PubMed Health⌕ Search

Biomedical subjects

R J Butkowski

Publications and source records attributed to R J Butkowski.

33 records · Page 2Linked to original sources
PubMed1984

Goodpasture antigen of the glomerular basement membrane: localization to noncollagenous regions of type IV collagen.

The glomerular basement membrane antigen in Goodpasture syndrome is a collagenase-resistant molecule with a monomer molecular weight of about 26,000. Type IV collagen isolated from glomerular basement membrane contains collagenase-resistant sequences within its structure. Polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay, and chemical analysis were used to demonstrate that the collagenase-resistant sequences of type IV collagen contain Goodpasture antigen.

Anti-Glomerular Basement Membrane Disease↗
PubMed1982

Amino acid sequence of rat alpha-lactalbumin: a unique alpha-lactalbumin.

The amino acid sequence of rat alpha-lactalbumin has been determined. Unlike other alpha-lactalbumins which contain 122 or 123 amino acids, rat alpha-lactalbumin is unique in that it contains 140 amino acids. The extra amino acids are a 17 amino acid extension at the carboxyl terminus. The amino acid sequence of this extension is Gly124-Ala-Pro-Ala-Leu-Val-Val130-Pro-Ala-Leu-Asp-Gly135-Glu-Thr-Pro-Val-Pro140 . The extension is proline rich, which may contribute to the anomalous structural properties of rat alpha-lactalbumin. The amino acid sequence from residues 1 to 123 is similar to that of other alpha-lactalbumins. One possible explanation for the 17 amino acid extension is a mutation at the termination codon.

Amino Acid Sequence↗
PubMed1982

Preparation and composition of mouse tubular and glomerular basement membranes.

Methods were developed to obtain tubules and glomeruli and their respective basement membranes from mouse kidneys. While the procedures are especially useful for preparing tubules, one method can be used to simultaneously prepare both tubules and glomeruli. Tubules can be obtained from single animals, while a minimum of five animals are required in order to prepare glomeruli. Either minced whole kidneys or dissected cortex tissue is dispersed using a polytron and the desired fractions are obtained by sucrose density gradient centrifugation. discontinuous gradients consisting of 49.5, 53, 57, and 60% sucrose are used in the first method. Following centrifugation, tubules are collected on the 49.5% sucrose layer and glomeruli are pelleted on the bottom of the tube. In the second method, disrupted tissue is mixed with 57% sucrose and after centrifugation a layer of pure tubules is obtained from the top of the sucrose solution. Basement membranes are then obtained by a sonication method and the average yields per kidney are 0.1 mg and 0.02 mg for tubular and glomerular basement membranes respectively. Their chemical compositions are similar to the respective basement membranes from other species.

Amino Acids↗
PubMed1981

Prothrombin.

Explore the source record for details and available documents.

Amino Acid Sequence↗
PubMed1979

Rabbit tubular basement membrane. Isolation and analysis of polypeptides.

Renal tubules from rabbit kidneys were isolated from thin shavings of the kidney surface. Basement membrane was then prepared following sonication of the isolated tubules. To insure preservation of the integrity of the basement membrane polypeptides, the protease inhibitors, diisopropyl fluorophosphate, ethylenediaminetetraacetic acid, N-ethylmaleimide, and epsilon-amino-caproic acid were used at all stages of the preparations. The optimal conditions of sonication and centrifugation were established and the chemical composition of basement membrane prepared under these conditions was examined in detail. Glycine, hydroxyproline, and hydroxylysine were found in concentrations of 206, 65, and 18 residues per thousand, respectively, in basement membrane from young kidneys. About 38% of the basement membrane was found to be soluble in sodium dodecyl sulfate upon incubation at 90 degrees C, and to possess relatively low amounts of the amino acids characteristic of collagen. Electrophoretic analysis of this fraction revealed that the major subunits ranged in approximate molecular weight from 18,500 to greater than 10(6). When analyzed with disulfide bonds reduced, a molecular weight range from 31,000 to 275,000 was observed for this fraction. The sodium dodecyl sulfate-insoluble fraction could be dissolved upon reduction and alkylation and its composition was enriched in the amino acids characteristic of collagen. Polypeptides from this fraction were analyzed by electrophoresis in agarose and in agarose-acrylamide gels. The approximate molecular weight of the smallest component was 164,000. Additional polypeptides were observed whose molecular weights occurred in multimers of this component, up to 1.1 x 10(6), possibly indicating covalent cross-linked multimers of a basic collagen-like polypeptide(s).

Amino Acids↗
PubMed1977

Thrombin: structural features related to specificity.

A comparison of the primary structure of human thrombin with the structures of chymotrypsin, trypsin, elastase and factor Xabeta reveals several structural features which may be involved in the specificity of thrombin toward macromolecular substrates. Among the major structural differences noted in such a comparison are the insertions of five extended peptide regions in the primary structure of alpha-thrombin when compared to chymotrypsin. These insertions, which we refer to as "loops", have been designated A, B, C, D, and E. The A, B and C "loops" in human thrombin appear to be large enough to interact at or near the active active site if an alpha-thrombin-chymotrypsin three-dimensional structural homology is assumed. In beta-thrombin, the configuration of the A and B "loops" may be perturbed by proteolysis, and the ability of beta-thrombin to clot fibrinogen is thus reduced. Perturbation of the configuration of the C "loops" by proteolysis in the formation of gamma-thrombin may further reduce the ability of thrombin to bind fibrinogen.

Amino Acid Sequence↗
PubMed1976

Effect of polylysine on the activation of prothrombin. Polylysine substitutes for calcium ions and factor V in the factor Xa catalyzed activation of prothrombin.

Polylysine has been demonstrated to dramatically accelerate the rate of the factor Xa catalyzed activation of both prothrombin and prethrombin 1. Under the present experimental conditions (pH 8.0, 23 C), no detectable activation of prothrombin or prethrombin 1 occurs with either factor Xa or polylysine alone. The activation of prethrombin 2, the direct precursor of alpha-thrombin, by factor Xa is not stimulated by polylysine. The activation of either prothrombin or prethrombin 1 by factor Xa in the presence of polylysine is partially inhibited by the presence of 5 mM CaCl2. Electrophoretic analysis in sodium dodecyl sulfate showed that the products that were formed in the above activation system comigrated with the reaction products derived from prothrombin activated by factor Xa in the presence of calcium ions and phospholipid. It is suggested that polylysine stimulates the factor Xa-catalyzes activation of prothrombin by replacing the combination of calcium ions and factor V.

Animals↗
PubMed1975

Human prothrombin activation.

Human prothrombin has been purified from American Red Cross Factor IX concentrates. Studies of the activation of the human prothrombin with the use of sodium dodecyl sulfate electrophoretic analysis of activation products indicated that human prothrombin activation is similar to bovine prothrombin activation. Molecular weight analysis of human prothrombin and intermediated by sodium dodecyl sulfate co-electrophoresis with bovine prothrombin and its intermediates resulted in molecular weights of 70,000 for prothrombin, 51,000 for intermediate 1, 41,000 for intermediate 2, 23,000 for intermediate 3, and 13,000 for intermediate 4. Amino acid compositions of human prothrombin and intermediates are similar to those for bovine prothrombin and intermediates. NH2-terminal sequence studies of human prothrombin, intermediates, and alpha-thrombin A and B chains placed the intermediates in the parent human prothrombin molecule as described for the bovine system. Intermediate 3 is the NH2-terminal of prothrombin, and intermediate 1 is the COOH-terminal segment of the zymogen. Intermediate 4 is the NH2-terminal of intermediate 1. Intermediate 2', the immediate precursor of alpha-thrombin, is the COOH-terminal segment of intermediate 1. In general, a high degree of homology in the primary structure of prothrombin and intermediates was observed between the human and bovine system. The NH2-terminal sequences of human intermediate 2' and alpha-thrombin A chain are identical. However, human intermediate 2' isolated in a manner identical with that used for the isolation of bovine intermediate 2 is homologous with bovine intermediate 2, beginning with residue 14.

Amino Acid Sequence↗
PubMed1975

Prothrombin fragments. Ca2+ binding and activation kinetics.

The binding of Ca2+ to prothrombin and the intermediates of prothrombin activation was investigated by equilibrium dialysis using 45Ca2+ as the ligand. Scatchard plots of these data indicate that prothrombin (Mr = 70,000) has 10 to 11 Ca2+ binding sites which can be differentiated in terms of their binding affinity. Six of these Ca2+ binding sites have log Kassoc = 3.5 and all are found intact in the NH2-terminal segment (activation intermediate 3, Mr = 23,000) of the prothrombin molecule. Four or five additional weaker binding sites for Cz2+ with log Kassoc = 2.7 present in prothrombin are found intact in the remaining COOH-segment (activation intermediate 1, Mr = 51,000) of the prothrombin molecule. Upon further activation the Ca2+ binding sites residing in intermediate 1 are found intact in activation intermediate 4 (which constitutes the NH2-terminal segment of the intermediate 1 molecule). The remaining COOH-terminal portion (activation intermediate 2, Mr = 41,000) of the intermediate 1 molecule has no affinity for Ca2+. The activation of prothrombin and activation intermediates 1 and 2 was studied using these activators: Factor Xa alone, Factor Xa-Ca+, AND Factor Xa-Ca2+-phospholipid. The rate of thrombin production from prothrombin was progressively increased as Ca2+ and phospholipid were added to the system, whereas no significant increase in the rates of activation of intermediate 1 and 2 was observed. When Factor V was added to the Factor Xa-Ca2+-phospholipid system, the rate of activation of intermediate 1 was greatly enhanced. In the absence of Ca2+, Factor V had no effect on the rate of thrombin formation from intermediate 1. Factor V had no stimulatory effects on the rate of intermediate 2 activation. However, in the presence of an equimolar amount of intermediate 4, Factor V accelerated the conversion of intermediate 2 to thrombin. These studies indicate that the Ca2+ binding sites of the prothrombin molecule are contained in the "pro" fragment (intermediates 3 and 4) of the prothrombin molecule. Intermediate 1 and intermediate 2, both of which lack the strong Ca2+ binding sites of prothrombin, are poor substrates for the Factor Xa-Ca2+-phospholipid complex activation when compared to prothrombin. The addition of Factor V to the catalyst results in acceleration of the activation rate of intermediate 1 and an equimolar mixture if intermediates 2 and 4. These results lead us to conclude that the strong Ca2+ binding sites are the sites of phospholipid binding (intermediate 3), whereas the seak binding sites are the sites of Factor V binding (intermediate 4).

Binding Sites↗