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Biomedical subjects

G A Homandberg

Publications and source records attributed to G A Homandberg.

At least 55 records · Page 3Linked to original sources
PubMed1991

Novel method of purification of human leukocytic elastase using adsorption on a high-performance liquid chromatography gel permeation column.

Neutrophil elastases are serine proteinases released during acute and chronic inflammatory states. We have developed a novel isolation method for neutrophil elastase, involving conventional gel chromatography followed by adsorption of protein at low ionic strength on a high-performance liquid chromatography gel permeation column. The bound elastase is then eluted by application of higher ionic strength. This adsorption step at low ionic strength, a step to be avoided in most purification methods, was used to advantage here to allow isolation of homogeneous material. This purification procedure should be useful for quick, simple bulk preparation of the enzyme.

Chromatography, Gel↗
PubMed1990

The amino-terminal 29- and 72-Kd fragments of fibronectin mediate selective monocyte recruitment.

Proteolytic fragments of fibronectin (Fn) can possess properties not inherent to intact Fn. Previously, only mixtures of low molecular weight Fn fragments, and the 120-Kd fibroblastic cell-binding segment, but not intact Fn, were shown to be selectively chemotactic for human monocytes (MOs). In order to determine if other structural domains of Fn were responsible, we tested six Fn fragments. The amino-terminal 72-Kd fragment at 1.5 microns was about 75% as potent as zymosan-activated serum (ZAS). Its amino-terminal 29-Kd degradation product at 1.0 micron was about one third as potent as ZAS. Checkerboard analysis confirmed chemotaxis. Complexing gelatin to 72-Kd fragments reduced MO chemotaxis by 28% to 30%. Reducing disulfide bonds in 29- and 72-Kd segments had no effect. A synthetic peptide containing the thrombin cleavage site between the 29- and 50-Kd segments of the 72-Kd fragment was chemotactic. The 50-, 190/170-, 35-, and 160/150/120-Kd fragments, and intact Fn were not chemotactic for MOs. The data suggest that the 72-Kd fragment and its 29-Kd subfragment are additional Fn fragments that mediate selective MO chemotaxis. We speculate that proteinases present at inflammatory sites can liberate such fragments that selectively recruit MOs.

Chemotaxis↗
PubMed1990

Insertion of fibrin peptides into urokinase enhances fibrin affinity.

Low molecular weight two-chain urokinase is a 33-kD plasminogen activator, which has no innate affinity for fibrin and consequently, its use to facilitate lysis of blood clots may lead to systemic activation of plasminogen. In order to impart clot affinities to this urokinase form (UK) we have generated two novel fibrin-binding derivatives by partially reducing UK and exchanging the native disulfide-linked peptide A with peptide A analogs. The peptide A analogs contained the fibrin-adherent fibrin-derived sequences, GPRP (derived from positions 17-20 of the fibrinogen alpha chain) or QAGDV (407-411 sequence of the fibrinogen gamma chain), each coupled through amino-hexanoic acid to a synthetic peptide, LKFQCGQK, containing the Leu 144-Lys 158 sequence of the urinary plasminogen activator A Chain. The resultant derivatives contained about 0.4 moles peptide analog/mole UK, were 75% active toward synthetic UK substrates, and were recovered in a nearly 80% yield. The two fibrin peptide derivatives had a five-fold greater affinity for the clots.

Amino Acid Sequence↗
PubMed1990

Reduction of disulfides in urokinase and insertion of a synthetic peptide.

Abbokinase is a commercially available two-chain lower molecular weight urinary plasminogen activator (u-PA) with low affinity for fibrin. Therefore, therapeutic use of this u-PA form (UK) may cause activation of not only clot-bound plasminogen but also systemic plasminogen. Since this form of UK contains a 13-residue peptide remnant of the A chain disulfide-linked to the catalytic B chain, disulfide-exchange could allow replacement of this native peptide with peptides of desired clot-directed properties. We report here a method in which native peptide was partially removed by subjecting UK to 1-100 mM dithiothreitol (DTT), in the presence of arginine. Arginine (250 mM) was found to slow the rate of release of the peptide by 2-fold, to slow the loss in enzymatic activity by about 9-fold and to limit the extent of disulfide reduction. Upon dialysis to remove DTT from the reduced UK mixture, the disulfides reformed and enzymatic activity was regained. Synthetic peptide added at this point became incorporated to the level of about 0.4 mol/mol UK. This method should be very useful for developing UK derivatives with altered affinities toward fibrin clots.

Amino Acid Sequence↗
PubMed1989

Comparison of affinities of urokinase and tissue plasminogen activator for fibrin clots.

Affinities of low molecular weight two-chain urokinase (UK) and tissue plasminogen activator (t-PA) for fibrin clots were investigated by using clot lysis rates to estimate an affinity (Kd) between activator and fibrin clots. Lysis rates were obtained using a simple spectrophotometric based clot lysis assay which is described here. Fibrin clots, containing residual plasminogen, were suspended in a 1 ml cuvette and the increase in absorbance at 280 nm due to release of soluble fibrin peptides measured over a 150 to 250 minute time period. Lysis rates were obtained from plots of time squared vs absorbance change. Plots of activator concentration vs reciprocal rates yielded regression coefficients of 0.999 and Kd values of nM for the affinity of both activators for fibrin clots. Although both activators are known to differ in affinity for fibrin, they nonetheless had similar affinities and lysis rates for the insoluble fibrin clots. This assay also suggested possible synergism; rates over twice that expected by an additive effect were observed when the two activators were mixed at 0.3 to 7.6 nM each.

Drug Synergism↗
PubMed1989

Carboxypeptidase inhibitors from Ascaris suum: the primary structure.

The carboxypeptidase A inhibitor from Ascaris suum was isolated from aqueous extracts by affinity chromatography toward immobilized carboxypeptidase A. The amino acid sequence is DQVRKCLSDT10DCTNGEKCVQ20KNKICSTIVE30IQRCEKEHFT40IPCKSNNDCQ50VWAHEKICN K60LPWGL65 . The carboxypeptidase A inhibitor is not homologous with the chymotrypsin/elastase or trypsin inhibitors from Ascaris, but shows homology in a 9-residue internal sequence with the 37/39-residue carboxypeptidase inhibitors from tomato and potato. The carboxy-terminal 5 (4) residues in the three inhibitors are similar, suggesting a common mechanism of inhibition.

Amino Acid Sequence↗
PubMed1989

Synthetic peptides of the amino-terminus of fibronectin inhibit endothelial cell growth.

Amino-terminal and carboxyl-terminal fragments of human plasma fibronectin, at nM concentrations, reversibly inhibit the growth of cultured bovine aortic endothelial cells. To define smaller active peptides, synthetic peptides corresponding to a carboxyl-terminal disulfide bonded loop segment of one of the fragments were tested for activity and found to be active at microM concentrations. The data suggest that the entire loop sequence is required for full expression of endothelial cell growth inhibitory activity in vitro.

Amino Acid Sequence↗
PubMed1987

Inaccessibility to ligands of the amino-terminal region of plasma fibronectin.

Fragments of plasma fibronectin can display properties not expressed by fibronectin. This work shows that the relative affinities of smaller fragments for gelatin or heparin increases with decreasing fragment size. When heparin or gelatin were added to fragments or to fibronectin, the affinities for the alternate ligand increased. The UV and CD spectra of a mixture of amino-terminal 29-kD and 50-kD fragments differed from that of the precursor, the intact 72-kD fragment, suggesting differences in secondary structure. Therefore, biologic activities of the 29-kD and 50-kD segments in fibronectin may be suppressed by structural constraints but may be expressed more fully by interaction with ligands.

Binding Sites↗
PubMed1987

Thrombospondin binds to amino-terminal fragments of plasma fibronectin.

Thrombospondin is a 420-kD trimeric glycoprotein that can bind type V collagen, heparin, fibrinogen and certain cells and may be one of the lectins responsible for platelet aggregation. Thrombospondin binds another glycoprotein, fibronectin, that is also released during platelet aggregation and also binds similar ligands. This work shows that the amino-terminal 29-kD segment of fibronectin binds thrombospondin, the interaction occurs within minutes, and one 29-kD molecule binds per thrombospondin subunit. The interaction was not inhibited by fibrinogen, type V collagen, or heparin. Two subfragments of the 29-kD fragment, an amino-terminal 20-kD and a carboxyl-terminal 8-kD subfragment, the latter containing a single disulfide-rigidified type I loop of the five homologous loops in the 29-kD fragment, reacted with thrombospondin while the reduced counterparts did not.

Binding Sites↗
PubMed1987

Studies on the ultrastructure of fibrin lacking fibrinopeptide B (beta-fibrin).

Release of fibrinopeptide B from fibrinogen by copperhead venom procoagulant enzyme results in a form of fibrin (beta-fibrin) with weaker self-aggregation characteristics than the normal product (alpha beta-fibrin) produced by release of fibrinopeptides A (FPA) and B (FPB) by thrombin. We investigated the ultrastructure of these two types of fibrin as well as that of beta-fibrin prepared from fibrinogen Metz (A alpha 16 Arg----Cys), a homozygous dysfibrinogenemic mutant that does not release FPA. At 14 degrees C and physiologic solvent conditions (0.15 mol/L of NaCl, 0.015 mol/L of Tris buffer pH 7.4), the turbidity (350 nm) of rapidly polymerizing alpha beta-fibrin (thrombin 1 to 2 U/mL) plateaued in less than 6 min and formed a "coarse" matrix consisting of anastomosing fiber bundles (mean diameter 92 nm). More slowly polymerizing alpha beta-fibrin (thrombin 0.01 and 0.001 U/mL) surpassed this turbidity after greater than or equal to 60 minutes and concomitantly developed a network of thicker fiber bundles (mean diameters 118 and 186 nm, respectively). Such matrices also contained networks of highly branched, twisting, "fine" fibrils (fiber diameters 7 to 30 nm) that are usually characteristic of matrices formed at high ionic strength and pH. Slowly polymerizing beta-fibrin, like slowly polymerizing alpha beta-fibrin, displayed considerable quantities of fine matrix in addition to an underlying thick cable network (mean fiber diameter 135 nm), whereas rapidly polymerizing beta-fibrin monomer was comprised almost exclusively of wide, poorly anastomosed, striated cables (mean diameter 212 nm). Metz beta-fibrin clots were more fragile than those of normal beta-fibrin and were comprised almost entirely of a fine network. Metz fibrin could be induced, however, to form thick fiber bundles (mean diameter 76 nm) in the presence of albumin at a concentration (500 mumol/L) in the physiologic range and resembled a Metz plasma fibrin clot in that regard. The diminished capacity of Metz beta-fibrin to form thick fiber bundles may be due to impaired use or occupancy of a polymerization site exposed by FPB release. Our results indicate that twisting fibrils are an inherent structural feature of all forms of assembling fibrin, and suggest that mature beta-fibrin or alpha beta-fibrin clots develop from networks of thin fibrils that have the ability to coalesce to form thicker fiber bundles.

Blood Coagulation↗
PubMed1987

ADP-induced platelet aggregation depends on the conformation or availability of the terminal gamma chain sequence of fibrinogen. Study of the reactivity of fibrinogen Paris 1.

Fibrinogen Paris I contains a mutant gamma chain that is longer than the normal chain, resulting in altered fibrin polymerization and cross-linking. Because these functions involve the carboxy-terminal region of the gamma chain, we decided to determine whether fibrinogen Paris I or the isolated Paris I gamma chain supports normal ADP-induced platelet aggregation, a function that requires the ultimate 12 residues of the normal gamma chain (400 through 411). Aggregation of ADP-stimulated normal platelets was defective with fibrinogen Paris I and markedly depressed with the gamma Paris I chain. These findings prompted us to characterize the carboxy-terminal structure of the region of the gamma Paris I chain responsible for this activity. The carboxy-terminal cyanogen bromide (CNBr) peptide of the normal gamma chain (385 through 411) or that from gamma Paris I was isolated by differential adsorption to triethylene-tetramine resin or by reverse-phase high-performance liquid chromatography (HPLC). The CNBr peptide from the Paris I gamma chain was identical to that of the normal gamma chain in its retention time on HPLC, its amino acid composition, and its sequence. Thus, the primary structure of the gamma Paris I chain from residue 384 through 411 is normal, indicating that a peptide insertion has occurred upstream from residue 384, resulting in an impairment of those physiologic functions attributable to the carboxy-terminal end of the gamma chain from position 384 (ie, cross-linking, ADP-induced platelet aggregation, and at least a portion of the gamma chain polymerization site). These observations demonstrate that the gamma chain platelet recognition site in the fibrinogen molecule is necessary but not alone sufficient to support normal ADP-induced platelet aggregation. There appears to be an additional requirement for normal conformation of the gamma chain or availability of its terminal sequence during the interaction of fibrinogen with platelets.

Adenosine Diphosphate↗
PubMed1986

Heparin-binding fragments of fibronectin are potent inhibitors of endothelial cell growth: structure-function correlations.

Heparin-binding fragments derived from the amino- and carboxyl-terminal regions of human plasma fibronectin appear to be at least relatively specific potent inhibitors of the growth of bovine aortic endothelial cells in culture by as yet unknown mechanisms. In order to understand better the sites which subserve this activity, we have compared the relative potency of other major fragments of fibronectin, most of which have dissimilar properties and do not bind heparin. We have also proteolytically digested and chemically modified the most potent of these fragments, the amino-terminal 29-kDa fragment, in order to test whether structural alterations that affect heparin-binding also affect the inhibitory property. Not all chemical modifications that abolished heparin-binding also abolished endothelial cell growth. Neither an amino-terminal 20-kDa nor a carboxyl-terminal 8-kDa subfragment of the 29-kDa fragment bound heparin; however, both were as inhibitory as native 29-kDa fragment. Reduction of the disulfides of the 20-kDa and 8-kDa fragments did not abolish inhibitory activity. We therefore conclude that the activity is not strictly conformation-dependent and that although the inhibitory activity is distributed throughout the 29-kDa segment, it can be expressed by an 8-kDa carboxyl-terminal segment containing residues of the last Type I loop structure.

Cells, Cultured↗
PubMed1986

Model of fibronectin tertiary structure based on studies of interactions between fragments.

Human plasma fibronectin aggregates in solution and is thought to form fibrils on cell surfaces, perhaps by self-associating and by interacting with other components such as proteoglycans. We have localized the self-association domains by testing the ability of various fragments of fibronectin to interact with each other. Complexation between fluorescamine-labeled fragments and unlabeled fragments or whole molecules was assessed by gel filtration high-performance liquid chromatography. The fragments studied included nonoverlapping fragments that are situated on the fibronectin polypeptide chain in the following order, beginning from the amino terminus: the 29-, 50-, 120-, 35-, and 25-kDa fragments, as well as multiple-domain fragments of 72 kDa containing the 29- and 50-kDa segments, a fragment of 150 kDa containing the 120- and 35-kDa segment, a fragment of 190 kDa containing the 120- and 35-kDa segments, a fragment of 190 kDa containing the 50-, 150-, and 25-kDa segments, and a 45-kDa fragment containing the 35-kDa segment. The amino-terminal 29-kDa fragment bound to the carboxyl-terminal heparin-binding (Hep II) 35-kDa fragment as well as the 150- and 190-kDa fragments that contain the 35-kDa segment. On the other hand, carboxyl-terminal 35- and 45-kDa Hep II containing fragments bound to each other as well as to amino-terminal 29- and 72-kDa fragments and to the 190-kDa fragment. Further, the 25-kDa carboxyl-terminal fibrin-binding fragment bound the 190-kDa fragment, the only fragment containing the 25-kDa segment.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Amino acid sequences of the carboxyl-terminal regions of rat plasma fibrinogen gamma A and gamma' chains.

Amino acid sequence analysis of human fibrinogen gamma' chains have shown that the larger size relative to the gamma A chains is due to differences at the COOH-terminus. As shown by DNA sequencing, these differences as well as those in rat fibrinogen gamma' and gamma A chains result from differential processing of the primary mRNA transcript. We isolated and sequenced carboxyl-terminal CNBr peptides from gamma' and gamma A chains. As predicted by DNA sequencing, the gamma A peptide has the carboxyl-terminal sequence Gly-Gly-Ser-Lys Gln-Val-Gly-Asp-Met-COOH. The gamma' peptide is identical through residue 6 and terminates with Ser-Val-Glu-His-Glu-Val-Asp-Val-Glu Tyr-Pro-COOH.

Amino Acid Sequence↗
PubMed1985

Evidence that rat platelet fibrinogen molecules lack the gamma' chain variant found in plasma fibrinogen molecules.

Heterogeneity of rat plasma fibrinogen gamma chains (gamma A, gamma') is due to differential splicing of the primary mRNA transcript. Human gamma' chains, which amount to 7% of the total gamma chain population of plasma (hepatic) fibrinogen, are not detectable in fibrinogen isolated from platelets. In this investigation, we extended our studies to rats and found that gamma' chains, representing 30% of the rat plasma gamma chain population, are not detectable in their platelet fibrinogen.

Animals↗
PubMed1985

Interaction between fluorescence-labeled fibronectin fragments studied by gel high-performance liquid chromatography.

Fibronectin is a large, adhesive glycoprotein which self-associates on many cell surfaces. We have begun to study this reaction by determining the domains of fibronectin which interact with each other. To avoid possible solid-phase artifacts of affinity chromatography, we have devised a solution-phase assay in which the smallest fibronectin fragment is labeled with fluorescamine, mixed with unlabeled fibronectin, and complexation is observed by the appearance of a new higher-molecular-weight peak on gel high-performance liquid chromatography columns. The assay allowed use of excess unlabeled reactant, high-sensitivity, low background without removal of reagent, and fast analysis. Our results show that the amino- and carboxyl-terminal fibronectin fragments bind the native molecule in solution.

Chromatography, High Pressure Liquid↗
PubMed1985

Preparation of functionally intact monomers by limited disulfide reduction of human plasma fibronectin dimers.

Most (90 to 95%) human plasma fibronectin (PFn) molecules exist as 450-kDa disulfide-rich dimers comprised of two major types of subunits (A, 220 kDa; B, 215 kDa) that are joined near the COOH terminus by two disulfide bonds. Smaller PFn species (Zone II; 190-235 kDa) consist mainly of monomers and/or a monomeric subunit joined covalently to a smaller peptide remnant presumably derived by proteolysis of a parent 450-kDa molecule. A relatively simple and selective method for preparing functionally active, partially reduced monomeric fibronectin subunits (PR-PFn) by limited and selective reduction of dimeric plasma fibronectin (PFn) has been developed. PR-PFn was prepared by incubating PFn in phosphate-buffered saline, pH 7.4, for 2 h at room temperature in the presence of 17 mM dithiothreitol (DTT). Following S-carboxymethylation or S-carboxyamidomethylation, the material was passed through a gelatin-Sepharose column and nonbinding material was discarded; gelatin-bound material was eluted using a 0 to 2 M KSCN gradient. Residual dimeric species (10-20%) could be separated from monomers in high yield by gel-sieving chromatography on a Sepharose 6B-Cl in the presence of a chaotropic salt, 0.3 M KSCN. Most new SH groups (74-81%) in that fraction of PR-PFn binding to gelatin were localized in proteolytic fragments containing the COOH terminus, thus suggesting that selective cleavage of the interchain disulfide bridges had taken place. The binding affinity of PR-PFn to gelatin- and fibrin-Sepharose was lower than that of dimeric PFn, but the same as that of Zone II PFn and other monomeric gelatin-binding proteolytic derivatives. PR-PFn also bound to heparin-Sepharose and promoted cell attachment and spreading. We conclude that PR-PFn monomers possess the same functional activities as those of the parent chains.

Chromatography, Affinity↗