Search PubMed⌕ Search

Biomedical subjects

K M Metters

Publications and source records attributed to K M Metters.

54 records · Page 3Linked to original sources
PubMed1993

Photoaffinity labelling and radiation inactivation of the leukotriene B4 receptor in human myeloid cells.

The leukotriene (LT) B4 receptor has been characterized in the human monocyte leukemia THP-1 cell line. Scatchard analysis of [3H]LTB4 specific binding to THP-1 cell membranes revealed a single population of high affinity (KD = 56 pM) and saturable (2000 receptors/cell) binding sites. [3H]LTB4 specific binding was enhanced by divalent cations, but inhibited by both monovalent cations and a non-hydrolysable GTP analogue. Treatment with GTP analogue resulted in a concentration-dependent reduction in the number of high affinity binding sites, accompanied by the appearance of an equal number of binding sites of lower affinity (KD = 1250 pM). In contrast, Scatchard analysis with human polymorphonuclear leukocyte (PMN) membranes consistently revealed two populations of LTB4 receptors (KD = 48 pM and 270 pM). Treatment with GTP analogue, however, converted all these detectable binding sites to the lower affinity state. These data suggest that the LTB4 receptor in both THP-1 cell and PMN membranes exists in interconverting affinity states modulated by G-protein coupling. The similarity between the LTB4 receptors present in these two cell types was also substantiated by target-size analysis by radiation inactivation, which estimated a comparable molecular mass of 56.5 kDa and 52.8 kDa for the THP-1 cell and PMN LTB4 receptors, respectively. Finally, the presence of a single LTB4 receptor in PMN was demonstrated by direct photolabelling. Irradiation of frozen [3H]LTB4 equilibrium binding assay incubations resulted in complete photolysis of [3H]LTB4. Subsequent resolution of the tritiated PMN proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) revealed one major radioactive peak migrating with an apparent molecular weight of 61,000. This peak was identified as the LTB4 receptor since radiolabelling could be completely inhibited by the presence of excess unlabelled LTB4 or the LTB4-receptor antagonist, L-662,328. Photolabelling was also partially inhibited by pretreatment with GTP analogue, consistent with G-protein uncoupling reagents reducing receptor affinity without complete inhibition. In summary, the LTB4 receptor identified in human myeloid cells is a G-protein coupled receptor with interconvertible high and low affinity states, having a molecular mass of 53-61 kDa.

Affinity Labels↗
PubMed1993

Identification of both NK1 and NK2 receptors in guinea-pig airways.

1. NK1 and NK2 receptors have been characterized in guinea-pig lung membrane preparations by use of [125I-Tyr8]-substance P and [125I]-neurokinin A binding assays in conjunction with tachykinin-receptor selective agonists ([Sar9Met(O2)11]substance P for NK1 and [beta Ala8]neurokinin A (4-10) for NK2) and antagonists (CP-99,994 for NK1 and SR48968 for NK2). 2. The presence of high affinity, G-protein-coupled NK1 receptors in guinea-pig lung parenchymal membranes has been confirmed. The rank order of affinity for competing tachykinins was as predicted for an NK1 receptor: substance P = [Sar9Met(O2)11]substance P > substance P-methyl ester = physalaemin > neurokinin A = neurokinin B >> [beta Ala8]neurokinin A (4-10). The novel NK1 antagonist CP-99,994 has a Ki of 0.4 nM at this NK1 site. 3. In order to characterize [125I]-neurokinin A binding to guinea-pig lung, the number of [125I]-neurokinin A specific binding sites was increased 3-4 fold by purification of the parenchymal membranes over discontinuous sucrose gradients. The rank order of affinity determined for NK1- and NK2-receptor agonists and antagonists in competition for these sites showed that the majority (80%) of [125I]-neurokinin A specific binding was also to the NK1 receptor. 4. Under conditions where the guinea-pig lung parenchymal NK1 receptor was fully occupied by a saturating concentration of either [Sar9Met(O2)11]substance P (1 microM) or CP-99,994 (2.7 microM), residual [125I]-neurokinin A specific binding was inhibited in a concentration-dependent manner by both [beta Ala8]neurokinin A and SR48968. This result shows that the NK2 receptor is also present in these preparations. 5. Similar studies using guinea-pig tracheal membranes demonstrated that [125I]-neurokinin A specific binding was composed of a NK1-receptor component (60%), inhibited by both [Sar9Met(02)11]substance P and CP-99,994, and a significant NK2-receptor component, inhibited by both [beta Ala 8]neurokinin A andSR48968.6. In summary, these data demonstrate that guinea-pig lung parenchyma and guinea-pig trachea express both NK1 and NK2 receptors.

Animals↗
PubMed1992

Purification of human leukotriene C4 synthase from dimethylsulfoxide-differentiated U937 cells.

Human leukotriene C4 (LTC4) synthase was purified > 10000-fold from dimethylsulfoxide-differentiated U937 cells. Steps included: (a) solubilization of membrane-bound LTC4 synthase from microsomal membranes by the anionic detergent taurocholate; (b) successive anion-exchange chromatography steps in the presence of taurocholate plus Triton X-100 (primary anion exchange) then taurocholate plus n-octyl glucoside (secondary anion exchange); and (c) LTC2-affinity chromatography on a matrix that was constructed by first biotinylating synthetic LTC2 then immobilizing the biotinylated LTC2 on streptavidin agarose. The purification of human LTC4 synthase was enabled by the finding that LTC4 synthase activity in preparations enriched > 500-fold was absolutely dependent on the presence in LTC4 synthase incubation mixtures of divalent cations (specifically Mg2+) and phospholipids (specifically phosphatidylcholine), and that reduced glutathione, which was required at 2-4 mM for stabilization of LTC4 synthase, irreversibly inactivated the enzyme when present at > or = 5 mM during freeze/thaw cycles. The > 10000-fold purified LTC4 synthase preparation was comprised of three polypeptides having molecular masses of 37.1, 24.5 and 18.0 kDa. An 18-kDa polypeptide in both microsomal membranes and in the LTC2-affinity purified fraction was specifically labelled by a radioiodinated LTC4 photoaffinity probe (azido 125I-LTC4). The Km values in the LTC2-affinity purified preparation for reduced glutathione and LTA4 were 1.83 mM and 19.6 microM (respectively), closely resembling the Km values in isolated human blood monocytes. The Vmax of LTC2-affinity purified LTC4 synthase was 2-4 mumol LTC4 formed .min-1 x mg-1.

Cell Differentiation↗
PubMed1991

Characterization of the leukotriene D4 receptor in hyperreactive rat lung.

A [3H]leukotriene D4 radioreceptor binding assay has been established in rat lung and has been used to fully characterize the leukotriene D4 receptor in lung membranes from an inbred strain of rats displaying non-specific bronchial hyperreactivity. [3H]leukotriene D4 specific binding in this tissue is of high affinity (KD 0.12 nM), saturable (Bmax 42 fmol/mg protein), inhibited by both guanine nucleotide analogues and sodium ions and increased by divalent cations. In addition, Ki values show that agonists, but not antagonists, compete for [3H]leukotriene D4 binding in rat lung with the same potency as they compete for [3H]leukotriene D4 binding in guinea-pig lung, the classical tissue for leukotriene D4 receptor studies. Finally, [3H]leukotriene D4 binding in hyperreactive rat lung has been compared with [3H]leukotriene D4 binding in lung tissue from Fischer rats, which are a less responsive strain.

Animals↗
PubMed1990

Detection of synenkephalin, the amino-terminal portion of proenkephalin, by antisera directed against its carboxyl terminus.

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.

Adrenal Gland Neoplasms↗
PubMed1988

Selective cleavage of proenkephalin-derived peptides (less than 23,300 daltons) by plasma kallikrein.

The ability of human plasma kallikrein to hydrolyze several proenkephalin-derived peptides has been studied, including the synthetic peptides BAM 12P and peptides E, F, and B as well as synenkephalin-containing peptides (8.6, 18.2, and 23.3 kDa) purified from bovine adrenal medulla chromaffin granules. All the identified cleavages occurred either COOH-terminal to or between pairs of basic amino acids, with plasma kallikrein recognizing Lys-Lys, Lys-Arg, and Arg-Arg as processing signals. Moreover, plasma kallikrein was found to cleave at the COOH terminus of the basic pairs of amino acids preceding enkephalin sequences thereby releasing the biologically active form of the peptide with the free NH2-terminal Tyr needed for receptor recognition.

Adrenal Medulla↗
PubMed1988

Enkephalin is liberated from metorphamide and dynorphin A1-8 by endo-oligopeptidase A, but not by metalloendopeptidase EC 3.4.24.15.

It has been previously reported that both the cysteinyl-endo-oligopeptidase A and the metalloendopeptidase EC 3.4.24.15 are able to generate enkephalin from a number of enkephalin-containing peptides, including dynorphin A1-8. The present study shows that only endo-oligopeptidase A is able to generate [Leu5]enkephalin and [Met5]enkephalin from dynorphin A1-8 and from metorphamide respectively. It is also shown that endo-oligopeptidase A neither hydrolyses the specific EC 3.4.24.15 substrate alpha-N-benzoyl-Gly-Ala-Ala-Phe p-aminobenzoate, nor is inhibited by the specific EC 3.4.24.15 inhibitor N-[1(RS)-carboxy-2-phenylethyl]-alpha-Ala-Ala-Phe p-aminobenzoate.

Chemical Precipitation↗
PubMed1988

Liberation of enkephalins from enkephalin-containing peptides by brain endo-oligopeptidase A.

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterised as a thiol endopeptidase. This enzyme, known to hydrolyse the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin has been shown to produce, by a single cleavage, [Leu]enkephalin or [Met]enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be 8-13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin and dynorphin B are of the same order. Taken together, these results indicate that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme. Finally, endo-oligopeptidase A and metalloendopeptidase EC 3.4.24.15 are two different enzymes. The latter is not able to liberate enkephalins from metorphamide and dynorphin.

Amino Acid Sequence↗
PubMed1987

Chromogranin A can act as a reversible processing enzyme inhibitor. Evidence from the inhibition of the IRCM-serine protease 1 cleavage of pro-enkephalin and ACTH at pairs of basic amino acids.

Bovine parathyroid chromogranin A inhibits the cleavage of Z-Ala-Lys-Arg-AMC by either trypsin or IRCM-serine protease 1 (IRCM-SP1), a putative novel processing enzyme originally isolated from porcine pituitary anterior and neurointermediate lobes. On larger substrates, chromogranin A is a reversible competitive inhibitor of the cleavage at pairs of basic amino acids by IRCM-SP1. The substrates tested included pituitary ACTH and adrenal medulla pro-enkephalin-derived peptides such as the 8.6 kDa synenkephalin-containing precursor and peptide B. Chromogranin A is itself selectively processed by IRCM-SP1, and ACTH was shown to compete for such cleavage. These data suggest that chromogranins as a class of acidic proteins could participate in the tissue-specific processing of pro-hormones.

Adrenocorticotropic Hormone↗
PubMed1987

Affinity purification of synenkephalin-containing peptides, including a novel 23.3-kilodalton species.

Affinity chromatography has been used for rapid and high-yield purification of synenkephalin (proenkephalin 1-70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN-Br-activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mM ammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7-3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low-molecular-weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high-molecular-weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6- and 12.6-kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182-203) and [Met5]-enkephalin-Arg6-Gly7-Leu8. The former column retains the 23.3-kilodalton species, whereas the latter column retains the 18.2-kilodalton species. The 23.3-kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin-1-206), containing [Leu5]-enkephalin at the C-terminus.

Animals↗
PubMed1987

Brain endo-oligopeptidase A, a putative enkephalin converting enzyme.

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterized as a thiol endopeptidase. This enzyme, known to hydrolyze the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin, has been shown to produce, by a single cleavage, Leu5-enkephalin or Met5-enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration-dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be eight to 13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin, and dynorphin B are of the same order, a result indicating that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme.

Animals↗
PubMed1986

A processing enzyme for prodynorphin derived peptides.

Endo-oligopeptidase A known to hydrolyse the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin has been shown to produce, by a single cleavage, leucine5-enkephalin from small prodynorphin derived enkephalin-containing peptides. The specificity constants (kcat/km) obtained for the hydrolysis of bradykinin, neurotensin and dynorphin B are of the same order, suggesting that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme.

Amino Acid Sequence↗
PubMed1985

Partial purification and characterisation of a 31 kDalton putative enkephalin precursor from guinea pig striatum.

The presence of several putative precursors for Met-enkephalin (ME) and Leu-enkephalin (LE) has been demonstrated in guinea pig striatal extracts. The major high molecular weight (Mr) species (31000) contained 9% of the total ME-immunoreactivity (IR) after digest. This species has been partially purified and characterized using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and nitrocellulose blotting techniques as well as highly specific radioimmunoassay (RIA) for ME, LE and ME-Arg6-Phe7.

Animals↗
PubMed1985

A comparison of opioid peptide precursors in guinea pig, rat and bovine striata and guinea pig adrenal.

In this study guinea pig, rat and bovine striatal extracts have been shown to contain putative opioid peptide precursors in the molecular weight (Mr) range 2000-greater than 70000. The material eluting greater than 70 kDalton contained bioassayable opioid activity but not Met- or Leu-enkephalin-like immunoreactivities (ME-IR and LE-IR) and may represent a novel endogenous opioid-peptide precursor.

Adrenal Glands↗
PubMed1985

Identification of a proenkephalin precursor in striatal tissue.

Recent studies have supported the suggestion that proenkephalin is the same in both adrenal medulla and brain. However, although previous investigations have characterized enkephalin-containing adrenal intermediates derived from proenkephalin, as yet no such intermediates have been isolated from the brain. This has led to the belief that the processing of proenkephalin in the brain is extremely rapid and enkephalin-containing intermediates do not accumulate. In this investigation Sephacryl-300 gel filtration chromatography of guinea pig striata, extracted in 8 M urea, demonstrated several peaks of both bioactive and immunoreactive enkephalin-like peptides after enzymatic digest (trypsin followed by carboxypeptidase B). Comparable profiles were obtained using rat and bovine striatal tissue. In guinea pig the major species emerging from gel filtration, eluting with an apparent molecular weight of 29,000, represented approximately 9% of the total (methionine) enkephalin immunoreactivity. It had an apparent pI of 5.0 when subjected to chromatofocusing. This species was further characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and nitrocellulose blotting techniques as well as highly specific radioimmunoassays to (Met5)-enkephalin, (Leu5)-enkephalin, and (Met5)-enkephalin-Arg6-Phe7. This species was found to contain these opioid peptides in an approximately 6:1:1 ratio, respectively, and to have an apparent molecular weight of 31,000. It was also indicated that (Met5)-enkephalin-Arg6-Phe7 constituted the C-terminal seven residues of this molecule.

Animals↗
PubMed1980

Binding of [3H]cimetidine to rat brain tissue.

Binding of [3H]cimetidine to rat brain tissue was investigated, and a saturable binding with dissociation constant 0.22 +/- 0.05 microM found. This binding is inhibited by a range of imidazole-derived histamine H2-receptor antagonists, but not by a number of non-imidazole H2-receptor antagonists. It is concluded that the [3H]cimetidine binding site in rat brain tissue that is labelled in these experiments is not the histamine H2-receptor.

Animals↗