Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Surfactant protein C (SP-C) is a lung-specific, hydrophobic peptide found in organic extracts of pulmonary surfactant. Alveolar SP-C (3.5 kD) is produced from proteolytic cleavage of a larger precursor molecule (pro-SP-C; 21 kD). While SP-C is synthesized by type II cells, the pathways for processing and secretion have remained elusive due, in part, to the lack of monospecific antibodies against SP-C or its precursors. This report describes production and characterization of a new antibody directed against pro-SP-C epitopes. Polyclonal antisera (anti-CPRO-SP-C) was prepared using a synthetic peptide corresponding to a portion of rat SP-C cDNA sequence (Ile26-Ser72). This contained amino acids 3-35 of mature SP-C plus additional C-terminal residues (His59-Ser72). On Western blots, anti-CPRO-SP-C competitively reacted to CPRO-SP-C but not to mature SP-C. Immunoblots of in vitro synthesized pro-SP-C confirmed that the antisera also recognized native protein. Immunocytochemistry with anti-CPRO-SP-C demonstrated staining for pro-SP-C peptides in isolated type II cells as well as in alveolar epithelial cells of rat lung sections. Pro-SP-C preferentially co-localized to cells that stained positive for Maclura pomifera antigen. Anti-CPRO-SP-C staining was not observed in lung interstitium, pulmonary vasculature, or several control tissues (brain, heart, and liver were negative). Western blotting of subcellular fractions demonstrated pro-SP-C peptides in plasma membrane (20 kD) and microsomal (20 and 21 kD) fractions with a 16 kD peptide present in lamellar bodies. No pro-SP-C peptides were detected in purified surfactant. These results demonstrate the use of a synthetic peptide to generate specific antiserum against more hydrophilic domains of pro-SP-C sequences and confirm that SP-C propeptides are unique to the lung.
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PMID:An antibody with specificity for surfactant protein C precursors: identification of pro-SP-C in rat lung. 138 9

Mammalian cells contain a microsomal vitamin K-dependent carboxylase activity which catalyzes the gamma-carboxylation of glutamate. While most cells have a limited ability to fully gamma-carboxylate proteins, it has been suggested that the ability of transformed cells to perform this complex post-translational modification may play a role in tumor biology. In this study, we examined the effect of transformation by adenovirus oncogenes on the ability of cells to efficiently gamma-carboxylate a vitamin K-dependent protein. Several morphologically transformed BHK-21 cell lines (BHK-Ad) were isolated following the chromosomal integration of the viral oncogenes E1A/E1B from human adenovirus type 12 (Ad12). The lines were capable of growing in soft agar and low serum and produced functional E1A as determined by promoter activation studies. Using a vector for the expression of the vitamin K-dependent recombinant human protein C (HPC), a regulator of the clotting cascade, Ad-transformed and nontransformed lines secreting rHPC were generated. The rHPC from the transformed and nontransformed cell lines displayed identical serine protease activities, and there were no apparent differences in the proteolytic processing of the proteins, although a minor difference in the proportion of each HPC glycoform was observed. However, the functional anticoagulant activity, which depends on the gamma-carboxyglutamic acid (Gla) content, was approximately 70% higher in the Ad-transformed lines. Approximately 90% of the rHPC from the Ad-transformed lines exhibited a calcium-dependent (high Gla) elution profile on anion-exchange resin, compared to only 15 to 26% from the nontransformed cell clones. By analyzing endogenous microsomal carboxylase, we determined that enzyme activity increased approximately 50% following transformation. Overall, our data demonstrate that transformation can increase the potential of a cell to efficiently gamma-carboxylate a protein and lend support to the suggested involvement of this post-translational modification in tumor cell function. Further, our results demonstrate a potential means of altering cells to enable full modification of vitamin K-dependent factors for structure/function studies and potentially for therapeutic use.
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PMID:Viral transformation increases vitamin K-dependent gamma-carboxylation of glutamate. 182 87

The L6 skeletal muscle cell line has been identified as a suitable model to study the action of insulin on glucose uptake in muscle [Klip, Li & Logan (1984) Am. J. Physiol. 247, E291-E296]. The signals that transfer information from occupied insulin receptors to glucose transporters remain unknown. Here we report that activation of protein kinase C by exogenous phorbol esters results in stimulation of glucose uptake. Protein C kinase activity was induced to migrate from the cytosolic fraction to the microsomal fraction after 40 min of exposure of intact cells to 4 beta-phorbol 12,13-dibutyrate. In contrast, incubation with insulin did not alter the subcellular distribution of the kinase. Prolonged preincubation of L6 cells with phorbol esters resulted in depletion of kinase C activity, whereas neither the basal rate of glucose uptake nor its stimulation by insulin were affected. This suggests that protein kinase C is expressed in L6 cells, and that insulin stimulation of hexose transport does not involve protein kinase C.
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PMID:Protein kinase C is not required for insulin stimulation of hexose uptake in muscle cells in culture. 329 42

Vitamin K is required as a cofactor for a microsomal enzyme that converts glutamyl residues in precursor proteins to gamma-carboxyglutamyl residues in completed proteins. These residues are essential for the biological function of prothrombin, factors VII, IX, and X, protein C, and protein S. Current data suggest that recognition of protein substrates by the carboxylase requires an unidentified protein-protein interaction in addition to the Glu substrate binding site. The primary vitamin K-dependent event has now been shown to be the abstraction of the gamma-hydrogen of the substrate Glu residue with the concurrent formation of vitamin K 2,3-epoxide. Coumarin anticoagulants appear to inhibit the microsomal vitamin K epoxide reductase and one of a number of microsomal quinone reductases. They therefore block vitamin K action by preventing the recycling of vitamin K epoxide to the quinone and to the active cofactor form, the hydroquinone. Excess vitamin K can reverse a coumarin anticoagulant effect as the nonsensitive quinone reductase can continue to furnish the active coenzyme.
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PMID:Studies of the vitamin K-dependent carboxylase and vitamin K epoxide reductase in rat liver. 353 Aug 99

The liver microsomal vitamin K-dependent carboxylase catalyzes the posttranslational conversion of specific glutamate residues to gamma-carboxyglutamate residues in a limited number of proteins. A number of these proteins have been shown to contain a homologous basic amino acid-rich "propeptide" between the leader sequence and the amino terminus of the mature protein. Plasmids encoding protein C, a vitamin K-dependent protein, containing or lacking a propeptide region were constructed and the protein was expressed in Escherichia coli. The protein products were assayed as substrates in an in vitro vitamin K-dependent carboxylase system. Only proteins containing a propeptide region were substrates for the enzyme. These data support the hypothesis that this sequence of the primary gene product is an important recognition site for this processing enzyme.
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PMID:Vitamin K-dependent carboxylase: possible role of the substrate "propeptide" as an intracellular recognition site. 354 32

Warfarin, an antagonist of vitamin K, is known to disrupt the microsomal vitamin K cycle, which results in a decrease in the plasma level of protein C, an anticoagulant factor, as well as some other vitamin K-dependent coagulation factors. Here, we examined the effect of warfarin on the secretion of recombinant protein C expressed in human kidney 293 or BHK cells. In transient expression, warfarin caused a 2-4-fold decrease in the quantity of protein C secreted, compared to findings with vitamin K-treated cells. Pulse-chase experiments using stable cells showed that, although recombinant protein C was secreted in the presence of vitamin K, the decrease in the total amount of radioactivity in the warfarin-treated cells suggested intracellular degradation. This degradation depended on the concentration of warfarin and was not inhibited by an endoplasmic reticulum (ER)-Golgi transport inhibitor (brefeldin A) or by lysosomotropic inhibitors (chloroquine and NH4Cl). Thus, protein C synthesized in the presence of warfarin is probably selectively degraded, and this degradation occurs in a pre-Golgi, nonlysosomal compartment. Among the protease inhibitors tested, N-alpha-acetyl-Leu-Leu-methioninal and N-alpha-acetyl-Leu-Leu-norleucinal blocked the degradation of protein C precursor synthesized in the presence of warfarin, and the precursor accumulated intracellularly, in a dose-dependent manner. Both inhibitors, however, did not disturb the secretion of protein C precursor in the vitamin K-treated cells. Thus, a cysteine protease(s) appeared to be responsible for the degradation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Warfarin causes the degradation of protein C precursor in the endoplasmic reticulum. 782 66

I have isolated glucose-6-phosphate dehydrogenase from rabbit liver microsomes and determined its complete amino acid sequence. Sequence determination was achieved by automated Edman degradation of peptides generated by chemical and enzymatic cleavages. The microsomal enzyme consists of 763 residues and is quite dissimilar from the previously characterized cytosolic enzymes. The N terminus of the microsomal enzyme is blocked by a pyroglutamyl residue. Carbohydrate is attached at Asn-138 and Asn-263, implying that the bulk of the protein is oriented on the lumenal side of the endoplasmic membrane. The amino acid sequence of the microsomal protein shows limited homology to the extensively sequenced cytosolic glucose-6-phosphate dehydrogenases. Clusters of up to six identical residues can be identified in four regions: peptide segments at residues 10-21, 154-163, and 173-261. In addition, another array of identical residues, requiring a 100-residue deletion in the sequence of the microsomal enzyme, spans residues 436-462 and corresponds to residues 348-373 of the cytosolic protein. Two segments with a Gly-Xaa-Gly-Xaa-Xaa-Gly motif, related to a coenzyme binding fold, were identified at Gly-399 and Gly-491. In the cytosolic enzymes, a variation of this sequence motif occurs at Gly-37 and Gly-241. The 300-residue C-terminal segment of the microsomal enzyme is unique and has no counterpart in the cytosolic or the bacterial enzymes. An unexpected finding with regard to the microsomal enzyme is that it lacks an identifiable membrane-spanning region or the lumenal-protein C-terminal consensus sequences Lys-Asp-Glu or His-Ile/Thr-Glu-Leu. Thus, the mode of transport and retention of this protein in the lumen of endoplasmic reticulum remains to be determined.
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PMID:Isolation and the complete amino acid sequence of lumenal endoplasmic reticulum glucose-6-phosphate dehydrogenase. 850 77

Warfarin is known to disrupt the microsomal vitamin K cycle, which results in a decrease of the plasma level of protein C, an anticoagulant factor, as well as some other vitamin K-dependent coagulation factors. We examined the effect of warfarin on secretion of recombinant protein C expressed in human kidney 293 or BHK cells. In transient expression, warfarin caused a two- to four-fold decrease in the quantity of protein C secreted, compared to findings with vitamin K-treated cells. Pulse-chase experiments using stable cells showed that, although recombinant protein C was secreted in the presence of vitamin K, the decrease in total amount of the radioactivity in the warfarin-treated cells suggested intracellular degradation. This degradation depended on the concentration of warfarin and was not inhibited by an endoplasmic reticulum (ER)-Golgi transport inhibitor or by lysosomotropic inhibitors. Thus, protein C synthesized in the presence of warfarin is selectively degraded and the degradation occurs in a pre-Golgi, nonlysosomal compartment. Among the protease inhibitors tested, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-norleucinal blocked the degradation of protein C synthesized in the presence of warfarin and the protein C accumulated intracellularly, in a dose-dependent manner. Both inhibitors, however, did not disturb the secretion of protein C in the vitamin K-treated cells. Thus, a cysteine protease(s) appeared to be responsible for the degradation. These results suggest that protein C synthesized in the presence of warfarin was selectively degraded by a cysteine protease(g) in the ER through a "quality control" mechanism.
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PMID:Quality control of protein C: protein C synthesized in the presence of warfarin is selectively degraded in the endoplasmic reticulum. 911 52

Irinotecan (CPT-11) is an anticancer prodrug. It is converted by carboxylesterase to yield an active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), which acts as a topoisomerase I inhibitor. Several oxidative metabolites of CPT-11 have been identified in humans, including 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (APC) and 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecin (NPC), generated by cytochrome P-450 3A4 (CYP3A4). Other minor metabolites in which metabolic pathways and biologic activities have not been identified also exist. To further investigate the metabolism of CPT-11 in human liver, we analyzed metabolites of CPT-11 in human hepatic microsomes using a high-performance liquid chromatography/mass spectrometry (HPLC/MS) system and detected a new metabolite that was the major one produced in the microsomal system. HPLC-tandem mass spectrometry (HPLC/MS/MS) analysis indicated that this compound was an oxidation product formed by the loss of two hydrogen atoms from the terminal piperidine ring. Kinetic analyses indicated that a single enzyme generated the metabolite, and we have identified this enzyme in two in vitro systems. The formation of the new metabolite was significantly inhibited by SKF525A, ketoconazole, and an anti-CYP3A4 antibody and catalyzed specifically by CYP3A4 expressed in insect microsomes. A significant correlation was observed between the generation of this metabolite and the CYP3A4 content in individual human hepatic microsomes. These findings indicate that this newly detected metabolite is a CYP3A4-generated product that may be produced in hepatic microsomes of patients treated with CPT-11.
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PMID:A new metabolite of irinotecan in which formation is mediated by human hepatic cytochrome P-450 3A4. 1160 29

A 54-year-old man of Persian origin presented to our department with a 1-year history of ulcers on the right leg that had been unresponsive to numerous topical treatments, accompanied by lymphedema of the right leg. Medical history included hypergonadotropic hypogonadism, which had not been further investigated. He was treated for 20 years with testosterone IM once monthly, which he stopped a year before the current hospitalization for unclear reasons. The patient reported no congenital lymphedema. Physical examination revealed two deep skin ulcers (Figure 1) on the right leg measuring 10 cm in diameter with raised irregular inflammatory borders and a boggy, necrotic base discharging a purulent hemorrhagic exudate. Bilateral leg pitting edema and right lymphangitis with lymphadenitis were noted. He had low head hair implantment, sparse hair on the body and head, hyperpigmentation on both legs, onychodystrophia of the toenails (mainly the large toe and less prominent on the other toes), which was atrophic lichen-planus-like in appearance and needed no trimming (Figure 2), normal hand nails, oral thrush, and angular cheilitis. Other physical findings were gynecomastia, pectus excavatum, small and firm testicles, long extremities, asymmetrical goiter, systolic murmur 2/6 in left sternal border, and slow and inappropriate behavior. The patient's temperature on admission was 39 degrees C. Blood cultures were negative for bacterial growth. Results of laboratory investigations included hemoglobin (11.2 g/dL), hematocrit (26.8%), normal mean corpuscular volume and mean corpuscular hemoglobin volume, and red blood cell distribution width (16%). Blood smear showed spherocytes, slight hypochromia, anisocytosis, macrocytosis, and microcytosis. Blood chemistry values were taken for iron (4 micro g/dL [normal range 40-150 micro g/dL]), transferrin (193 mg/dL [normal range 220-400 mg/dL]), ferritin (1128 ng/mL [normal range 14-160 ng/mL]), transferrin saturation (1.5% [normal range 20%-55%]), serum folate (within normal limits), and vitamin B12 (within normal limits). Direct Coombs' test equaled positive 2 + IgG. All these values indicated anemia of chronic diseases combined with hemolytic anemia. Further blood work-up tested antinuclear antibody (positive <1:80 homogeneous pattern), rheumatoid factors (143 IU/mL [positive >8.5 IU/mL]), C-reactive protein (286 mg/L [normal range 0-5 mg/L]), anticardiolipin IgM antibody (9.0 monophosphoryl lipid U/mL [normal range 0-7.00 MPL U/mL]) and antithrombin III activity (135% [normal range 74%-114%]). Results of other blood tests were within normal limits or negative, including lupus anticoagulant, beta2 glycoprotein, anticardiolipin IgG Ab, anti-ss DNA Ab, C3, C4, anti-RO, anti-LA, anti-SC-70, anti-SM Ab, P-ANCA, C-ANCA, TSH, FT4, anti-T microsomal, antithyroglobulin, protein C activity, protein S free, cryoglobulins, serum immunoelectrophoresis, VDRL, hepatitis C antibodies, hepatitis B antigen, and human immunodeficiency virus. Endocrinological work-up examined luteinizing hormone (22.9 mIU/mL [normal range for adult men 0.8-6 mIU/mL]), follicle stimulating hormone (49.7 mIU/mL [normal range for adult men 1-11 mIU/mL]), testosterone (0.24 ng/mL [normal range for adult men 2.5-8.0 ng/mL]), bioavailable testosterone (0.02 ng/mL [normal range for adult men >0.6 ng/mL]), and percent bioavailable test (8.1% [normal value >20%]). These results indicate hypergonadotropic hypogonadism. Plasminogen activator inhibitor 1 was 6 U (normal value 5-20 U/mL). Karyotyping performed by G-banding technique revealed a 47 XXY karyotype, which is diagnostic of Klinefelter's syndrome. Doppler ultrasound of the leg ulcers disclosed partial thrombus in the distal right femoral vein. X-rays and bone scan displayed osteomyelitis along the right tibia. Histological examination of a 4-mm punch biopsy from the ulcer border revealed hyperkeratosis, acanthosis, hypergranulosis, and mixed inflammatory infiltrate containing eosinophils compatible with chronic ulcer. Multiple vessels were seen, compatible with a healing process. Direct immunofluorescence of the biopsy revealed granular IgM in the dermo-epidermal junction. Indirect immunofluorescence was negative. Thyroid function tests showed normal thyroid stimulating hormone and free throxine4. Multinodular goiter was seen on thyroid scan and ultrasound. Thyroid fine needle aspiration was compatible with multinodular goiter (normal follicular cells, free colloid, macrophages with pigment). IV treatment with amoxicillin-clavulanic acid 1 g t.i.d. was administered for 2 weeks, with a decrease in temperature and normalization of the leukocyte level. Oral antibiotic treatment with amoxicillin-clavulanic acid was continued for 10 more days, followed by 25 days of ciprofloxacin for the osteomyelitis. Local treatment included saline soakings followed by application of Promogran (Johnson & Johnson, New Brunswick, NJ) and Kaltostat (ConvaTec Ltd., a Bristol-Myers Squibb Company, New York, NY) with slight improvement. At the same time, the patient was treated with warfarin sodium due to deep vein thrombosis under international normalized ratio 2-3. The patient was treated with IM testosterone once monthly for 1 year, which resulted in a reduction in the diameter and depth of the leg ulcers (Figure 3). Blood tests were not performed for follow-up of the immune state.
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PMID:Klinefelter's syndrome presenting with leg ulcers. 1536 65


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