ABSTRACT
Substance P has been implicated as a mediator of inflammation. The involvement of this neuropeptide in carrageenan-induced hind paw edema in the rat was assessed. Subcutaneous injection of carrageenan into the rat paw caused a significant increase in substance P levels, which preceded the onset of inflammation. While injection of substance P alone caused mild edema, coadministration of submaximal doses of carrageenan and substance P resulted in a synergistic exacerbation in the degree of inflammation. This synergistic response was not detected when the nonamidated precursor of substance P was coinjected with carrageenan. The effects of substance P depletion on inflammation were also evaluated. In animals pretreated with capsaicin followed by injection with carrageenan, no significant increase in either the levels of substance P or the extent of edema was observed when compared to capsaicin-treated controls. These results indicate that substance P may play an important role in the early stages of carrageenan-induced paw edema and that a reduction in the biosynthesis of substance P may lessen the severity of this inflammatory response.
Subject(s)
Carrageenan , Edema/chemically induced , Foot , Substance P/pharmacology , Animals , Capsaicin/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Edema/metabolism , Edema/pathology , Female , Foot/pathology , Organ Size/drug effects , Rats , Rats, Sprague-Dawley , Substance P/metabolismSubject(s)
Ditiocarb/pharmacology , Hypothalamus/metabolism , Multienzyme Complexes , Spinal Cord/metabolism , Substance P/metabolism , Animals , Hypothalamus/drug effects , Mixed Function Oxygenases/metabolism , Mixed Function Oxygenases/pharmacology , Rats , Rats, Inbred Strains , Reference Values , Spinal Cord/drug effects , Substantia Nigra/drug effects , Substantia Nigra/metabolismABSTRACT
A sensitive alpha-amidating enzyme (alpha AE) assay using C-terminal glycine-extended substance P (SP-Gly) as a substrate was developed. The product, substance P (SP), was measured by a radioimmunoassay with specific polyclonal antibodies which recognize SP with an affinity 10,000-fold higher than that of SP-Gly. The sensitivity of the radioimmunoassay was 5 fmol. Enzyme activity could be readily detected with 25 ng alpha AE partially purified from the conditioned medium of rat medullary thyroid carcinoma CA-77 cells. The Km and Vmax values were 2.0 +/- 0.2 microM and 1.7 +/- 0.1 nmol/mg/min (mean +/- SE, n = 3), respectively. The assay enabled the kinetic characterization of alpha AE from a single rat pituitary homogenate. Optimal Cu2+ required was 30 microM and greater than 3 mM of ascorbate was needed for maximal enzyme activity. The sensitivity of this assay will aid efforts to examine the regulation of in vivo alpha AE activity.
Subject(s)
Mixed Function Oxygenases , Multienzyme Complexes , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Animals , Carcinoma/pathology , Culture Media , Goats , Kinetics , Male , Pituitary Gland/enzymology , Rabbits , Radioimmunoassay , Rats , Rats, Inbred Strains , Substance P/analogs & derivatives , Substance P/metabolism , Thyroid Neoplasms/pathology , Tissue Extracts/metabolism , Tumor Cells, CulturedABSTRACT
Conditioned medium from cultures of rat medullary thyroid carcinoma CA-77 cells was used as a source for purification of the secreted forms of peptidyl alpha-amidating enzyme. The alpha-amidating enzyme activity was partially purified using a combination of weak anion exchange and gel filtration chromatography. Subsequent strong anion exchange chromatography at pH 6.0 resolved this partially pure enzyme into four distinct peaks of activity, termed Ia, Ib, II, and III. Peaks Ia and Ib exhibited broad pH optima between pH 6.0-8.5, whereas peaks II and III both exhibited pH optima at approximately pH 5.0. The peak III activity was further purified to electrophoretic homogeneity using hydrophobic interactive chromatography followed by strong anion exchange chromatography at pH 8.0. The enzyme exhibited an apparent molecular mass of 75K, a pH optimum of approximately pH 5.0, and a maximal turnover number of 580 min-1 in the presence of L-ascorbate. Kinetic studies demonstrated that the enzyme probably functions through a ping-pong mechanism with respect to the binding of the glycine-extended peptide substrate and the L-ascorbate cofactor. The peak III enzyme exhibits several distinctive characteristics compared to amidating enzymes isolated and characterized by other laboratories.
Subject(s)
Isoenzymes/metabolism , Mixed Function Oxygenases , Multienzyme Complexes , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Thyroid Neoplasms/enzymology , Animals , Cell Line , Chromatography, Gel , Chromatography, Ion Exchange , Culture Media , Isoenzymes/isolation & purification , Kinetics , Oxidoreductases Acting on CH-NH Group Donors/isolation & purification , RatsABSTRACT
A peptidyl alpha-amidating enzyme has been partially purified from conditioned medium derived from cultured medullary thyroid CA-77 cells. The interactions of this enzyme with a series of tripeptides, pentapeptides, and mature glycine-extended prohormones has now been studied using a competition assay that features the enzymatic alpha-amidation of N-dansyl-Tyr-Val-Gly. While a peptide C-terminal glycine was obligatory for tight binding to the alpha-amidating enzyme, other peptide structural elements modulated the interaction. Thus, a greater than 1300-fold range in apparent inhibitor constants was observed by substitution at the -1 (penultimate) position in a C-terminal glycine-containing tripeptide with each of the 20 common L-amino acids. Peptide inhibitory potency decreased through the following amino acid groupings: sulfur containing greater than aromatic greater than or equal to histidine greater than nonpolar greater than polar greater than glycine greater than charged. This pattern was qualitatively dissimilar to that observed for a more limited series of substitutions at the -2 position, demonstrating the positional selectivity of these structural requirements. The structure-activity relationships observed with the tripeptides at the -1 position were consistent with the apparent inhibitor constants obtained for a collection of prohormones and their pentapeptide mimics. Finally, selected prohormones and their pentapeptide mimics were equipotent inhibitors, demonstrating that the peptide structural elements important for alpha-amidating enzyme recognition are located entirely within the C-terminal pentapeptide segment.
Subject(s)
Carcinoma/enzymology , Mixed Function Oxygenases , Multienzyme Complexes , Oxidoreductases Acting on CH-NH Group Donors/isolation & purification , Peptides/metabolism , Thyroid Neoplasms/enzymology , Animals , Binding Sites , Binding, Competitive , Cell Line , Glycine/metabolism , Growth Hormone-Releasing Hormone/pharmacology , Kinetics , Neuropeptides/pharmacology , Oxidoreductases Acting on CH-NH Group Donors/antagonists & inhibitors , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Rats , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
A rapid and sensitive method for the determination of peptidyl alpha-amidation activity has been developed and is based on reverse-phase high-performance liquid chromatographic separation and fluorometric detection. A dansylated tripeptide, N-dansyl-Tyr-Val-Gly-OH, is used as the substrate in the assay and the amount of alpha-amidation activity is determined by quantitating the extent of its conversion to product, N-dansyl-Tyr-Val-NH2. Both product and substrate can be detected in a single assay in quantities as low as 5 fmol by isocratic elution using C-18 reverse-phase columns. The method yields highly reproducible results and requires less than 3 min per sample for separation and quantitation. The assay procedure is applicable to the screening of a large number of samples under different pH conditions and is readily adaptable for use in a variety of studies. For example, the procedure is ideal for detecting alpha-amidation activity in various tissues, monitoring activity at the different stages during purification of a particular alpha-amidation enzyme, determining kinetic parameters of the purified enzyme, and identifying both competitive and noncompetitive inhibitors.