed TRAP-positive multinucleate cells that were active in bone resorption. Eel calcitonin (eCT) decreased the number of pits in a dose-dependent manner, and its half maximal inhibition dose (ID50) was 1.08 x 10(-11) M. Even after having been frozen in liquid nitrogen for 5 months, upon thawing, these cells were capable for forming pits; and this pit formation was inhibited by eCT. Since no appropriate osteoclastic cell line for evaluating bone resorption is available at present, this system can provide a useful, practical means for assaying osteoclastic bone-resorbing activity.
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PMID:Establishment of a rapid bone resorption in vitro assay using previously frozen mouse unfractionated bone cells pretreated with parathyroid hormone. 815 5

The development of satisfactory cell culture models for the study of parathyroid hormone (PTH)-induced inhibition of Pi transport has proven difficult. Using subcellular fractionation techniques we investigated the response of primary cultures of rat proximal tubular cells to PTH-(1-34). Specific binding of 125I-bPTH-(1-34) occurred at 2 degrees C. After 5 min of rewarming, trypsin-releasable radioactivity decreased from 90 to 50%, indicating internalization of the ligand. Cell disruption, followed by density centrifugation with 17% Percoll either directly after binding at 2 degrees C or post-rewarming for 20 min, showed a shift of 125I label from the plasma membrane (5'-nucleotidase) to lysosomal fractions (beta-D-glucosaminidase), confirming the sequential occurrence of cell surface binding, internalization and transport to lysosomes of 125I-bPTH-(1-34). Reculture at 37 degrees C revealed steady accumulation of trichloroacetic acid-soluble radioactivity in the medium, indicating degradation of 125I-bPTH-(1-34). Phosphate transport in the absence of sodium was minimal. Incubation of the cells with bPTH-(1-34) resulted in up to 50% inhibition of sodium-dependent phosphate transport. Prior phosphate depletion abrogated the response to PTH.
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PMID:Parathyroid hormone transport effects and hormonal processing in primary cultured rat proximal tubular cells. 834 17

Oncogenic osteomalacia is a condition where renal phosphate wasting occurs causing defective mineralisation, in the presence of a tumor. Cultures of cells were established from a hemangiopericytoma resected from a patient with oncogenic osteomalacia. Conditioned media from the cells inhibited phosphate uptake in opossum kidney cells and stimulated of cAMP in rat osteosarcoma cells, a standard parathyroid hormone (PTH)-like assay. This cAMP stimulation was suppressed by the PTH analogue, 3-34 bPTH and also by heat and trypsin treatment of the media. Tests of conditioned media for PTH and parathyroid hormone related protein (PTHrP) immunoreactivity were negative, however, and no hybridisation to probes for PTH, PTHrP or human stanniocalcin was detected in tumor cell RNA on Northern blot. These data support the hypothesis that tumors responsible for oncogenic osteomalacia produce a humoral substance that reduces renal phosphate reabsorption and provide evidence that the factor may act via PTH/PTHrP receptors.
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PMID:Characteristics of tumor cell bioactivity in oncogenic osteomalacia. 902 20

The neonatal Bartter syndrome (NBS) is associated with a complex disorder of mineral metabolism in children, including hypercalciuria, nephrocalcinosis, and diminished bone mineral density. Although cyclooxygenase inhibition usually brings about improvement in these findings, there is a variable component which is resistant to such therapy in many children. The factor mediating this disorder has not been identified. Blood and urine from 12 children with NBS were examined. When compared with samples from normal children and adults, all (NBS) sera reduced bone calcium uptake in a bone disc bioassay. This effect persisted in the presence of parathyroid hormone (PTH) antibody and PTH receptor blockade, indicating that neither PTH nor PTH related peptide was responsible. It was eliminated by indomethacin, suggesting that prostanoid generation was essential. Protamine was also inhibitory, as was the addition of ecteola, an anion binder. Activity could be recovered from ecteola by elution with hypertonic buffer. Urine samples from children with NBS had the same calcitropic effect. The agent was removed by ecteola and recovered by hypertonic elution. Activity was eliminated by protamine and by heparinase, but not by trypsin digestion. Size exclusion centrifugation showed that the activity was associated with a material between 10 and 30 kilodaltons. Finally, urine ecteola eluates from NBS patients raised serum concentrations of calcium after intraperitoneal injection in rats. These data suggest that children with NBS have a calcitropic substance in their serum and urine which is not found in normal individuals. The substance is heparin like, and mediates its effects through prostanoid production. These studies provide additional evidence against a direct renal cause of the urinary calcium disturbance characteristic of the disorder.
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PMID:Humoral factor in children with neonatal Bartter syndrome reduces bone calcium uptake in vitro. 968 54

Oncogenic osteomalacia (OOM) is characterized by renal phosphate wasting and abnormal metabolism of vitamin D, somewhat similar to the phenotype of X-linked hypophosphatemic rickets (HYP). DNA from OOM tumor cells was analyzed for mutations in the PHEX gene, which is mutated in HYP. Screening for mutations by single-strand conformation polymorphism analysis and subsequent sequencing of all the exons revealed no mutations. Conditioned media from long-term cultures of OOM tumor cells were used to further characterize the physical properties of the phosphate-regulating factor and its mechanism of action. Inhibition of OK 3B2 cell renal phosphate transport by conditioned media was dose-dependent and maximal after 20 h. This time course differed from that of parathyroid hormone (PTH). The bioactivity was stable to mild acid and alkali treatment and freeze drying and was retained in the aqueous phase following organic solvent extraction. The activity was not suppressed by heat or by treatment with trypsin but was suppressed by the protease papain and had an apparent molecular weight of < 5000. No change was detected in the expression of type II sodium/phosphate cotransporter (NaPi) mRNA in OK 3B2 cells in response to conditioned media, unlike the reduction seen in Hyp mice. In the presence of colchicine or cytochalasin D, the inhibitory response to conditioned media was reduced, similar to the effect of these agents on the response to PTH. Cycloheximide also suppressed the inhibitory response of conditioned media, but not the response to PTH. These studies indicate that mutations in the PHEX gene are unlikely to be responsible for OOM and suggest that the tumor-derived factor that inhibits phosphate uptake is a small protein that does not downregulate type II NaPi mRNA, and requires an intact cytoskeleton and protein synthesis for activity.
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PMID:Phosphate wasting in oncogenic osteomalacia: PHEX is normal and the tumor-derived factor has unique properties. 1133 25

Active cathepsin B has been found in cell extract and medium of human osteoblast-like cells and MG-63 cells. The released form is stable at neutral and alkaline pH and, in both cell types, intracellular and extracellular cathepsin B activities are increased by interleukin-1 beta (IL-1beta) and parathyroid hormone (PTH). To evaluate the physiological role of cathepsin B in osteoblasts, we investigated the production and secretion of this enzyme in normal human synovial fibroblasts and modulation by IL-1beta and PTH. Lactate secretion concurrent with release of cathepsin B and comparable responses in osteoblasts were also examined. Our data show that synovial fibroblasts respond differently to treatment with the two agents, suggesting a cell-specific regulation of cathepsin B and possible involvement in osteoblast physiology. Cathepsin B involvement was then evaluated in the activation of plasminogen activator (PA) in MG-63 cells using two specific inhibitors of cathepsin B, CA074 and CA074-Me, in constitutive conditions and after treatment with IL-1beta. As results of PA activity obtained in the presence of IL-beta were in contrast with previous reports, we examined the activities of PA, pro-PA activated with trypsin, and plasmin in cell extract and media of MG-63 cells after 24-h treatment with IL-1beta. Results show that in normal conditions and in the presence of IL-1beta, cathepsin B is involved in the activation of PA. Moreover, IL-1beta stimulates PA, pro-PA activated by trypsin, and plasmin activity in medium, whereas in cell extract it stimulates pro-PA activated by trypsin and plasmin activity. IL-1beta has no effect on cell extract-associated PA.
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PMID:Cathepsin B in osteoblasts. 1272 91

Teriparatide, a recombinant parathyroid hormone (1-34) is the first approved agent for the treatment of osteoporosis that stimulates new bone formation. Currently, the drug is administered daily by s.c. injection. Because of the obvious advantages of oral teriparatide administration, the development of such a delivery system would be of great benefit. Besides other barriers, the enzymatic barrier caused by gastro-intestinal (GI) proteolytic enzymes is believed to be responsible for negligible teriparatide oral bioavailability. It was therefore the aim of the study to evaluate the stability of teriparatide towards a variety of GI proteases under physiological conditions. Results indicate that teriparatide is entirely degraded by trypsin, chymotrypsin and pepsin within 5 min. In contrast, even after 3 h of incubation with elastase about 85% of undegraded teriparatide could still be detected. Within an incubation period of 3 h in the presence of rat small intestinal mucosa, approximately half of the teriparatide was degraded. Experiments with isolated aminopeptidase N demonstrated that this membrane bound peptidase is primarily involved in the degradation process. Results gained from and recorded in this study provide a precise characterisation of the enzymatic barrier for oral teriparatide administration and represents a prerequisite for the development of oral teriparatide delivery systems.
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PMID:Degradation of teriparatide by gastro-intestinal proteolytic enzymes. 1675 24

Daily injection of parathyroid hormone (PTH) is a clinically approved treatment for osteoporosis. It suppresses apoptosis of bone-forming osteoblasts although its exact anti-apoptotic mechanism(s) is incompletely understood. In this study, PTH treatment of cultured osteoblasts blocked the pro-apoptotic effects of serum withdrawal and nutrient deprivation; hydrogen peroxide induced oxidative stress, and UV irradiation. We hypothesized that PTH might suppress osteoblast apoptosis by enhancing DNA repair. Evidence is provided showing that post-confluent, non-proliferating osteoblasts treated with PTH exhibited a protein kinase A-mediated activation of two proteins that regulate DNA repair processes (proliferating cell nuclear antigen and forkhead box transcription factor 3a) as well as a suppression of the pro-apoptotic growth arrest and DNA damage protein 153. Additional proof of a connection between DNA damage and osteoblast apoptosis came from an unexpected finding whereby a majority of fixed PTH-treated osteoblasts scored weakly positive for Terminal Deoxynucleotidyl dUTP Nick-End Labeling (TUNEL), even though similar cultures were determined to be viable via a trypsin replating strategy. TUNEL identifies DNA excision repair, not just apoptotic DNA fragmentation, and the most likely explanation of these TUNEL results is that PTH's activation of DNA repair processes would permit nucleotide incorporation as a result of enhanced excision repair. This explanation was confirmed by an enhanced incorporation of bromodeoxyuridine in PTH-treated cells even though a majority of the cell population was determined to be non-replicating. An augmentation of DNA repair by PTH is an unreported finding, and provides an additional explanation for its anti-apoptotic mechanism(s).
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PMID:Parathyroid hormone suppresses osteoblast apoptosis by augmenting DNA repair. 1945 Jul 16


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