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Biomedical subjects

R L Lundblad

Publications and source records attributed to R L Lundblad.

At least 37 records · Page 2Linked to original sources
PubMed1991

Linkage between blood coagulation and inflammation: stimulation of neutrophil tissue kallikrein by thrombin.

There has been major interest in the potential interaction between blood coagulation and inflammation. Most of the effort has focused on cellular interactions involving platelets and polymorphonuclear leukocytes (PMNS). The recent discovery of tissue kallikrein(TK) activity in PMNs prompted the study of the possible role of thrombin(IIa) in this process. Human PMNs were isolated by density gradient centrifugation. Human IIa was compared with fMLP with respect to chemotaxis and enzyme release. Results from the challenges by IIa and fMLP were compared to a NaCl control using Student's paired t-test. IIa was a potent chemotactic agent for PMNs (p less than or equal to 0.0121) and stimulated the release of TK (p less than or equal to 0.0001) as determined by hydrolysis of S-2266. FMLP significantly stimulated PMN chemotaxis (p less than or equal to 0.0028) but had no effect on TK release. Release of TK was confirmed by Western Blot analysis and 35S-methionine incorporation into a 35 KD protein after IIa challenge. These results demonstrate that IIa is chemotactic for PMNs and can cause release of tissue kallikrein demonstrating a direct role for blood coagulation in the regulation of the inflammatory response.

Blood Coagulation↗
PubMed1991

Differentiation of myeloperoxidase and glandular peroxidase in biological fluids: application to human saliva.

A modification of the assay system for peroxidase activity in human mixed saliva using 2,2'-azinobis-(-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as the substrate is reported. The modifications permit differentiation between glandular peroxidase (SPX) and myeloperoxidase (MPX) activity. In addition, factors endogenous to saliva such as thiocyanate did not adversely affect the modified assay system. The basic peroxidase assay utilized 1.0 mM ABTS and 0.1 mM H2O2 in 0.1 M sodium acetate, pH 4.7. The addition of 100 mM NaCl to the assay inhibited 90% of the activity of purified MPX but only 40% of the SPX activity in parotid saliva presumed to be free of MPX. Addition of Cl- to the assay had varied effects on the mixed saliva SPX activity (44-68% inhibition), suggesting that MPX is present in varying amounts in mixed saliva. Assay of mixed saliva from patients with different states of periodontal health indicated that the amount of Cl- inhibition is dependent on amount of inflammation and thus amount of MPX present. The results suggest that the modified assay yields a reliable estimate of relative SPX activity in mixed saliva.

Benzothiazoles↗
PubMed1989

Functional consequences of an arginine180 to glutamine mutation in factor IX Hilo.

Factor IX Hilo is a variant factor IX molecule that has no detectable coagulant activity. The defect in factor IX Hilo arises from a point mutation in the gene such that in the protein Arg180 is converted to a Gln. Activation of factor IX Hilo by factor Xla was monitored using the fluorescent active site probe p-aminobenzamidine. Normal factor IX showed complete activation in one hour as determined by measuring the increase in fluorescence when p-aminobenzamidine bound to activated factor IX. Factor IX Hilo showed no increase in fluorescence even after 24 hours, indicating that the active site was not exposed. Polyacrylamide gel electrophoresis showed that factor IX Hilo was cleaved to a light chain plus a larger peptide with a molecular weight equivalent to a heavy chain covalently linked to an activation peptide. Amino terminal amino acid sequencing of factor IX Hilo cleaved by factor Xla showed cleavage only at Arg145-Ala146, indicating that the Gln180-Val181 bond was not cleaved and that the active site was thus not exposed. The presence of factor IX Hilo in patient plasma was responsible for the patient having a very long ox brain prothrombin time characteristic of severe hemophilia Bm. Patient plasma had an ox brain prothrombin time of 100 seconds using a Thrombotest kit, significantly prolonged over the normal control value of 45 seconds. When factor IX Hilo was depleted from patient plasma using an immunoaffinity column, the ox brain prothrombin time decreased to 41 seconds. When factor IX Hilo was added back to depleted patient plasma, to normal plasma depleted of factor IX by the same affinity column, or to plasma from a CRM- hemophilia B patient, the ox brain prothrombin time was significantly prolonged. We conclude that the Arg180 to Gln mutation in factor IX Hilo results in a molecule that cannot be activated by factor Xla. Further, our data suggest that the mutation results in a molecule that interacts with components of the extrinsic pathway to give a prolonged ox brain prothrombin time.

Amino Acid Sequence↗
PubMed1988

A hydrophobic site in human prothrombin present in a calcium-stabilized conformer.

Human prothrombin and prothrombin fragment 1 were demonstrated to bind to Phenyl-TSK columns in the presence of 5.0 mM calcium ions but not in the presence of either magnesium ions or manganese ions. The calcium-dependent interaction of prothrombin fragment 1 is markedly reduced upon oxidation of approximately one mole of tryptophan per mole of protein. The ability of prothrombin fragment 1 to inhibit prothrombin activation by factor Xa in the presence of calcium ions and phospholipid is also markedly reduced by reaction with N-bromosuccinimide. These results provide the first demonstration of a calcium-specific site in prothrombin outside of the "GLA domain".

Binding Sites↗
PubMed1988

The reaction of bovine alpha-thrombin with tetranitromethane. Characterization of the modified protein.

Previous studies from several laboratories have shown that thrombin is inactivated by tetranitromethane with the formation of nitrotyrosine. The inactivation is characterized by an apparently greater loss of fibrinogen-clotting activity than activity toward synthetic ester substrates, suggesting that the residues modified by tetranitromethane are involved in the interaction of thrombin with fibrinogen. This study was designed 1) to determine the effect of solvent conditions on the rate of modification and the stoichiometry of the reaction of tetranitromethane with bovine alpha-thrombin; 2) to identify the residue(s) modified; and 3) to characterize the modified enzyme with respect to its interaction with peptide nitroanilide substrates and fibrinogen. The inactivation of thrombin by tetranitromethane proceeded more rapidly in 50 mM Tris, pH 8.0, than in 50 mM sodium phosphate, 100 mM NaCl, pH 8.0. Approximately 10% fibrinogen-clotting activity remained at maximal inactivation. A study of the effect of tetranitromethane concentration on the rate of inactivation suggested that the loss of activity was the result of the modification of 1 mol of tyrosine/mol of thrombin. A similar result was obtained from the analysis of the extent of inactivation as a function of the extent of protein modification. Structural analysis of the modified protein showed substantial modification at both Tyr71 and Tyr85. Enzyme kinetic studies were performed with the modified protein and a control thrombin with N2-tosylglycylprolylarginine p-nitroanilide. H-D-phenylalanylpipecolylarginine p-nitronailide, and purified bovine fibrinogen. With all three substrates, a substantial decrease in kcat was observed, whereas there was essentially no change in Km. These results suggest that, contrary to previous suggestions, the modification of Tyr71 and Tyr85 in thrombin does not influence the binding of substrates, but rather influences active site reactivity.

Animals↗
PubMed1988

Influence of divalent and monovalent cations on some active site properties of human factor Xa.

The effect of divalent and monovalent cations on the hydrolysis of BzIleGlu(OR)GlyArgpNA(S-2222) was compared to the rate of inactivation of factor Xa by dansyl-GluGlyArg-chloromethylketone(DERG-CK). At substrate concentrations below Km, an approximate four-fold increase in amidase activity was observed in the presence of manganese ions while a three-fold increase was observed with calcium ions. The presence of magnesium ions resulted in a two-fold increase in amidase activity. Similar increases in the rate of inactivation of factor Xa by DERG-CK were observed. Na+ ions had a marked enhancing effect of both factor Xa amidase activity and inactivation by DERG-CK. Kinetic parameters for the hydrolysis of S-2222 by factor Xa were obtained in the presence and absence of Ca++ and Na+. Vmax values increased in the presence of either Ca++ or Na+. Km values increased in the presence of Ca++ while there was a modest decrease in Km in the presence of Na+. It is suggested that the enhanced activity of factor Xa is a reflection of changes in the reactivity of active site residues.

Amino Acid Chloromethyl Ketones↗
PubMed1988

The assay of glandular kallikrein and prekallikrein in human mixed saliva.

The measurement of glandular kallikrein in biological fluids most often utilizes a synthetic substrate, H-D-valylleucylarginine-p-nitroanilide (S-2266), which assesses amidase activity. Although this substrate has reasonable specificity for glandular kallikrein, other tryptic-like proteases found in mixed saliva may also cause hydrolysis. The primary purpose of this study was to assess the accuracy of the use of this substrate for the measurement of glandular kallikrein in human mixed saliva. An additional objective was to determine the presence of prekallikrein in mixed saliva. The addition of soybean trypsin inhibitor (SBTI), which inhibits other tryptic-like enzymes but not glandular kallikrein, resulted in an approx. 30 per cent decrease in the hydrolysis of S-2266 by centrifuged mixed human saliva. A correlation of 0.918 was obtained between the biological assays for kinin release and amidase activity in 19 subject samples. Amidase activity increased following treatment of saliva with trypsin, indicating the presence of prekallikrein in human mixed saliva. It is concluded that S-2266 is an accurate substrate for the assay of glandular kallikrein in human mixed saliva; that the inclusion of SBTI in the assay mixture is needed to inhibit non-kallikrein proteases that may also hydrolyse the synthetic substrate; and that prekallikrein is present in mixed saliva. Thus any future studies of changes in the level of kallikrein in saliva may wish to consider the presence of both active and total levels of glandular kallikrein.

Amidohydrolases↗
PubMed1988

Effects of amines and polyamines on turbidimetric and lysoplate assays for lysozyme.

The lysoplate and turbidimetric assays have often been used to measure lysozyme in biological fluids, such as blood and saliva. When the same purified lysozyme standard is used in both assays, results obtained with the lysoplate assay are much higher than those obtained for the same sample in the turbidimetric assay. It is likely, therefore, that other components in the biological fluid sample influence the expression of lysozyme activity in one or both assays to produce such divergent results. Certain amines or polyamines are found in various biological fluids and have the potential to influence the reactions in these two assay systems. It was the purpose of this study to incorporate selected amines and polyamines into purified lysozyme preparations and saliva or into the assay preparations to compare their effects on observed lysozyme activity between the two assay systems. Results showed that when the amine or polyamine was included in the purified sample, lysozyme activity was considerably greater than that of controls in the lysoplate assay, whereas a negligible effect was observed in the turbidimetric assay. If the amine or polyamine was incorporated into the assay preparation, results were more dramatic, with large increases in activity in the lysoplate assay and concomitant inhibition in the turbidimetric assay. Similar effects were observed in the assay of lysozyme in saliva, suggesting a potential mechanism by which the two assay systems produce markedly different results for the same sample.

Amines↗
PubMed1987

Interaction of polylysine with bovine factor Xa: effect of divalent cations.

Poly-L-lysine has been demonstrated to partially replace biological cofactors in the activation of prothrombin by factor Xa. The present study was initiated to determine if poly-L-lysine has an effect on the enzymatic activity of factor Xa in the absence of prothrombin. At low ionic strength (50 mM Tris-Cl, pH 8.0, ambient temperature), poly-L-lysine inhibits amidase activity (S-2222) of bovine factor Xa with high affinity (Ki = 7 nM). The inhibition was readily reversed by 100 mM NaCl. The inhibition was also markedly reduced by the addition of 1.0 mM CaCl2 but not by MnCl2 or MgCl2. All three metal ions enhance amidase activity in the absence of poly-L-lysine. Poly-L-lysine also inhibits the amidase activity of factor Xa from which the gamma-carboxyglutamic acid domain has been removed by limited proteolysis with chymotrypsin (factor Xa-GD) but with somewhat lower avidity (Ki = 35 nM). As with native factor Xa, calcium ions reduce the observed inhibition while either manganese or magnesium ions are much less effective. The amidase activity of factor Xa-GD is enhanced with any one of the three divalent cations. These results provide additional support for the existence of a functionally significant binding site for calcium ions outside of the gamma-carboxyglutamic domain of factor Xa.

Amidohydrolases↗
PubMed1987

Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions.

The purpose of the current study is to present further evidence for prothrombin self-association as assessed by chemical crosslinking. When the self-association (evaluated by covalent crosslinking with dithiobis(succinimidylpropionate) of prothrombin or fragment 1 was evaluated at the same molar concentration of protein, similar rates of dimer formation were observed for either protein. When prothrombin and fragment 1 were incubated together with the crosslinking reagent and calcium ions, a heterodimer consisting of prothrombin and fragment 1 was observed in addition to prothrombin dimer and fragment 1 dimer. Similar experiments with prethrombin 1 showed neither significant self-association nor effect on prothrombin self-association. Comparison of the formation of prothrombin fragment 1 heterodimer formation with the effect of fragment 1 on prothrombin activation by factor Xa suggests that the anticoagulant activity of fragment 1 is not solely a result of the formation of a heterodimer between prothrombin and fragment 1.

Animals↗
PubMed1986

Mechanism of the calcium-dependent self-association of bovine prothrombin. Use of a covalent cross-linking reagent to study the reaction.

The present study has made use of a covalent cross-linking agent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin and has demonstrated that the covalent dimerization reaction involves the gamma-carboxyglutamic acid region of prothrombin (1-42 of 582). An essential role for the gamma-carboxyglutamic acid residues of prothrombin in the association reaction was demonstrated by experiments that converted gamma-carboxyglutamic acid residues to gamma-methylene glutamic acid or glutamic acid and resulted in a prothrombin species that was inactive in our cross-linking assay. Other experiments showed that very high concentrations of calcium ion inhibit the cross-linkage of prothrombin. This result is most consistent with an essential gamma-carboxyglutamic acid-calcium ion-gamma-carboxyglutamic acid bridge(s) in the calcium-dependent self-associated form of prothrombin.

1-Carboxyglutamic Acid↗
PubMed1986

Comparison of five techniques for the determination of protein content in mixed human saliva.

This study was conducted to assess the relative accuracy of five different assay techniques for the determination of protein concentration in human mixed saliva. The protein concentration of paraffin-stimulated saliva from 20 individuals was determined using the biuret reaction, the Lowry assay, a modified Lowry technique using bicinchoninic acid, and two dye-binding assays. Using bovine serum albumin as the standard, mean values ranged from 0.67 to 2.37 mg/ml. The use of bovine serum albumin, trypsinogen, lysozyme, bovine pancreatic ribonuclease, and poly-L-lysine as standards with the five different assay techniques to measure protein concentration of pooled mixed saliva from the above subjects produced results ranging from 0.74 to 65.5 mg/ml. The protein concentration obtained for this saliva sample by amino acid analysis was consistent with the value obtained for the biuret reaction using any of the five different standard proteins. Thus, the protein concentration obtained for human saliva depends upon both the technique used and the protein standard.

Amino Acids↗
PubMed1986

Factors influencing measurement of human salivary lysozyme in lysoplate and turbidimetric assays.

The use of different assay conditions has complicated the evaluation of studies relating salivary lysozyme levels to oral or systemic disease. The purpose of this study was to compare values obtained for lysozyme activity in mixed saliva of 104 healthy subjects by using two assay techniques and four variations in sample preparation. Lysozyme activity was assayed by the turbidimetric and lysoplate methods with human colostrum lysozyme as the standard. Lysozyme activity in saliva samples made 0.5 M with respect to NaCl was compared with that in untreated samples with and without centrifugation. Mean values for lysozyme concentration in centrifuged saliva were 2.2 micrograms/ml with the turbidimetric assay and 5.9 micrograms/ml with the lysoplate assay. In samples which were salt treated before centrifugation, mean concentrations increased to 17.3 and 72.9 micrograms/ml, respectively. The results for uncentrifuged saliva were four to five times higher than the results for centrifuged saliva in each of the assay systems. Salt treatment without centrifugation produced values comparable to those obtained with centrifugation. The addition of salt to human colostrum or hen egg white lysozyme generally resulted in a 20 to 25% increase in expressed activity. These results indicate that the measurement of lysozyme in the supernatant of centrifuged saliva is of questionable value, most of the lysozyme in whole saliva is inactive and may be activated by markedly increasing the ionic strength, and values for lysozyme activity in whole saliva are much greater in the lysoplate assay than in the turbidimetric assay when the same standard is used.

Biological Assay↗
PubMed1986

Levels of glandular kallikrein in whole saliva obtained from patients with solid tumors remote from the oral cavity.

Glandular kallikrein activity in whole saliva was determined in 16 patients with malignant tumors distant from the oral cavity and in an equal number of healthy control subjects of similar age and sex distribution. The level of glandular kallikrein was significantly greater in saliva obtained from the patients with solid tumours as determined with a tripeptide nitro-anilide substrate, D-Val-Leu-ArgpNA. A basis for the higher levels of glandular kallikrein in saliva obtained from the solid tumor patients has not been established, nor is it possible to establish a role for glandular kallikrein in the pathogenesis of malignant disease. The higher level of glandular kallikrein observed in whole saliva obtained from patients with solid tumors suggests the need for further investigation of the role of kallikrein in malignant disease and the usefulness of salivary measurements of this enzyme as a reflection of systemic changes.

Breast Neoplasms↗
PubMed1985

Effect of divalent cations on the limited proteolysis of prothrombin by thrombin.

The inhibitory influence of divalent cations on the ability of bovine alpha-thrombin to hydrolyze prothrombin showed the trend Mn2+ much greater than Ca2+ greater than or equal to Mg2+ greater than Sr2+ much greater than Ba2+. This effect was not due to an inhibition of thrombin's catalytic activity as measured by hydrolysis of a specific synthetic substrate, H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-PhePipArgNA). The presence of divalent cations did not inhibit thrombic proteolysis of gamma-carboxyglutamic acid (Gla)-domainless prothrombin. Prothrombin and Gla-domainless prothrombin were used as competitive inhibitors in the thrombic hydrolysis of D-PhePipArgNA. The apparent Ki value calculated for prothrombin was 18 microM. When either Ca2+ or Mn2+ were present, there was no inhibition. The apparent Ki value determined for Gla-domainless prothrombin was 28 microM in either the absence or presence of Ca2+. Addition of divalent cations to prothrombin, but not to Gla-domainless prothrombin, resulted in an altered protein conformation as measured by high-performance size-exclusion chromatography and ultraviolet difference spectroscopy. These results suggest that a conformational change secondary to the interaction of divalent cations with the Gla-containing domain of prothrombin is required for cation-dependent inhibition of thrombin hydrolysis.

Animals↗
PubMed1985

Modification of histidines in human prothrombin. Effect on the interaction of fibrinogen with thrombin from diethyl pyrocarbonate-modified prothrombin.

Diethyl pyrocarbonate (ethoxyformic anhydride) was used to modify histidyl residues in prothrombin. Diethyl pyrocarbonate inactivated the potential fibrinogen-clotting activity of prothrombin with a second-order rate constant of 70 M-1 min-1 at pH 6.0 and 25 degrees C. The difference spectrum of the modified protein had a maximum absorption at 240 nm which is characteristic of N-carbethoxyhistidine. The pH dependence for inactivation suggested the participation of a residue with a pKa of 6.2. Addition of hydroxylamine to ethoxyformylated prothrombin reversed the loss of fibrinogen-clotting activity. No structural differences were detected between the native and modified proteins using fluorescence emission and high-performance size-exclusion chromatography. The tyrosine and tryptophan content was not altered, but approximately 1-2 amino groups were modified. Statistical analysis of residual enzyme activity and extent of modification indicates that among 7 histidyl residues modified per molecule, there is 1 essential histidine (not in the active site) involved in the potential fibrinogen-clotting activity of prothrombin. To further examine its properties, the modified prothrombin was activated to thrombin using Echis carinatus venom protease. There was no difference in the catalytic activity of thrombin obtained from either native or ethoxyformylated prothrombin, as measured by H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-Phe-Pip-Arg-NA) hydrolysis. However, thrombin produced from the modified protein showed a loss of fibrinogen-clotting activity but had a comparable apparent Ki value (about 20 microM) to thrombin from native prothrombin when fibrinogen was used as a competitive inhibitor during D-Phe-Pip-Arg-NA hydrolysis. The similarity in Ki values indicated that thrombin derived from diethyl pyrocarbonate-modified prothrombin does not have an altered fibrinogen-binding site. Although the histidyl residue involved during inactivation has not been identified, the results suggest that a histidyl residue in the thrombin portion of prothrombin is essential for interaction with fibrinogen.

Diethyl Pyrocarbonate↗
PubMed1985

Effect of monovalent cations on bovine alpha- and beta-thrombin: importance of substrate structure.

The relative ability of bovine alpha- and beta-thrombin to hydrolyze selected anilide and ester substrates in presence and absence of monovalent cations has been evaluated. The hydrolysis of both TosGlyProArgpNA and H-D-PhePipArgpNA by either alpha-thrombin or beta-thrombin is more effective in the presence of 0.1 M NaCl than in the presence of 0.1 M KC1. The hydrolysis of BzArgpNA by alpha-thrombin was less effective in the presence of sodium ions than in the presence of potassium ions. The hydrolysis of this latter substrate by beta-thrombin was similar in the presence of either sodium ions or potassium ions. These results suggest that the ionic composition of the solvent affects that catalytic efficiency of alpha-thrombin and beta-thrombin in different ways dependent on the nature of the substrate.

Animals↗