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Biomedical subjects

R J Baugh

Publications and source records attributed to R J Baugh.

8 recordsLinked to original sources
PubMed2000

Exosite interactions determine the affinity of factor X for the extrinsic Xase complex.

The initiation of coagulation results from the activation of factor X by an enzyme complex (Xase) composed of the trypsin-like serine proteinase, factor VIIa, bound to tissue factor (TF) on phospholipid membranes. We have investigated the basis for the protein substrate specificity of Xase using TF reconstituted into vesicles of phosphatidylcholine, phosphatidylserine, or pure phosphatidylcholine. We show that occupation of the active site of VIIa within Xase by a reversible inhibitor or an alternate peptidyl substrate is sufficient to exclude substrate interactions at the active site but does not alter the affinity of Xase for factor X. This is evident as classical competitive inhibition of peptidyl substrate cleavage but as classical noncompetitive inhibition of factor X activation by active site-directed ligands. This implies that the productive recognition of factor X by Xase arises from a multistep reaction requiring an initial interaction at sites on the enzyme complex distinct from the active site (exosites), followed by active site interactions and bond cleavage. Exosite interactions determine protein substrate affinity, whereas the second binding step influences the maximum catalytic rate for the reaction. We also show that competitive inhibition can be achieved by interfering with exosite binding using factor X derivatives that are expected to have limited or abrogated interactions with the active site of VIIa within Xase. Thus, substrate interactions at exosites, sites removed from the active site of VIIa within the enzyme complex, determine affinity and binding specificity in the productive recognition of factor X by the VIIa-TF complex. This may represent a prevalent strategy through which distinctive protein substrate specificities are achieved by the homologous enzymes of coagulation.

4-Aminobenzoic Acid↗
PubMed1998

Regulation of extrinsic pathway factor Xa formation by tissue factor pathway inhibitor.

Tissue factor (TF) pathway inhibitor (TFPI) regulates factor X activation through the sequential inhibition of factor Xa and the VIIa.TF complex. Factor Xa formation was studied in a purified, reconstituted system, at plasma concentrations of factor X and TFPI, saturating concentrations of factor VIIa, and increasing concentrations of TF reconstituted into phosphatidylcholine:phosphatidylserine membranes (TF/PCPS) or PC membranes (TF/PC). The initial rate of factor Xa formation was equivalent in the presence or absence of 2.4 nM TFPI. However, reaction extent was small (<20%) relative to that observed in the absence of TFPI, implying the rapid inhibition of VIIa.TF during factor X activation. Initiation of factor Xa formation using increasing concentrations of TF/PCPS or TF/PC in the presence of TFPI yielded families of progress curves where both initial rate and reaction extent were linearly proportional to the concentration of VIIa.TF. These observations were consistent with a kinetic model in which the rate-limiting step represents the initial inhibition of newly formed factor Xa. Numerical analyses of progress curves yielded a rate constant for inhibition of VIIa.TF by Xa.TFPI (>10(8) M-1.s-1) that was substantially greater than the value (7.34 +/- 0.8 x 10(6) M-1.s-1) directly measured. Thus, VIIa.TF is inhibited at near diffusion-limited rates by Xa.TFPI formed during catalysis which cannot be explained by studies of the isolated reaction. We propose that the predominant inhibitory pathway during factor X activation may involve the initial inhibition of factor Xa either bound to or in the near vicinity of VIIa.TF on the membrane surface. As a result, VIIa.TF inhibition is unexpectedly rapid, and the concentration of active factor Xa that escapes regulation is linearly dependent on the availability of TF.

Animals↗
PubMed1996

Role of the activation peptide domain in human factor X activation by the extrinsic Xase complex.

The activation of factor X by the extrinsic coagulation system results from the action of an enzyme complex composed of factor VIIa bound to tissue factor on phospholipid membranes in the presence of calcium ions (extrinsic Xase complex). Proteolysis at the Arg52-Ile53 peptide bond in the heavy chain of factor X leads to the formation of the serine protease, factor Xa, and the generation of a heavily glycosylated activation peptide comprising residues 1-52 of the heavy chain. The role of the activation peptide region in mediating substrate recognition and cleavage by the extrinsic Xase complex is unclear. The protease Agkistrodon rhodostoma hydrolase gamma (ARHgamma), from the venom of the Malayan pit viper, was used to selectively cleave human factor X in the activation peptide region. Three cleavage sites were found within this region and gave products designated Xdes1-34, Xdes1-43, and Xdes1-49. The products were purified to yield Xdes 1-49 and a mixture of Xdes 1-34 and Xdes 1-43. Reversed phase high pressure liquid chromatography analysis indicated that the cleaved portion of the activation peptide was likely removed during purification. All cleaved species were inactive and could be completely activated to factor Xa by the extrinsic Xase complex or by a purified activator from Russell's viper venom. Steady state kinetic studies using tissue factor reconstituted into membranes yielded essentially equivalent kinetic constants for the activation of intact factor X and the cleaved derivatives under a wide range of conditions. Since Xdes 1-49 lacks all but three residues of the activation peptide and is devoid of the carbohydrate present in this region, the data suggest that the specific recognition of human factor X by the extrinsic Xase complex is not achieved through specific interactions with residues 1-49 of the activation peptide or with carbohydrate structures attached to these residues.

Amino Acid Sequence↗
PubMed1984

Sexuality education for the visually and hearing impaired child in the regular classroom.

With more than two million handicapped children being "mainstreamed" into the regular classroom, teachers are confronted for the first time with assisting visual- and hearing-impaired children. Sensory-impaired children usually will demonstrate a deficiency in psychosocial-sexual adjustment, and teachers may doubt their ability to help in this area. These children have the same emotional and social needs as others, and their maladjustment is due usually to a lack of sufficient information concerning their sexuality. With a better understanding and an early recognition of the impediments these individuals experience in acquiring sexuality information, teachers can modify their methods of presentation and provide opportunities and assistance in the child's psychosocial-sexual adjustment.

Child↗
PubMed1979

A rapid procedure for the large scale purification of elastase and cathepsin G from human sputum.

A procedure is described which permits the rapid isolation of large amounts of elastase and cathepsin G from purulent sputum. This procedure involves: (1) digestion of sputum with DNase, (2) extraction of the insoluble residue that remains with 1 M NaCl, pH 8, (3) affinity chromatography on Sepharose-bound Trasylol, and (4) separation of the two enzymes by chromatogrphy on CM-Sephadex. Starting with 500 g of sputum it was possible to isolate 175 mg of each of these two enzymes within 7 to 10 days. Active site titration indicated both enzymes to be at least 97% pure. Disc gel electrophoresis in the presence and absence of SDS and amino acid sequence of the N-terminal region support the conclusion that the elastase and cathepsin G isolated from sputum are identical to the same enzymes isolated directly from the leukocytes of human blood.

Amino Acid Sequence↗
PubMed1976

Human leukocyte granule elastase: rapid isolation and characterization.

Human granulocytic elastases have been purified by a two-step procedure involving affinity chromatography of crude extracts of leukocytic granules on Sepharose-Trasylol, followed by ion-exchange chromatography on CM-cellulose to resolve the isoelastases. All of these enzymes were found to be glycoproteins with the carbohydrate content of the major form being composed essentially of only neutral sugars. The molecular weight of this form was found to be near 30 000 daltons with the other forms being slightly higher. Preliminary structural analyses indicate that all of the elastase isozymes have identical NH2-terminal sequences suggesting that the differences in mobility of the four proteins are not due to different degrees of activation from a common zymogen but, more likely, from minor changes in carbohydrate content. Human granulocytic elastases are less active on ligament elastin than porcine pancreatic elastase, but both are inhibited by synthetic elastase active-site directed low molecular weight compounds (Tuhy, P. M., and Powers, J. C. (1975), FEBS Lett. 50, 359) as well as by plasma alpha-1-proteinase inhibitor (formerly called alpha-1-antitrypsin). In the latter case a stable complex with mol wt of 78 000 daltons is formed indicating the formation of a 1:1 complex.

Amino Acid Sequence↗