Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe an amplified enzyme-linked immunoassay of human proinsulin in serum that detects intact proinsulin and both the 32/33 and 65/66 split forms. The method uses the IgG fraction of a polyclonal antibody raised in a guinea pig against intact proinsulin, which we used to coat plastic microtiter plates. A sandwich was formed with proinsulin by using a monoclonal antibody against C-peptide labeled with alkaline phosphatase. We quantified the reaction by using the enzyme amplification procedure, which detected as little intact proinsulin as 0.1 pmol/L. We found no cross-reactivity with C-peptide in the assay, and decreased recovery attributable to the presence of insulin could be demonstrated only with a 30-fold excess of this hormone over proinsulin.
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PMID:Amplified enzyme-linked immunoassay of human proinsulin in serum (detection limit: 0.1 pmol/L). 154 Oct 5

Immunoenzymometric assays (IEMAs) for human insulin and intact proinsulin were developed using the amplification system developed by Johannsson et al. (Clin. Chim. Acta 148 (1985) 119-124) for the detection of the enzyme alkaline phosphatase. The detection limit of the assays was 0.8 pmol/l for proinsulin and 0.8 pmol/l for insulin whereas it was 1.8 pmol/l and 2.3 pmol/l respectively for the homologous immunoradiometric assays (IRMA). These assays are superior to immunoradiometric assays in terms of sensitivity, shelf-life of the labelled antibody and suitability for automation.
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PMID:Sensitive amplified immunoenzymometric assays (IEMA) for human insulin and intact proinsulin. 157 36

A 'sandwich-type' enzyme immunoassay for the measurement of serum insulin is described in which a monoclonal antibody-alkaline phosphatase conjugate and an antibody-immobilized polystyrene solid phase are used. Serum samples of 50 microliters can be analyzed and the enzyme immunoassay is as sensitive as the conventional radioimmunoassay for insulin. The results obtained with ELISA correlate well with those of the radioimmunoassay (r = 0.9844) and the between-assay and within-assay coefficients of variation are less than 15% over the useful ranges of the assay (2-200 microIU/ml). The sensitivity is 2 microIU/ml and this can be increased by longer incubation times. The crossreaction with porcine insulin is 45%, with bovine insulin 30% and with human proinsulin 20%.
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PMID:Evaluation of a solid-phase monoclonal antibody-based enzyme immunoassay for insulin in human serum. 265 25

The gene encoding human proinsulin has been fused in-frame with the E. coli alkaline phosphatase gene (pho A) (EC 3.1.3.1). Two constructions are described. One construction consists of the entire proinsulin gene fused to the 5'-terminal end of pho A. In the other construction a 42 base pair DNA fragment has been deleted from the 3'-terminal end of the proinsulin gene. The two purified fusion proteins are enzymatically active showing a specific activity of 10-15 U/mg and 18-25 U/mg, respectively. The first construction exhibited insulin antigenicity and was used to design a simple competitive ELISA for insulin. The lower detection limit was found to be at least 2.5 ng/ml. Both fusion proteins were also shown to have potential for use in a competitive ELISA for proinsulin.
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PMID:The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion. 332 79

Osteoporosis is a known complication of diabetes mellitus, suggesting a role for insulin in bone homeostasis. We studied insulin receptors and insulin action in the osteoblast-like rat osteogenic sarcoma cell line ROS 17/2.8. These cells share many common features with the osteoblast, such as 1,25-dihydroxyvitamin D3 receptors, PTH receptors, and 1,25-dihydroxyvitamin D3-induced modulation of alkaline phosphatase activity and osteocalcin. Competition binding studies revealed high affinity insulin receptors, with an ED50 for insulin of 1 nM. The receptors were highly specific for insulin, with 60% inhibition of insulin binding by an antireceptor antibody, no competition by epidermal growth factor, and an ED50 of 300 nM for proinsulin. Steady state maximal insulin binding was obtained by 40 min at 37 C, and insulin degradation, as measured by trichloroacetic acid solubility, was 1%/h at 37 C. ROS cells readily internalized insulin, and under steady state binding conditions at 37 C, 56% of the cell-associated radioactivity consisted of intracellular material. Chloroquine (100 microM) inhibited intracellular processing of insulin, leading to a 300% increase in cell-associated insulin by 2 h (37 C). Photoaffinity labeling of the insulin receptor with the photosensitive analog of insulin, B2 (2-nitro-4-azidophenyl-acetyl)des-pheB1-insulin, followed by solubilization and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed specific bands of 125K and 430K mol wt under reducing and nonreducing conditions, respectively. Thus, the structure of insulin receptors in ROS cells appears comparable to that of insulin receptors of known target tissues. Insulin action was also examined. Insulin did not stimulate [2-3H]deoxyglucose uptake or [1-14C]leucine incorporation into protein. In contrast, physiological concentrations of insulin inhibited alkaline phosphatase activity in nonconfluent cells. After exposure to insulin for 24 h, alkaline phosphatase activity was decreased compared to basal by 39.5% and 50% with 5 and 50 ng/ml insulin, respectively. In conclusion, ROS cells bind insulin to specific receptors that are similar to insulin receptors on other target tissues; receptors internalize insulin, which is then processed through a chloroquine-sensitive pathway; insulin does not affect membrane substrate transport; and insulin does inhibit the activity of an enzyme that is important in bone metabolism. ROS cells represent a model for studying insulin effects on bone.
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PMID:Demonstration of insulin receptors and modulation of alkaline phosphatase activity by insulin in rat osteoblastic cells. 353 Jul 24

Enzyme amplification has proved to be a highly sensitive quantification technique for immunoassays. We have shown that by using a fluorescent end-point, even more sensitive enzyme amplification assays can be generated than hitherto reported. We describe some general properties of this system and demonstrate its application in an assay for human proinsulin in plasma. The detection system can be used to measure less than one thousandth of an attomole (1 zeptomole) of alkaline phosphatase, equivalent to about 350 molecules of alkaline phosphatase per well of a microtiter plate. We have used this system to construct a proinsulin assay with a sensitivity of 0.017 pmol/L.
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PMID:Determination of one thousandth of an attomole (1 zeptomole) of alkaline phosphatase: application in an immunoassay of proinsulin. 850 65

A synthetic DNA encoding human proinsulin was inserted in frame in the bacterial alkaline phosphatase gene. A homogeneous recombinant human proinsulin-alkaline phosphatase conjugate was obtained directly from the periplasm of Escherichia coli transformed with a plasmid carrying the hybrid gene. The recombinant conjugate was stable and could be produced in the bacteria. The immunological properties of the recombinant conjugate and those of the human insulin and human proinsulin were compared using a panel of six different human insulin-specific monoclonal antibodies. Three immunological groups were thus distinguished and one of them indiscriminately recognized all of the insulin-like molecules. One monoclonal antibody from this group was used in combination with the recombinant conjugate to develop successfully a competitive immunoenzymatic assay for detecting insulin.
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PMID:Engineering of a recombinant colorimetric fusion protein for immunodiagnosis of insulin. 889 Aug 93

Here we report a case of acute myelogenous leukemia (M2, FAB classification) presenting with cytogenetic abnormalities of ins(21;8), +del(8) without t(8;21). A 8;21 chromosome translocation is frequently found in acute myelogenous leukemia, especially in the M2 subtype. The translocation results in a fusion transcript between AML1 and MTG8 (ETO), assigned on chromosomes 21 and 8, respectively. Among patients with a t(8;21) abnormality, solid leukemic tumor deposits outside the marrow or good response to chemotherapy are observed frequently. Decrease in neutrophil alkaline phosphatase score and positive rate, and eosinophilia in bone marrow or the blast cells with Auer rods expressing CD19, CD56 antigens occur at a relatively high rate. Although our case lacked these clinical, cytological and cytochemical features, expression of chimeric AML1-MTG8 mRNA was detected. AML1-MTG8 fusion transcript may play a critical role in leukemogenesis of AML M2. Studies on this case may help to reveal the oncogenic function of the AML1-MTG8 fusion gene in AML M2.
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PMID:[Acute myelogenous leukemia with ins(21;8) expressing AML-1-MTG8 fusion transcript]. 896 Jun 65

Osteopenia has been ascribed to diabetics without residual insulin secretion and high insulin requirement. However, it is not known if this is partially due to disturbances in the IGF system, which is a key regulator of bone cell function. To address this question, we performed a cross-sectional study measuring serum levels of IGF-I, IGF-binding protein-1 (IGFBP-1), IGFBP-3, IGFBP-4 and IGFBP-5 by specific immunoassays in 52 adults with Type 1 (n=27) and Type 2 (n=25) diabetes mellitus and 100 age- and sex-matched healthy blood donors. In the diabetic patients, we further determined serum levels of proinsulin, intact parathyroid hormone (PTH), 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and several biochemical bone markers, including osteocalcin (OSC), bone alkaline phosphatase (B-ALP), carboxy-terminal propeptide of type I procollagen (PICP), and type I collagen cross-linked carboxy-terminal telopeptide (ICTP). Urinary albumin excretion was ascertained as a marker of diabetic nephropathy. Bone mineral density (BMD) of hip and lumbar spine was determined by dual-energy X-ray absorptiometry. Data are presented as means+/-s.e.m. Differences between the experimental groups were determined by performing a one-way analysis of variance (ANOVA), followed by Newman-Keuls test. Correlations between variables were assessed using univariate linear regression analysis and partial correlation analysis. Type 1 diabetics showed significantly lower IGF-I (119+/-8 ng/ml) and IGFBP-3 (2590+/-104 ng/ml) but higher IGFBP-1 levels (38+/-10 ng/ml) compared with Type 2 patients (170+/-13, 2910+/-118, 11+/-3 respectively; P<0.05) or healthy controls (169+/-5, 4620+/-192, 3.5+/-0.4 respectively; P<0.01). IGFBP-5 levels were markedly lower in both diabetic groups (Type 1, 228+/-9; Type 2, 242+/-11 ng/ml) than in controls (460+/-7 ng/ml,P<0. 01), whereas IGFBP-4 levels were similar in diabetics and controls. IGF-I correlated positively with IGFBP-3 and IGFBP-5 and negatively with IGFBP-1 and IGFBP-4 in all subjects. Type 1 patients showed a lower BMD of hip (83+/-2 %, Z-score) and lumbar spine (93+/-2 %) than Type 2 diabetics (93+/-5 %, 101+/-5 % respectively), reaching significance in the female subgroups (P<0.05). In Type 1 patients, BMD of hip correlated negatively with IGFBP-1 (r=-0.34, P<0.05) and IGFBP-4 (r=-0.3, P<0.05) but positively with IGFBP-5 (r=0.37, P<0. 05), which was independent of age, diabetes duration, height, weight and body mass index, as assessed by partial correlation analysis. Furthermore, biochemical markers indicating bone loss (ICTP) and increased bone turnover (PTH, OSC) correlated positively with IGFBP-1 and IGFBP-4 but negatively with IGF-I, IGFBP-3 and IGFBP-5, while the opposite was observed with bone formation markers (PICP, B-ALP) and vitamin D3 metabolites. In 20 Type 2 patients in whom immunoreactive proinsulin could be detected, significant positive correlations were found between proinsulin and BMD of hip (r=0.63, P<0.005), IGF-I (r=0.59, P<0.01) as well as IGFBP-3 (r=0.49, P<0.05). Type 1 and Type 2 patients with macroalbuminuria showed a lower BMD of hip, lower IGFBP-5 but higher IGFBP-4 levels, suggesting that diabetic nephropathy may contribute to bone loss by a disturbed IGF system. In conclusion, the findings of this study support the hypothesis that the imbalance between individual IGF system components and the lack of endogenous proinsulin may contribute to the lower BMD in Type 1 diabetics.
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PMID:Serum levels of insulin-like growth factor system components and relationship to bone metabolism in Type 1 and Type 2 diabetes mellitus patients. 979 71

Autosomal dominant diabetes in the Akita mouse is caused by mutation of the insulin 2 gene, whose product replaces a cysteine residue that is engaged in the formation of an intramolecular disulfide bond. These heterozygous mice exhibit severe insulin deficiency despite coexpression of normal insulin molecules derived from three other wild-type alleles of the insulin 1 and 2 genes. Although the results of our previous study suggested that the mutant proinsulin 2 is misfolded and blocked in the transport from the endoplasmic reticulum to the Golgi apparatus, its dominant negative nature has not been fully characterized. In the present study, we investigated the possible pathogenic mechanisms induced by the mutant proinsulin 2. There is no evidence that the mutant proinsulin 2 attenuates the overall protein synthesis rate or promotes the formation of aberrant disulfide bonds. The trafficking of constitutively secreted alkaline phosphatase, however, is significantly decreased in the islets of Akita mice, indicating that the function of early secretory pathways is nonspecifically impaired. Morphological analysis has revealed that secretory pathway organelle architecture is progressively devastated in the beta-cells of Akita mice. These findings suggest that the organelle dysfunction resulting from the intracellular accumulation of misfolded proinsulin 2 is primarily responsible for the defect of coexisting wild-type insulin secretion in Akita beta-cells.
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PMID:Dominant negative pathogenesis by mutant proinsulin in the Akita diabetic mouse. 1254 Jun 15


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