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J A Koehn

Publications and source records attributed to J A Koehn.

16 recordsLinked to original sources
PubMed1999

Rapid kinetics of tyrosyl radical formation and heme redox state changes in prostaglandin H synthase-1 and -2.

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34-35 G) was formed by 4 ms, followed by transition to a wide singlet (33-34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25-26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.

Animals↗
PubMed1998

The expression, refolding, and purification of the catalytic domain of human collagenase-3 (MMP-13).

We have cloned, expressed, and purified a recombinant C-terminal truncated form (residues Glu103-Asn274) of human collagenase-3 (MMP-13) in Escherichia coli. The molecule contains the catalytic domain of the enzyme and is expressed almost exclusively as inclusion bodies. Using a combination of rapid dilution and diafiltration, the enzyme has been successfully refolded from these inclusion bodies. The protein was purified to homogeneity using cation-exchange and size-exclusion chromatography. The purified enzyme is a monomer with a Mr of approximately 19,600 and was characterized using a variety of techniques including, SDS-PAGE, RP-HPLC, LC-MS, amino acid analysis, and dynamic light scattering. Microheterogeneity at the NH2-terminus of the refolded, purified protein disappeared after incubating for 30-60 min at 37 degreesC. The enzyme was highly active using a fluorescent peptide substrate and was found to release S-GAG from bovine nasal cartilage chips.

Amino Acids↗
PubMed1989

Rat vascular tissue contains a neutral endopeptidase capable of degrading atrial natriuretic peptide.

Neutral endopeptidase (NEP, EC 3.4.24.11), purified from renal brush border, cleaves the Cys7-Phe8 amide bond of rat atrial natriuretic peptide (ANP), to generate the inactive metabolite (ANP cleaved at the Cys7-Phe8 bond; x-ANP). To determine if NEP contributes to the inactivation of circulating ANP, we investigated the degradation of rat ANP (rANP, 1-28) in the vasculature. Formation of x-ANP from exogenous ANP was studied in a mesenteric arterial preparation by perfusion in a single pass system in the presence and absence of the NEP inhibitors, thiorphan or phosphoramidon. In addition, a purified membrane fraction was prepared from mesenteric arterial homogenate and compared with an equivalent renal membrane fraction. Formation of x-ANP was quantified by a specific immunoassay (ELISA). Renal and vascular membranes shared the same pH optima for x-ANP formation (pH 7.5), although x-ANP generation was considerably greater in renal vs. vascular membranes (31.6 and 0.4 nmol min-1 mg-1 of protein, respectively). Both preparations were inhibited in a similar, dose-dependent manner by phosphoramidon, thiorphan or a polyclonal antibody to NEP. In perfused mesenteric arteries, 1.6 +/- 0.8 pmol of x-ANP were generated from 1 microgram of ANP; this formation was inhibited 51% by 10 microM phosphoramidon or thiorphan. Plasma levels of x-ANP after bolus i.v. administration of rANP in rats, were inhibited (70-80%) by thiorphan at comparable doses to those used in perfused mesenteric arteries. These studies indicate that ANP is degraded in the vasculature by NEP or an "NEP-like" enzyme(s).

Animals↗
PubMed1987

Degradation of atrial natriuretic factor by kidney cortex membranes. Isolation and characterization of the primary proteolytic product.

Synthetic rat atrial natriuretic factor (r-ANF, 1-28) was incubated with rat kidney cortex membranes, and a predominant degradation product was identified by reverse-phase high performance liquid chromatography. The degradation product was subjected to amino acid analyses and found to have a composition identical to r-ANF. Amino-terminal sequence analyses identified two distinct amino-terminal residues and suggested that cleavage had occurred between the cysteine-phenylalanine bond (residues 7 and 8) of r-ANF. This degradative process could be inhibited by 1,10-phenanthroline and EDTA, suggesting that the enzyme responsible for proteolysis is a metalloendoprotease. The enzyme exhibits a Michaelis-Menten constant of approximately 10 microM for the metabolism of r-ANF and has a broad pH optimum between 8.5 and 9.5. These findings suggest that ANF may be initially degraded in the kidney at a single cleavage site within the 17-residue ring structure.

Amino Acid Sequence↗
PubMed1986

Development of a radioimmunoassay for the fibrinogen-derived peptide B beta 1-42.

The peptide B beta 1-42 is the initial cleavage product of plasmin-mediated proteolysis of the NH2-terminal region of fibrinogen or fibrin I, while beta 15-42 is the major fragment released by plasmin degradation of fibrin II. Numerous studies have described the measurement of plasma B beta 1-42 levels as an index of plasmin activity. Previous assays were indirect and included quantitation of thrombin-increasable fibrinopeptide B immunoreactivity (TIFPB) or measurement of beta 15-42 with an antiserum (132) which cross-reacted with B beta 1-42. We report on a new antiserum (R142) directed against B beta 1-42 which does not cross-react with beta 15-42 or fibrinopeptide B. Employing this antiserum, a specific assay for B beta 1-42 was developed. This assay was used to measure plasmin-mediated B beta 1-42 release from fibrinogen and its subsequent proteolysis by thrombin. The selectivity constant (Kcat/Km) for thrombin cleavage of the B beta 14-15 bond of B beta 1-42 was 10(5)-fold less than that for proteolysis of the same bond in the intact fibrinogen molecule, thus explaining the stability of this peptide in the presence of thrombin activity in the blood. Similarly, the selectivity constant for plasmin cleavage of the B beta 21-22 bond of B beta 1-42 was 140-fold less than that for proteolysis of the B beta 42-43 bond of fibrinogen, indicating that secondary plasmin attack of the B beta 1-42 molecule is not physiologically important. The specific B beta 1-42 assay provided excellent recovery of peptide added to blood or plasma. Comparison of B beta 1-42 levels with TIFPB values in 37 patient samples yielded good correlation over a wide range of levels (r2 = 0.91). The median plasma B beta 1-42 level in 15 normal individuals was 1.2 pmol/mL. This is similar to the previously reported normal range for TIFPB (1 to 4 pmol/mL) but is higher than the normal level of 0.4 pmol/mL reported with the assay employing antiserum 132. This discrepancy reflects rapid removal of Arg (B beta 42) by plasma carboxypeptidase activity resulting in 50% loss of B beta 1-42 immunoreactivity with antiserum 132 but no loss with R142. To circumvent this problem, we have developed a beta 15-42 antiserum (R154) which, like antiserum 132, cross-reacts with B beta 1-42. However, B beta 1-42/beta 15-42 immunoreactivity with R154 is stable in the presence of carboxypeptidase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Drug Stability↗
PubMed1985

Characterization of fibrinogen New York 1. A dysfunctional fibrinogen with a deletion of B beta(9-72) corresponding exactly to exon 2 of the gene.

Fibrinogen New York 1 (NY-1) was identified in a family with a thrombotic tendency. Studies on fibrinogen NY-1 and the fibrinogen from her brother, designated NY-1a, showed that both have abnormal thrombin-nonclottable fibrinogen (50% of the total fibrinogen in NY-1 and 35-40% in NY-1a) and that the trait is heterozygous and autosomal codominant. The abnormal fibrinogen polymerizes in the presence of calcium and can be further cross-linked by Factor XIIIa. The release rates of fibrinopeptides A and B by thrombin from both (NY-1 and NY-1a) were slower than those from normal fibrinogen. Two mol of fibrinopeptide A but only 0.6-1.0 (NY-1) or 1.0-1.3 (NY-1a) mol of fibrinopeptide B were released per mol of fibrinogen. Additionally, only 1.0 (NY-1) or 1.3 (NY-1a) mol of the B beta(1-42) peptide were released by plasmin/mol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reduced fibrinogen revealed two protein bands in the B beta-chain region (Mr = 54,000 as compared with 57,300 for the normal). When NY-1a fibrinogen was treated with CNBr, two sizes of the NH2-terminal disulfide knot were obtained (Mr = 59,000 and 49,000). The Mr = 49,000 component is consistent with an abnormal NH2-terminal disulfide knot with two defective NH2-terminal B beta-chains. Amino acid sequence analyses demonstrated that the abnormal B beta-chain is the result of a deletion in the sequence from residues 9 to 72. This deletion corresponds exactly to exon 2 of the gene. Since this family has a thrombotic tendency, the defect in the fibrinogen may be important in the pathogenesis of thrombosis in this family.

Amino Acid Sequence↗
PubMed1983

The release of B beta 1-42 from fibrinogen and fibrin by plasmin.

The balance between thrombin and plasmin action has been postulated to be an important determinant of thrombosis. Measurement of plasma concentrations of fibrinopeptide A (FPA), which reflect thrombin action on the NH2-terminal end of the A alpha chain, and of B beta 1-42 (thrombin-increasable fibrinopeptide B immunoreactivity-TIFPB) which reflect plasmin action on the NH2-terminal end of the B beta chain have shown systematic changes in the relative concentrations of the two peptides in thrombotic states. This paper reports kinetic data for TIFPB release by plasmin using fibrinogen, fibrin I monomer, and fibrin I polymer as substrates. For fibrinogen and fibrin I monomer the data fit the Michaelis-Menten equation. Experiments were performed with human proteins in 0.15M Tris-buffered saline at pH 7.4 and at 37 degrees C. With fibrinogen as substrate the Km was calculated to be 0.87 microM and the Vmax 3.75 X 10(-5) M/min/unit of plasmin. With fibrin I monomer as the substrate the Km was calculated to be 1.25 microM and the Vmax 5.5 X 10(-5) M/min/unit of plasmin. With fibrin I polymer as substrate the data did not fit the Michaelis-Menten equation but there appeared to be no dramatic differences in rates from those obtained with the other two substrates. The influence of factor XIIIa-induced cross-linking of fibrin was not examined. It is concluded from these findings that fibrinogen and non-cross-linked fibrin I are equally good substrates for plasmin cleavage of the NH2-terminal end of the B beta chain.

Batroxobin↗
PubMed1983

Sequence of plasmin proteolysis at the NH2-terminus of the b beta-chain of human fibrinogen.

Employing high-performance liquid chromatography (HPLC), we have isolated and quantified the peptides that are released from the NH2-terminus of human fibrinogen B beta-chains by plasmin proteolysis. The peptides were identified by amino acid composition and by a radioimmunoassay developed for fibrinopeptide B detection. B beta 1-42 was the earliest fragment released during limited plasmin proteolysis. The level of this peptide reached a maximum and then began to decline during the course of the digestion. In addition, increasing levels of B beta 1-21 and of FPB followed the production of B beta 1-42. Using purified B beta 1-42 as a substrate, preferential cleavage was shown to occur at the 21-22 bond, with a minor cleavage at the 14-15 bond. Exhaustive digestion yielded two major components which were separated by HPLC: B beta 1-14 (FPB) and beta 22-42. The rate of cleavage at the 14-15 bond, which is the customary site of thrombin proteolysis, was not affected by the addition of hirudin indicating that this was not the result of trace contamination with thrombin. We have also examined plasmin proteolysis at the NH2-terminal region of the B beta-chains of a variety of fibrinogen derivatives and have found similar patterns of B beta 1-42 release. Using HPLC data, we have estimated the Km for plasmic cleavage of the beta 21-22 bond to be 1.8 X 10(-5) M and of the beta 14-15 bond to be 2.8 X 10(-5) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1982

Alpha chain crosslinks of human fibrin: purification and radioimmunoassay development for two A alpha chain regions involved in crosslinking.

Rapid and efficient purification methods that include hydrophobic chromatography on Phenyl Sepharose CL-4B have been developed for the peptides that span residues 241-476 (CNBr VIII), and 518-584 (CNBr X) in the A alpha chain of human fibrinogen. Amino acid analysis, NH2-terminal sequence determination, and SDS-PAGE indicated that greater than 95% purity of each peptide was achieved. Sensitive and specific radioimmunoassays capable was achieved. Sensitive and specific radioimmunoassays capable of detecting antigen in the range of 0.1-10.0 pmol/ml have also been developed for CNBr VIII and CNBr X. These assays have been characterized and successfully applied in studies designed to localize the two COOH-terminal A alpha chain regions in column effluents of CNBr-digested fibrinogen and crosslinked fibrin. When these immunoassays were used to study purified preparations of a high molecular weight, crosslink-containing CNBr derivative of a alpha polymer, the data provided immunologic confirmation for the involvement of CNBr VIII and CNBr X in alpha chain crosslinking.

Amino Acid Sequence↗
PubMedDate not supplied

Identification of protease 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat kidney.

We have identified a metalloendoprotease from rat kidney cortex that cleaves the cysteine-phenylalanine bond (Cys7-Phe8) within the 17 amino acid ring structure of atrial natriuretic factor (ANF). Cleavage at this site represents the major ANF degradative activity in rat kidney, and is inhibited by the known metalloendoprotease inhibitors, thiorphan, phosphoramidon and zincov with IC50 values in the nanomolar range. Since these are specific inhibitors of protease 3.4.24.11, both protease 3.4.24.11 and ANF degrading activities were monitored during purification. Both activities copurified at each chromatographic step. Furthermore, purified protease 3.4.24.11 cleaved ANF specifically at the Cys7-Phe8 bond. It is concluded from this work that the major ANF degrading enzyme in rat kidney is protease 3.4.24.11.

Animals↗