Purification and properties of parathyroid hormone-related peptide isolated from milk.

The occurrence and properties of PTH-related peptide (PTH-RP) in milk was investigated. PTH-RP was purified to homogeneity from human and bovine milk using heat and acid to precipitate milk proteins followed by ion exchange chromatography and reverse-phase HPLC. The peak of PTH-RP from HPLC was detected using a sensitive bone cell bioassay. A single band of peptide was detected on silver-stained polyacrylamide gels, which migrated as a 20-21-kDa macromolecule. PTH-RP isolated from either human or bovine milk had similar electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The partially purified bovine PTH-RP stimulated cAMP production in UMR106-01 and OK cell lines and elicited a concentration-dependent inhibition of sodium-dependent phosphate transport in OK cells. Incubation of milk extracts with an anti-PTH antiserum did not affect their bioactivity, whereas an antihuman PTH-RP 1-34 antiserum markedly reduced the cAMP response of UMR106-01 cells to the immunoabsorbed milk extracts. A PTH antagonist, norleu PTH 3-34, blocked the stimulation of cAMP production in UMR106-01 cells treated with milk extracts. PTH-RP immunoreactivity and bioactivity occurred in milk extracts of diverse animals from both eutherian and metatherian (marsupial) species. Porcine colostrum also had immunoreactive PTH-RP, although the levels were lower than the immunoreactive PTH-RP concentrations observed in milk samples collected at 7 and 14 days of lactation. Thus, a 20-21-KDa PTH-RP is secreted into milk where it could play a role in the development of suckling, newborn animals.

Ethanol modulates epidermal growth factor-stimulated tyrosine kinase and phosphorylation of PLC-gamma 1.

A431 cells have an abundance of Epidermal Growth Factor (EGF) receptors which possess intrinsic tyrosine kinase activity. Treatment of membranes isolated from A431 cells with EGF caused a 2-3 fold increase in phosphorylation of a synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly) which is a substrate for tyrosine kinase. Treatment of these membranes with 0.1 to 100 mM ethanol altered basal tyrosine kinase activity in a biphasic manner; increase at 10 mM and decrease at 100 mM ethanol. The treatment of the membranes with the same concentrations of ethanol also altered EGF's ability to stimulate tyrosine kinase activity: increase at 0.1 mM ethanol and decrease at 10 mM. Strikingly, EGF-stimulated tyrosine kinase was more sensitive to ethanol than the basal activity. Experiments with other alcohols showed a relationship between chain length and the inhibitory ability of the alcohol. These data demonstrate a biochemical effect of low concentrations of ethanol on tyrosine kinase. Interestingly, ethanol treatment of A431 cells inhibited EGF-stimulated phosphorylation of PLC-gamma 1 which is a substrate for EGF receptor tyrosine kinase. It is concluded that ethanol at low concentrations has significant modulatory effect on basal and EGF-stimulated tyrosine kinase, as well as PLC-gamma 1 phosphorylation.

Role of guanine nucleotide-binding protein and tyrosine kinase in platelet-activating factor activation of phospholipase C in A431 cells: proposal for dual mechanisms.

A431 cells, a human epidermoid carcinoma, possess specific [3H]platelet-activating factor (PAF) and [3H]WEB 2086 binding sites indicating the presence of PAF receptors. PAF-stimulated PLC as determined by the increase in inositol phosphate levels. Pretreatment of A431 cells with genistein, a putative tyrosine kinase inhibitor, abolished the ability of PAF to activate PLC, whereas pretreatment with staurosporine, a protein kinase C inhibitor, potentiated the ability of PAF to activate PLC. Pretreatment of A431 cells with phorbol-12-myristate-13-acetate, a protein kinase C activator, blocked PAF-stimulated PLC. Overnight exposure of cells to pertussis toxin (PT) partially blocked the ability of PAF to stimulate PLC. Based on these observations the involvement of PT-sensitive and -insensitive guanine nucleotide-binding protein(s) (G-protein) as well as the role of tyrosine kinase in the activation of PLC by PAF was considered further. PT treatment of A431 cell membranes obliterated PAF-stimulated GTPase and indicated that PT-insensitive membrane-associated G-proteins were not involved in PAF actions. In alpha-toxin permeabilized cells, PT blocked GTP-gamma-S potentiation of PLC activation by PAF, thus suggesting that PT-insensitive G-proteins were not involved in PAF activation of PLC in A431 cells. PAF stimulated tyrosine kinase activity as observed with the increase in radioactivity associated with proteins immunoprecipitated with polyclonal antibodies to phosphotyrosine residues. This increase was blocked by PAF receptor antagonists, CV 6209 and TCV 309, and by pretreatment with genistein. PAF also activated the phosphorylation of pp60c-src and Src associated proteins in A431 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of testosterone and 6beta-hydroxytestosterone by gas chromatography-selected ion monitoring-mass spectrometry for the characterization of cytochrome p450 3A activity.

A method for the determination of testosterone and its metabolite, 6beta-hydroxytestosterone, in liver microsomal incubates employing gas chromatography with selected ion monitoring mass spectrometric detection (GC-SIM-MS) has been developed. The method is more rapid than previously reported methods. Testosterone and its metabolites are extracted from the incubation mixture in a single step with methylene chloride. The method does not require derivatization and testosterone and its metabolites are separated on a HP-5MS fused-silica capillary column in less than 15 min. The retention times of testosterone (m/z 288), methyltestosterone (m/z 302), and 6beta-hydroxytestosterone (m/z 304) are approximately 12.7, 12.8, and 13.4 min, respectively. There are no interferences from other known CYP450 metabolites of testosterone. In addition, the selectivity and specificity of the mass spectrometer helps eliminate possible interferences from drugs and new chemical entities evaluated using this methodology. Calibration curves for testosterone and 6beta-hydroxytestosterone are linear from 0.25 to 100 microM. Extraction recoveries are better than 92% for both analytes and the internal standard, methyltestosterone. Over the course of five separate runs, within-day and inter-day precision (expressed as relative standard deviation) was less than 5% for all concentrations of testosterone and 6beta-hydroxytestosterone. Accuracies ranged from 95.8 to 105.8% for testosterone and 94.6 to 104.2% for 6beta-hydroxytestosterone. The assay has been used to characterize the CYP3A metabolic activity of multiple preparations of human, rat, and dog liver microsomes.