Search PubMed⌕ Search

Biomedical subjects

R M Stroud

Publications and source records attributed to R M Stroud.

At least 163 records · Page 9Linked to original sources
PubMed1977

Cleavage of C2 by C1s into the antigenically distinct fragments C2a and C2b: demonstration of binding of C2b to C4b.

The activation of complement component C2 by C1s is a major reaction step leading to the assembly of two related macromolecular enzymes in the classical complement pathway C3 convertase and C5 convertase. The present studies clearly document the smaller fragment, C2b, that results when human C2 reacts with C1s. We have identified and characterized C2b (34,000 daltons) as a single protein on disc electrophoresis and immunoelectrophoresis. C2a (73,000 daltons), the larger fragment from this reaction, has a more acidic nature and C2b is more basic. These fragments can also be detected by their different antigenic determinants. When the C2-C4b complex is activated in the fluid phase by C1s and allowed to decay, it dissociates into C2a and the C2b-C4b complex. Furthermore, when C2 is bound to C4b-Sepharose and then reacted with C1s, only the C2a fragment is released from the solid phase C2-C4b-Sepharose into the fluid phase, and the C2b fragment remains noncovalently bound to C4b-Sepharose. These results suggest that the C2b portion of C2 contains a stable binding site for C4b and, after the decay release of C2a from this C3 convertase, the C2b fragment remains bound. Thus, the decay release of C2a may represent a temperature-dependent dissociation from C2b.

Binding Sites↗
PubMed1977

Human factor D of the alternative complement pathway: purification and characterization.

D was purified to homogeneity from outdated human plasma by successive chromatography on Bio Rex 70, Sephadex G-200, Bio Rex 70, and Sephadex G-75. Column fractions were monitored for D activity by a hemolytic diffusion plate assay. The overall yield was approximately 4% by activity. A m.w. of 22,900 daltons was established by sedimentation equilibrium. Amino acid analyses have been obtained and Isoleucine has been determined as the NH2-terminus. Incubation of D with purified B and CoVF in the presence of Mg++ resulted in cleavage of B, as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. D hydrolyzed certain synthetic amino acid esters of arginine, lysine, and tyrosine. Benzoyl-L-arginine methyl esters (BAME) was the most sensitive substrate for D among those tested. The substrate profile of D was dinstinct when compared to that of CIs, CIr, plasmin, urokinase, and trypsin. Both the enzymatic and hemolytic activity of D were irreversibly inhibited by treatment with 10 mM DFP as well as by reduction and alkylation.

Blood Proteins↗
PubMed1976

Mechanism of hydrolysis by serine proteases: direct determination of the pKa's of aspartyl-102 and aspartyl-194 in bovine trypsin using difference infrared spectroscopy.

The pKa of aspartyl-102 in trypsin is shown to be 6.8+/-0.2 by difference infrared titration. All but 2.5 of the carboxyls in bovine trypsin were first modified with semicarbazide. The modified enzyme still retains full activity toward nonspecific substrates. The remaining free carboxyls include one equivalent each of Asp-102 and Asp-194. The absorbances associated with the C=O and C=O stretching modes at 1570 and 1710 cm-1 were used to monitor the proportion of ionized or protonated carboxyl present in the enzyme as a function of pD. The pKa of 6.8 was assigned to Asp-102 using copper ions that bind to trypsin between Asp-102 and histidine-57, so lowering the pKapp of Asp-102. The implication of this result for the ionization of the active site, and for the mechanism of serine proteases, is discussed. Asp-194 and the C terminus are shown to titrate with an average pKa of 2.9.

Amino Acid Sequence↗
PubMed1976

pH dependence of tritium exchange with the C-2 protons of the histidines in bovine trypsin.

At pH 8.9 and 37 degrees C the half-times for tritium exchange with the C-2 protons of the histidines of trypsin are 73 days for His-57, and greater than 1000 days for His-40 and His-91. These half-times are much longer than the half-life of exchange for the C-2 proton of free histidine (2.8 days at pD 8.2), and longer than any previously reported half-time of exchange at pH greater than 8. These very low rates of exchange are discussed with reference to the refined structure of trypsin. The tritium exchange of His-57 depends on an apparent pKa of 6.6. This pKa may represent the pKa of the imidazole of His-57 in an inactive conformation of the enzyme.

Amino Acid Sequence↗
PubMed1976

Free and total insulin levels in a patient with insulin-resistant diabetes mellitus and chronic lymphatic leukemia: effect of prednisone therapy.

An insulin-resistant diabetic patient who also has chronic lymphocytic leukemia and very high plasma levels of free and total insulin along with high levels of insulin antibodies is described. In response to prednisone therapy, his insulin requirement decreased, but the total and free insulin concentrations increased as insulin antibody measured as the maximal insulin-binding capacity of plasma remained unchanged. In insulin resistance, persistent hyperglycemia, in spite of high levels of immunoreactive free insulin, presumably reflects peripheral tissue unresponsiveness to insulin. The beneficial effect of prednisone treatment in this patient is discussed, and it is postulated to be the result of either increased availability of free insulin or an increased responsiveness of the tissues to insulin or both.

Adolescent↗
PubMed1976

Evidence that C1s participates in the alternative complement pathway.

Purified C1s, subcomponent of C1, induced electrophoretic conversion of factor B and consumption of hemolytic C3 and C5 in sera genetically deficient in C2 or C4. Blocking of the hemolytic activity of C1s in C2-deficient serum by F(ab')2 anti-C1s resulted in inhibition of the alternative pathway, as indicated by the failure of zymosan or cobra venom factor to induce comsumption of C3 and C6. Zymosan also failed to activate the alternative pathway when C1s was absorbed from C1r-deficient serum using a solid immunoabsorbent. These data, showing that C1s participates in the alternative pathway under certain experimental conditions, suggest the interesing possibility that C1s is important in the activation sequence of both the classical and the alternative pathway more generally.

Animals↗
PubMed1976

A comparative analysis of the C1-binding ability of fragments derived from complement-fixing and noncomplement-fixing IgM proteins.

The purpose of this study was to examine the molecular parameters necessary for initiation of complement fixation by IgM proteins. To determine why some IgM molecules are capable of complement fixation while others are not, several different Waldenström IgM proteins were examined for their ability to fix total hemolytic complement in the CH(50) assay. Subsequently, the C1 fixing ability of a 56-residue fragment derived from the Cmu4 domain of each of these IgM molecules was studied with C1 fixation assay. One of the three Waldenström IgM proteins (Gr) used in the present study was found unable to consume complement in a CH(50) assay when tested at the same concentration as the two complement-consuming IgM molecules (Dau and Bus). However, when the 56-residue C(H)4 fragment from the Cmu4 domain of each IgM molecule was tested for C1-fixing ability, all three were found to bind C1. On the basis of these observations, it is proposed that a C1 binding site exists within the Cmu4 domain of both complement-fixing and noncomplement-fixing IgM molecules. Presumably, the latter molecules are unable to interact in their native state with C1 in the manner required for initiation of the classical complement pathway, possibly due to the configurational inaccessibility of the entire C1 binding site.

Complement C1↗
PubMed1975

C1 fixation and classical complement pathway activation by a fragment of the Cmu4 domain of IgM.

A 56 residue fragment derived from a Waldenströme IgM protein and consisting of 24 residues of the amino-terminal portion of the Cmu4 domain disulfide bonded to 32 residues of the carboxy-terminal region of the loop has been shown to fix active C1 (C1) in a C1-fixation assay. Cleavage of the disulfide bond within the CH4 fragment resulted in a marked decrease of C1-fixing ability, although the isolated A and B fragments did retain a limited ability to fix C1. Upon incubation with normal human serum the intact CH4 fragment and equal molar amounts of the isolated A and B peptides consumed C4 suggesting that the C1-activating determinant of IgM remains intact in these three fragments. Furthermore, on a molar basis the intact or the reduced CH4 fragment consumed C4 as effectively as each of its component chains suggesting that transient binding of C1 by the individual A and B peptide chains is sufficient to activate C1. On the basis of these observations it is proposed that a classical complement fixation function, i.e. C1 binding and activation, can be localized within a region of the IgM molecule corresponding to the Cmu4 domain.

Amino Acid Sequence↗
PubMed1975

Detection and quantitation in plasma and synovial fluid of a fragment of human C4 with alpha mobility generated during the activation of the complement system.

A newly identified fragment of human C4 was detected, using a particular antiserum, in human serum after activation by heat-aggregated immunoglobulins, but not after activation of the complement alternative pathway. This fragment was shown to have a sedimentation velocity of approximately 2.5S, to be heat stable, and to exhibit alpha mobility in immunoelectrophoresis. This C4alpha mobility fragment was not generated in human C4 deficient serum but was generated in human C2 deficient serum after incubation with heat-aggregated immunoglobulin. After precipitation of native C4 and its higher molecular weight fragments from serum by polyethylene glycol, it was possible to quantitate the lower molecular weight C4 alpha mobility fragment by radial immunodiffusion. In kinetic experiments, it was shown that the C4alpha mobility fragment was generated after some delay when compared to the disappearance of C4 hemolytic activity. Quantitation of the C4alpha mobility fragment may be of further use in human diseases for the evaluation of the catabolism of C4: joint fluids of patients suffering from rheumatoid arthritis contained high levels of the C4alpha mobility fragment, and low concentrations were found in patients with degenerative joint disease.

Animals↗
PubMed1974

The structural basis for binding of complement by immunoglobulin M.

An insight into the structural features of human IgM that are responsible for its capacity to bind the first component of complement (C) has been obtained by examining the ability of IgM subfragments to bind active C1 (C1). The smallest two fragments found to bind C1 were the major CNBr fragment of the Fc portion of IgM and the C(H)4 fragment of the carboxy-terminal domain. The smallest fragment which fixes C1 has a disaggregated mol wt of 6,800, consists of 60 residues, and contains no carbohydrate. Structural considerations and sequence overlaps suggest that the amino-terminal side of the C(H)4 domain (24 amino acid residues) might be responsible for fixing C1.

Amino Acid Sequence↗