Modification of fibrin network ultrastructure by Fab fragments specific for different domain of fibrinogen

Kinetics of inhibition of fibrin monomer polymerization produced by Fab fragments prepared from immunochemically purified monospecific antibodies to the surface epitopes of different domains of fibrinogen molecule has been correlated with electron microscopic observations of resulting specimens. Fab fragments prepared from anti FgD antisera were the most efficient inhibitors of thrombin-catalysed conversion of fibrinogen to fibrin; polymerization of fibrin monomers as detected spectrophotometrically was abolished at 2:1 molar ratio of anti FgD Fab fragments to fibra monomer. These Fab fragments acting as a steric hindrance of polymerization sites inhibited the first stage of fibrin monomer aggregation. Interaction of Fab fragments derived from antibodies specific for alpha 239-476 with corresponding segment of fibrinogen molecule resulted in a weak inhibition of fibrin monomer polymerization. However, fibrin obtained in the presence of these Fab fragments was significantly modified and showed no periodicity. This observation may suggest that anti alpha 239-476 Fab impaired the course of the second stage of fibrin monomer polymerization, i.e. lateral association of fibrin fibrils.     

Isolation and characterization of proteoglycans from the swarm rat chondrosarcoma.

Proteoglycan monomer (D1) and aggregate (A1) preparations were isolated from 4 M guanidinium chloride extracts of the Swarm rat chondrosarcoma. When EDTA, 6-aminohexanoic acid, and benzamidine were present in the solutions, the D1 preparation contained a single component (SO = 23 S), and the A1 preparation contained 30% monomer (SO = 23 S) and 70 percent aggregate (SO = 111 S). In the absence of EDTA, 6-aminohexanoic acid, and benzamidine, the A1 preparations contained only small proteoglycan fragments, indicating that extensive enzymatic degradation had occurred. The composition of the proteoglycan monomer was different from that of proteoglycan monomer preparations from normal hyaline cartilages in that it did not contain keratan sulfate and chondroitin 6-sulfate; only chondroitin 4-sulfate was found. The A1 preparation from the chondrosarcoma contained only one link protein, which was like the smaller (molecular weight of 40,000) of the two link proteins present in A1 preparations from bovine nasal cartilage. When the A1 preparation from the chondrosarcoma was treated with chondroitinase ABC and trypsin and the digest was chromatographed on Sepharose 2B, a complex was isolated which contained the link protein and the segments of the protein core from the hyaluronic acid-binding region of the proteoglycan molecules.     

Expression of primary polymerization sites in the D domain of human fibrinogen depends on intact conformation

Fragments D1 and DD, plasmic degradation products of human fibrinogen and cross-linked fibrin, respectively, originate from the COOH-terminal domain of the parent molecule. Since a specific binding site for fibrin resides in the COOH-terminal region of the gamma chain, the primary structure of the two fragments was compared and their affinity for fibrin monomer measured. Fragments D1 and DD contained the same segments of the three fibrinogen chains, corresponding to the sequences alpha 105-206, beta 134-461, and gamma 63-411. Fragment DD had a double set of the same chain remnants. Fragments D1 and DD inhibited polymerization of fibrin monomer in a dose-dependent manner; 50% inhibition occurred at a molar ratio of fragment to monomer of 1:1 and 0.5:1, respectively. To prevent fibrin monomer polymerization and render it suitable for binding studies in the liquid phase, fibrinogen was decorated with Fab fragments isolated from rabbit antibodies to human fragment D1. Fibrinogen molecules decorated with 6 molecules of this Fab fragment did not clot after incubation with thrombin, and the decorated fibrin monomer could be used to measure binding of fragments D1 and DD in a homogeneous liquid phase. The data analyzed according to the Scatchard equation and a double-reciprocal plot gave a dissociation constant of 12 nM for fragment D1 and 38 nM for fragment DD. There were two binding sites/fibrin monomer molecule for each fragment. After denaturation in 5 M guanidine HCl, the inhibitory function on fibrin polymerization was irreversibly destroyed. Denatured fragments also lost binding affinity for immobilized fibrin monomer. The preservation of the native tertiary structure in both fragments was essential for the expression of polymerization sites in the structural D domain.     

Depolymerization of legume seed galactomannan by Celloviridin G20x

The depolymerization of legume galactomannans by the commercial preparation Celloviridin G20x was studied with the aim of obtaining macromolecular fragments of a constant composition. Four galactomannans, representative of the entire range of monomer ratios characteristic of this class of phytopolysaccharides, were hydrolyzed. The galactomannans were isolated from oriental goat’s rue (Galega orientalis Lam., Man: Gal 1.1), guar (Cyamopsis tetragonoloba L., Man: Gal 1.6), honeylocust (Gleditsia triacanthos f. inermis L., Man: Gal 2.4), and sophora (Styphnolobium japonicum (L.) Schott., Man: Gal 5.1). Fragments with a monomer ratio close to that in the original polysaccharides were obtained with a high yield (75–80%). The degree of substitution of the 1,4-β-D-mannopyranose chain at position C-6 with α-galactose residues influenced the molecular weight of the final reaction product.     

Cause of increase in the efficiency of Ca2+ transport by fragments of sarcoplasmic reticulum from fast skeletal muscles induced by protein kinase

The effect of cAMP-dependent protein kinases from rabbit skeletal muscles on Ca2+ uptake by fragments of skeletal muscle sarcoplasmic reticulum was studied. It was shown that incubation of the sarcoplasmic reticulum fragments with protein kinase increases the rate of Ca2+ uptake without changing the activity of Ca2+-dependent ATPase. This phenomenon is not accompanied by phosphorus incorporation into the protein components of the reticulum membranes. The protein kinase preparation subjected to "self-phosphorylation" is also capable to increase the rate of Ca2+ uptake. Using (14C) -oleic acid, it was shown that the increase of the rate of Ca2+ transport under effects of the "self-phosphorylated" protein kinase occurs due to the binding of free fatty acids present in the sarcoplasmic reticulum membranes. It was found that the effect observed is due to phosphofructokinase (ATP : D-fructose-6-phosphate-1-phosphotransferase) present in the protein kinase preparation.     

Subunit structure of submitochondrial particle membrane transhydrogenase

The subunit structure of membrane-bound mitochondrial transhydrogenase was investigated. Chemical modification of bovine heart submitochondrial particles with the cleavable bifunctional cross-linking reagent, dithiobis(succinimidyl propionate), resulted in the formation of three dimeric "cross-link isomers" of the enzyme, identified by immunoautoradiography, that are characteristic of cross-linked purified transhydrogenase. A limited amount of cross-linking of transhydrogenase monomer to Mr = 25,000 polypeptide was also observed. At high concentration of the cross-linker, a small amount of a higher molecular weight species was formed with both purified and membrane enzyme. Reductive cleavage of the dimeric and higher molecular weight species resulted in the regeneration of transhydrogenase monomer and several other proteolytically derived fragments. It is concluded that transhydrogenase exists in the native membrane primarily as a dimeric species.     

The ATP-operated clamp of human DNA topoisomerase IIalpha: hyperstimulation of ATPase by "piggy-back" binding

We have constructed a series of clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. All fragments exhibit DNA-dependent ATPase activity. Fragment 1-420 shows hyperbolic dependence of ATPase on DNA concentration, whereas fragment 1-453 shows hyperstimulation at low ratios of DNA to enzyme, a phenomenon found previously with the full-length enzyme. The minimum length of DNA found to stimulate the ATPase activity was approximately 10 bp; fragments >or=32 bp manifest the hyperstimulation phenomenon. Molecular mass studies show that fragment 1-453 is a monomer in the absence of nucleotides and a dimer in the presence of nucleotide triphosphate. The results are consistent with the role of the N-terminal domain of topoisomerase II as an ATP-operated clamp that dimerises in the presence of ATP. The hyperstimulation effect can be interpreted in terms of a "piggy-back binding" model for protein-DNA interaction.     

Allotype suppression in rabbits: the requirement for CH3 domain of anti-allotype antibody.

Facb fragments of rabbit anti-allotype antibody were prepared by plasmin digestion and isolated by gel chromatography. The antibody preparation was used in an attempt to induce allotype suppression in newborn rabbits. The Facb fragments were found to be ineffective in inducing the allotype suppression. Administration of Facb fragments caused a "burst" of immunoglobulin synthesis almost immediately after the administration of the antibody. It was concluded that the CH3 domain, which is responsible for the cytophilic activity of the antibody, is essential in induction of allotype suppression.     

Arylsulfatase from Pseudomonas sp. strain C12B: purification to homogeneity, immunological analysis, and physical properties.

Arylsulfatase was purified 219-fold from Pseudomonas sp. strain C12B. The final preparation was homogeneous by electrophoretic and immunological analysis. The enzyme is a monomer of molecular weight about 51,000, with a Stokes radius of 3.0 X 10(-7) cm, a frictional ratio of 1.2, and a sedimentation coefficient of 4.1S.     

Isolation and characterization of a conserved actin-binding domain from rat hepatic actinogelin, rat skeletal muscle, and chicken gizzard alpha-actinins

A common protease-resistant fragment (Mr = 27,000) was generated from purified rat hepatic actinogelin, and rat skeletal muscle and chicken gizzard alpha-actinins by limited proteolysis using thermolysin. A monoclonal antibody (A-1) which was raised against the rat hepatic actinogelin and has a cross-reactivity with rat skeletal muscle and chicken gizzard alpha-actinins was found to bind to all of the 27-kDa fragments selectively. Furthermore, one-dimensional peptide maps of the 27-kDa fragments showed a close similarity indicating the presence of some conservation in primary structure of the fragments. The 27-kDa fragments were purified to homogeneity by the same procedure using ammonium sulfate fractionation and hydrophobic chromatography. As the fragments were easily separated from other peptides during purification, they might be present as an independent structural domain. Purified 27-kDa fragments were found to bind to F-actin in a Ca2+-insensitive manner. The fragments failed to affect the low-shear viscosity of F-actin up to a molar ratio to actin monomer of 1:3.2, indicating that gelation activity of the parental molecules was lost and the fragments have only a single binding site on F-actin. Binding of the fragments to F-actin was almost completely inhibited by respective parental molecules, while binding of the whole molecules was blocked partly by their 27-kDa fragments. Thus, the interaction of the fragments with F-actin seemed to be specific, although apparent affinity was lower than the parental molecules. Considering these results, we concluded that the 27-kDa fragments are a conserved, monovalent, and Ca2+-insensitive actin-binding domain of the actinogelin and muscle alpha-actinins.     

Isolation and biochemical characterization of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density.

Relatively homogeneous fractions of proteoglycan fragments were prepared from tryptic digests of the 4M-guanidinium chloride extract of bovine nasal cartilage. Glycosaminoglycan-containing fragments were separated from non-proteoglycan contaminants by ion-exchange chromatography and fractionated by equilibrium density-gradient centrifugation under dissociative conditions. The fractions of highest buoyant density were chromatographed on a column of Sepharose 4B, digested with chondroitinase ABC and chromatographed on a column of Sepharose 6B, yielding two distinct fractions: fraction B/6B-4 contained fragments from the chondroitin sulphate-bearing region of the proteoglycan monomer, and fraction B/6B-2 fragments from the keratan sulphate-rich region, most probably including a chondroitin sulphate-bearing monomer segment. By dansyl chloride analysis, fraction B/6B-2 had alanine and leucine as sole and fraction B/6B-4 had isoleucine and leucine as greatly predominant N-terminal amino acids, indicative of the relative homogeneity of these preparations of cartilage proteoglycan monomer fragments.     

High precision immunoscanning electron microscopy using Fab fragments coupled to ultra-small colloidal gold.

Ultra-small colloidal gold (less than 1 nm), bound to Fab fragments provides the shortest practical specific marker system to date and can be used in concert with field emission scanning electron microscopes to precisely locate antigenic sites. An "in-lens" FE-SEM equipped with a highly sensitive single crystal YAG-detector for backscattered electrons, as well as the use of advanced specimen preparation techniques based on cryofixation, are among the indispensible prerequisites. A T-even type Escherichia coli bacteriophage, Tu II*-46, was chosen to study properties of the immunogold labeling system. Distinct regions on the tail fibers of this phage were labeled with Fab fragments derived from antibodies against the related phage Tu II*-6. The tail fibers are composed of pairs of homologous proteins, thus offering two identical antigenic sites at the same locus on the tail fibers. Fab fragments can be visualized in the SEM at high accelerating voltage (30 kV) without any additional marker. This permits comparison of the labeling characteristics of unmarked and colloidal gold-marked Fab fragments. Unmarked Fab fragments often bind by pairs (two singlet Fab fragments bound opposed to each other along the axis of the tail fiber). The labeling efficiency of unmarked Fab fragments was greater than that of ultra-small gold-labeled Fab fragments. Binding by pairs was not seen after labeling with ultra-small gold-Fab fragments. The conjugates used in this study exhibited one colloidal gold per Fab fragment.     

Synthesis Of RNA by the Rapid Phosphotriester Method Using Azido-Based 2′-O-Protecting Groups

The azidomethyl and 2-(azidomethyl)benzoyl as 2′-OH protecting groups are reported for preparation of oligoribonucleotides by the phosphotriester solid-phase method using O-nucleophilic intramolecular catalysis. The procedures for the synthesis of the corresponding monomer synthons were developed and the usefulness of the application of 2′-O-azidomethyl and 2′-O-2-(azidomethyl)benzoyl groups was examined in the synthesis of different RNA fragments with a chain length of 15–22 nucleotides. The azidomethyl group was found to be more preferable for effective synthesis of oligoribonucleotides. Hybridization properties of RNAs toward their complementary oligonucleotides were examined before and after the removal of 2′-O-azidomethyl groups.     

Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12.

Lipopolysaccharide (LPS) bound to isolated porin was detected on polyacrylamide gels by using a carbohydrate-specific silver stain and on Western blots by using anti-lipid A monoclonal antibodies. Porin was isolated from Escherichia coli JF733 (Ra chemotype) and D21f2 (Re chemotype). Isolated porin was separated from loosely associated LPS by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Unheated porin traveled on gels as aggregates, presumably trimers, with an apparent molecular weight of 78,000 to 83,000. After heating to 100 degrees C for 2 min in SDS, the porin traveled as a monomer with a molecular weight of 36,000. The unheated, high-molecular-weight trimer band reacted in the gel with the carbohydrate-specific silver stain, while the heated monomer band showed no staining. In contrast, lipid A-specific monoclonal antibodies showed reactivity on Western blots to the 36,000-molecular-weight band but not to the trimer. Finally, both monomer and trimer bands were isolated from gels and rerun by SDS-PAGE. LPS was released from the trimer preparation when the sample was heated, but the monomer band that was formed by heating the trimer isolate still reacted with anti-lipid A antibodies. Quantitative Limulus amebocyte lysate analysis revealed an approximately equal molar ratio of LPS to protein in the electroeluted porin monomer. Thus, some but not all of the LPS could be released from trimer complexes by boiling in SDS. The isolated monomer did not release more LPS on boiling in SDS a second time but still had LPS tightly bound, as detected by lipid A-specific monoclonal antibodies.     

Purification and Characterization of Poly(γ-glutamic acid) Hydrolase from a Filamentous Fungus,Myrothecium sp. TM-4222

Poly(γ-glutamic acid) (PGA) hydrolase was purified from the culture filtrate of a filamentous fungus, Myrothecium sp. TM-4222 and its general properties, especially the mode of hydrolytic action on the γ-glutamyl bond of PGA, were investigated. The purified preparation demonstrated a homogeneous band on an acidic slab gel of pH 4.3 with polyacrylamide gel electrophoresis. The enzyme showed its maximum activity at 37°C and at pH 5.0, being stable up to 40°C. The molecular mass was estimated to be 68 kDa by gel filtration. The hydrolytic action of the enzyme was specific for PGA, but not for other γ-glutamyl peptides or amides. The enzyme converted 38% of the original PGA with an average molecular mass of 500 kDa to smaller peptides, and then depolymerized these fragments to a mixture of γ-oligopeptides which consisted of only L-glutamic acid. L-Glutamic acid monomer was negligible in the reaction mixture. The remaining 62% of PGA was resistant to the enzyme action, in which D-glutamic acid was mainly detected. This study demonstrated a novel endo-type specificity of hydrolysis on PGA by the enzyme.     

Interaction of cationic bilayer fragments with a model oligonucleotide

The interaction between cationic bilayer fragments and a model oligonucleotide was investigated by differential scanning calorimetry, turbidimetry, determination of excimer to monomer ratio of 2-(10-(1-pyrene)-decanoyl)-phosphatidyl-choline in bilayer fragment dispersions and dynamic light scattering for sizing and zeta-potential analysis. Salt (Na?HPO?), mononucleotide (2'-deoxyadenosine-5'-monophosphate) or poly (dA) oligonucleotide (3'-AAA AAA AAA A-5') affected structure and stability of dioctadecyldimethylammonium bromide bilayer fragments. Oligonucleotide and salt increased bilayer packing due to bilayer fragment fusion. Mononucleotide did not reduce colloid stability or did not cause bilayer fragment fusion. Charge neutralization of bilayer fragments by poly (dA) at 1:10 poly (dA):dioctadecyldimethylammonium bromide molar ratio caused extensive aggregation, maximal size and zero of zeta-potential for the assemblies. Above charge neutralization, assemblies recovered colloid stability due to charge overcompensation. For bilayer fragments/poly (dA), the nonmonotonic behavior of colloid stability as a function of poly (dA) concentration was unique for the oligonucleotide and was not observed for Na?HPO? or 2'-deoxyadenosine-5'-monophosphate. For the first time, such interactions between cationic bilayer fragments and mono- or oligonucleotide were described in the literature. Bilayer fragments/oligonucleotide assemblies may find interesting applications in drug delivery.     

Disulfide bonding of secretory component to a single monomer subunit in human secretory IgA.

The arrangement of disulfide bonds joining secretory component (SC) to the alpha chains in secretory IgA was studied by determining the molecular size of the principal fragments resulting from CNBr digestion of secretory dimeric Fc fragments from IgA (Fc)2alpha fragments). In vitro complexes formed by incubating 125I-free SC and myeloma 131I-(Fc)2alpha fragments were isolated by gel filtration and subsequently digested with cyanogen bromide. The CNBr digests of SC-(Fc)2alpha fragments were analyzed by gel filtration in 5 M guanidine. Two principal fragments were obtained, one containing a monomeric Fc fragment from IgA (Fcalpha) associated with SC (m.w. congruent to 110,000) and a second containing the second Fcalpha monomer (m.w. congruent to 50,000) from the dimeric SC-(Fc)2alpha. Similar results were obtained when secretory (Fc)2alpha fragments isolated from native secretory IgA dimer were subjected to CNBr digestion. The data indicate that SC is disulfide bonded to a single monomer subunit in secretory IgA dimer.     

Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (myeloma protein Tro), II. Cleavage of the monomer IgA-molecule and the reduced and alkylated H- and L-chains by cyanogen bromide (author's transl)]

The monomer of myeloma protein Tro as well as the reduced and alkylated H- and L-chains were cleaved by cyanogen bromide. All cyanogen-bromide fragments were isolated and characterized by amino acid analyses, end-group and molecular weight determinations. The 4 smaller fragments of the 5 H-chain fragments were split with trypsin. The peptides were isolated and their primary structure was determined.     

Separation of Adenosine Triphosphatase of HK and LK Sheep Red Cell Membranes by Density Gradient Centrifugation

Membrane fragments from high potassium (HK) and low potassium (LK) sheep red cells were separated by density gradient centrifugation. Three preparations were studied: (1) HK membranes sonicated for 20 minutes, (2) HK membranes sonicated for 3 minutes, and (3) LK membranes sonicated for 3 minutes. The adenosine triphosphatase (ATPase) activity in the maximally disrupted preparation (1) was not sensitive to Na + K and was recovered in relatively small but heavy (specific gravity 1.19) fragments which made up no more than 8 per cent of the total membrane. Both Na + K-sensitive (S) and Na + K-insensitive (I) ATPase activity were found in the more gently broken up preparations (2) and (3) but the ratio of S- to I-ATPase was much greater in HK than in LK membrane fragments. S-ATPase activity in preparation (2) was about 50 per cent that observed in HK membranes prior to sonication. S-ATPase activity was recovered from the density gradient in relatively large but light (specific gravity 1.10) fragments. As was the case with the maximally disrupted preparation (1), I-ATPase activity in both preparations (2) and (3) was recovered in small but heavy (specific gravity > 1.20) fragments. The possibility that sensitivity of sheep red cell membrane ATPase to Na + K depends on the association between units containing the enzyme(s) and large, light, phospholipid-containing components is discussed.     

Properties of chromatin subunits from developing trout testis.

When a sample of trout testis nuclei is digested with micrococcal nuclease, the DNA is cleaved almost entirely to discrete fragments approximately 200 base pairs long and multiples thereof. The same DNA fragments can be obtained when isolated chromatin, as opposed to intact nuclei, is nuclease digested. These DNA fragments can also be found in discrete chromatin "subunits" isolated from nuclease-digested nuclei. Sedimentation through sucrose gradients or velocity sedimentation in an analytical ultracentrifuge separates these chromatin subunits into 11 S (monomer), 16 S (dimer), and 22 S (trimer) etc. species. Subunits can also be fractionated on a Sepharose 2B column equilibrated and run in low salt. High salt (greater than 40 mM NaCl) or divalent cations (congruent to 5 mM) cause subunit precipitation. Chromatin subunits have a protein to DNA ratio of approximately 1.2 and contain all the histones, including the trout-specific histone T. There are, however, no detectable nonhistone chromosomal proteins. Mg-2+ precipitates of the 11 S chromatin monomers, when pelleted, are thin and clear, while oligomer Mg-2+ pellets are thick and white. This could reflect a more symmetrical or ordered packing of 11 S monomers, which are deficient in histone I. This histone may cross-link the larger oligomers, resulting in a disordered Mg-2+ complex. These results are consistent with the subunit model of chromatin structure, based on 200 base pair long regions of DNA associated with histones. These subunits would be separated by nuclease-sensitive DNA spacer regions and cross-linked by histone I.