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PubMed1989

Intracellular cleavage of newly synthesized low affinity Fc epsilon receptor (Fc epsilon R2) provides a second pathway for the generation of the 28-kDa soluble Fc epsilon R2 fragment.

It has been reported that the 45-kDa low affinity Fc epsilon R (Fc epsilon R2) on B cells is cleaved spontaneously from the cell surface to release a 28-kDa soluble fragment (sFc epsilon R2). This study demonstrates an additional mechanism by which B cells generate this fragment. Data from 35S methionine pulse-chase experiments with the Fc epsilon R2 bearing human B lymphoblastoid cell line, RPMI 8866, and immunoprecipitations of cell lysates and culture supernatants with an Fc epsilon R2 specific mAb, mAb 25, demonstrates the existence of a cell-associated 28-kDa Fc epsilon R2 fragment. This fragment was shown by partial amino(NH2)-terminal sequence analysis to be identical to the previously described 28-kDa sFc epsilon R2. The resistance to cell treatment with trypsin indicated that it was located intracellularly. Its appearance early in the biosynthesis of the Fc epsilon R2 (within a 10-min pulse), before the Fc epsilon R2 reached the cell surface, suggested that some of this fragment was generated intracellularly. Neutralization of acidic organelles with NH4Cl inhibited the formation of this intracellular fragment, strongly suggesting that it was a produce of intracellular cleavage of the Fc epsilon R2. Finally, this 28-kDa intracellular fragment was shown to be released into the culture supernatant, suggesting an intracellular mechanism by which the cells generate sFc epsilon R2.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1989

Regular fragmentation of hydrogen peroxide-treated fibronectin.

In the presence of low concentrations (less than 0.5 mM) of hydrogen peroxide Mr 350,000 and 170,000 fragments were generated from plasma and fibroblast medium fibronectins (Fns). No other major fragments were detected when H2O2 concentration was raised or the incubation time prolonged. A 200-300-fold concentration of H2O2 was needed for a complete degradation of the protein. The degradation was inhibited or completely prevented by deferoxamine, diethylene-triaminepentaacetic acid, and thiourea or by Chelex-pretreatment of the Fn solution suggesting a Fenton-type reaction to produce .OH radicals from H2O2. In immunoblotting the Mr 170,000 fragment reacted with monoclonal antibodies against the NH2 terminus and mid-molecule but not with those against the cell-binding site and the COOH terminus of Fn. Reduction of the Mr 350,000 fragment produced alpha- and beta-monomers of Fn as well as Mr 95,000 and 85,000 fragments which reacted with monoclonal antibodies against the cell-binding site and the COOH terminus of Fn. These results suggest that the Mr 170,000 fragment is derived from the NH2-terminal part of both subunits of Fn. The rest of the subunits, the Mr 95,000 (from alpha-chain) and Mr 85,000 (from beta-chain), thus remain disulfide-bonded to an intact Fn subunit to form the nonreduced Mr 350,000 polypeptide. The results show that oxygen radical action may generate defined and reproducible fragments from Fn. The high susceptibility of Fn to the radical induced degradation makes it plausible to occur also in vivo.

Binding Sites↗
PubMed1989

Tryptic fragments of the Escherichia coli DNA gyrase A protein.

Treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two large fragments which are stable to further digestion. The molecular masses of these fragments are 64 and 33 kDa, and they are shown to be derived from the N terminus and the C terminus of the A protein, respectively. These fragments could represent structural and/or functional domains within the A subunit of DNA gyrase. The trypsin-cleaved A protein (A'), in combination with the B subunit of gyrase, can support ATP-dependent supercoiling of relaxed DNA and other reactions of DNA gyrase. The isolated 64-kDa fragment will also catalyse DNA supercoiling in the presence of the B protein, but the 33-kDa fragment shows no enzymic activities. We conclude that the N-terminal 64-kDa fragment represents the DNA breakage/reunion domain of the A protein, while the 33-kDa fragment may contribute to the stability of the gyrase-DNA complex.

DNA Topoisomerases, Type II↗
PubMed1988

Lymphocyte-virus interactions. Identification of a restriction fragment permitting virus replication in lymphocytes.

We are interested in understanding the effects of virus infection on lymphocyte function. To approach this question, we used a unique system of two genetically related leporipoxviruses to generate recombinants. One of these viruses, malignant fibroma virus (MV), replicates in many different cell types, including lymphocytes. The other, Shope fibroma virus (SFV), replicates principally in fibroblasts, but cannot replicate in lymphocytes. Fibroblasts infected with SFV received restriction fragments from MV by transfection. Recombinant viruses were selected in vitro for their ability to replicate in lymphocytes. By these means we have identified one restriction fragment, the 10.8-kb BamHI "C" fragment, capable of transferring from MV to SFV the ability to replicate in lymphocytes. A family of recombinants bearing different sized inserts of this restriction fragment has been isolated and is being characterized. Lymphocytotropic recombinants bearing portions of this restriction fragment produce colony morphology in vitro intermediate between MV's plaques and SFV's foci. On the basis of their ability to grow in and suppress mitogen responsiveness of lymphocytes, these recombinants may be classified into four different groups. Group 1 viruses are the most immunosuppressive, whereas those of group 4 are least. These traits correlate with ability to replicate in lymphocytes. Genetic analysis of recombinants indicates that the most immunosuppressive recombinants do not necessarily contain the most fragment C DNA. Therefore, we have identified a restriction fragment, one or more of the genes of which are sufficient to allow an otherwise nonlymphocytotropic virus to replicate in lymphocytes. Additional genetic and immunologic analysis should permit us to determine the structure and function of the protein responsible for this effect.

Animals↗
PubMed1988

Intragenic deletions in 21 Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) families studied with the dystrophin cDNA: location of breakpoints on HindIII and BglII exon-containing fragment maps, meiotic and mitotic origin of the mutations.

Following the strategy outlined in an accompanying paper, we studied 32 X-linked muscular dystrophy families (29 Duchenne [DMD] and three Becker [BMD] type) for abnormalities of HindIII and BglII fragments detected by the entire dystrophin cDNA. Twenty-one different single-intragenic deletions, and no duplications, were identified. The deletion endpoints were precisely mapped on the published HindIII fragment map. Detailed analysis of overlapping deletions led to clarification of the fragment order for some previously unsettled regions of the HindIII map and to the construction of a partial map of exon-containing BglII fragments. For the regions involved in deletions, the corresponding HindIII and BglIII fragments could be identified. Noncontiguous comigrating fragments were detected in two regions by careful analysis of the patterns in deletion patients. Four of the 21 deletions generated novel restriction fragments that facilitated detection of female carriers in these families. Twelve of the deletions had a breakpoint in one of the two large introns known to be the sites of breakpoint clusters. By combining deletions and RFLP analyses, we unequivocally identified the gamete that first carried the mutation in 13 families: eight oocytes and five sperm. Germ-line mosaicism previously detected in one male was confirmed by cDNA studies. In two additional families gonadal mosaicism was found in females. As evidence is accumulating for frequent mitotic origin of these deletion mutations, this phenomenon has to be considered when postulating mutational mechanisms and in genetic counseling of DMD/BMD families.

Bacterial Proteins↗
PubMed1989

Resistance of heparinase-derived heparin fragments to biotransformation.

The biotransformation of heparinase-derived heparin fragments was examined via a combined approach using 35S-labeled heparin fragments as well as unlabeled chemically defined heparin fragments. Rats dosed with either [35S]di-, tetra-, hexa-, or octasaccharide fragments (2 mg/kg body weight, intravenously) excreted 63-69% of the injected radioactivity into the urine within 24 h with two-thirds being excreted during the first 6 h. Gel permeation chromatography of the urinary material shows that the tetra- and octasaccharides have undergone minor (approximately 5%) depolymerization whereas no change was observed for the di- and hexasaccharides. No N-desulfation was demonstrated for any of the substances. The hexa- and octasaccharide metabolites present in the urine 24 h after dosing exhibited the same antifactor Xa activity as that of the injected material. A chemically defined trisulfated disaccharide and a hexasulfated tetrasaccharide were prepared and dosed in a similar manner. Only one metabolite was recovered from animals dosed with disaccharide. This compound was characterized by anion exchange chromatography, proton nuclear magnetic resonance spectroscopy, Fourier transform infrared spectrometry, and mass spectrometry and shown to be identical to the injected disaccharide. Five metabolites were isolated from the urine of rats dosed with the hexasulfated tetrasaccharide. The major metabolite, consisting of at least 65% of the total, was characterized as described for the disaccharide and shown to be identical to the injected compound. The remaining material appeared to be disaccharides and, possibly, a tetrasaccharide conjugate. Taken together, our results show that the heparinase-derived heparin fragments are very resistant to biotransformation compared with heparin and endogenous heparin fragments. These fragments may therefore be useful in defining structure activity relationships in vivo.

Animals↗
PubMed1987

Tryptic digestion of dynein 1 in low salt medium. Origin and properties of fragment A.

Dynein 1 was extracted from sperm flagella of the sea urchin Tripneustes gratilla with 0.6 M NaCl and dialyzed against 0.5 mM EDTA, 14 mM 2-mercaptoethanol, 5 mM imidazole/HCl buffer, pH 7.0, for 24-48 h. In some cases, fractions containing the alpha heavy chain and the beta/intermediate chain 1 complex (beta/IC1) were separated by density gradient centrifugation in the same solution. Treatment of the samples at a trypsin:protein ratio of 1:10 w/w for 32 min at room temperature yields a crude digest from which Fragment A is purified by density gradient centrifugation. The purified Fragment A consists of two principal peptides (Mr = 195,000 and 130,000) that cosediment with the peak of ATPase activity at 12.5 S, which is slightly faster than the 11 S of the original beta/IC1 complex. When digests of the separated alpha chain and of the beta/IC1 complex are followed as a function of time, the early cleavages of the two heavy chains (Mr = 428,000) resemble each other in that both lead to similarly sized peptides of Mr 316,000 and 296,000, but only in the beta/IC1 fraction does the digestion proceed to form Fragment A. The remainder of the beta chain, termed Fragment B, occurs as an Mr 110,000 peptide sedimenting at 5.7 S with no associated ATPase activity. Fragment A has a specific ATPase activity of 4.3 mumol Pi X min-1 X mg-1, with a Km of 29 microM in 0.1 M NaCl medium, and an apparent Ki for inhibition by vanadate of 1.2 microM in the absence of salt, and 22 microM in 0.6 M NaCl. Photoaffinity labeling with [alpha-32P]8-azidoadenosine 5'-triphosphate indicates that the ATP binding site on the beta chain of dynein 1 is located on the Mr 195,000 peptide of Fragment A. The possibility that Fragments A and B of the beta/IC1 complex may correspond to the head and tail regions of the tadpole-shaped particle seen by electron microscopy is discussed.

Adenosine Triphosphatases↗
PubMed1988

Increase of the DNA strand assimilation activity of recA protein by removal of the C terminus and structure-function studies of the resulting protein fragment.

A proteolytic fragment of recA protein, missing about 15% of the protein at the C terminus, was found to promote assimilation of homologous single-stranded DNA into duplex DNA more efficiently than intact recA protein. This difference was not found if Escherichia coli single-stranded DNA binding protein was present. The ATPase activity of both intact recA protein and the fragment was identical. The difference in strand assimilation activity cannot be due to differences in single-stranded DNA affinity, since both the fragment and intact proteins bind to single-stranded DNA with nearly identical affinities. However, the fragment was found to bind double-stranded DNA more tightly and to aggregate more extensively than recA protein; both of these properties may be important in strand assimilation. Aggregation of the fragment was extensive in the presence of duplex DNA under the same condition where recA protein did not aggregate. The double-stranded DNA binding of both recA protein and the fragment responds to nucleotide cofactors in the same manner as single-stranded DNA binding, i.e. ADP weakens and ATP gamma S strengthens the association. The missing C-terminal region of recA protein includes a very acidic region that is homologous to other single-stranded DNA binding proteins and which has been implicated in DNA binding modulation. This C-terminal region may serve a similar function in recA protein, possibly inhibiting double-stranded DNA invasion. The possible role of the enhanced double-stranded DNA affinity of the fragment protein in the mechanism of strand assimilation is discussed.

Adenosine Diphosphate↗
PubMed1987

Polypeptide fragments of horse cytochrome c activate a small subset of secondary B lymphocytes primed against the native protein.

Horse cytochrome c (cyt c) and two large, overlapping cyanogen bromide-cleaved fragments (1-80 and 66-104), together encompassing the entire length of the polypeptide chain, were examined for their abilities to stimulate into antibody production individual secondary B lymphocytes primed against the intact protein. T cell help was provided against the carrier protein, hemocyanin, to which cyt c and its peptides were conjugated by using glutaraldehyde. All the B cells activated by both of the fragments elicited antibodies that reacted with intact cyt c in enzyme-linked immunosorbent assay, whereas only a fraction of the antibodies elicited by the intact protein reacted with the peptides. However, in general, antibodies reactive with the polypeptide fragments, whether elicited by the intact protein or by the fragments, could not be effectively inhibited from binding plate-bound cyt c in enzyme-linked immunosorbent assay in the presence of soluble native cyt c. This indicates that these antibodies are specific for denatured forms of cyt c that apparently arise during the chemical coupling of cyt c to carrier molecules for immunization and/or during emulsification of the immunogen in adjuvant. Whereas, at most, 5% of the secondary B cells specific for native cyt c could be activated by the 1-80 fragment, even fewer were activated by the 66-104 fragment. Therefore, it is unlikely that smaller peptides which fail to assume native conformation would be effective. Antibodies elicited in vivo in a primary response to the 1-80 fragment also failed to bind native cyt c. These results suggest that linear peptides intended to mimic epitopes on globular proteins, and which have not been engineered to adopt native conformation, will not be very effective either as primary or as secondary vaccines for B cell activation.

Animals↗
PubMed1988

Conformation and model membrane interactions of diphtheria toxin fragment A.

Low pH is believed to play a critical role in the penetration of membranes by diphtheria toxin in vivo. In this report, the pH dependence of the conformation of fragment A of diphtheria toxin has been studied using fluorescence techniques. As pH is decreased, fragment A in solution undergoes a reversible conformational change beginning below pH 5. The conformational change occurs rapidly upon exposure to low pH. It involves both an increase in the exposure of tryptophanyl residues to solution and a switch from a hydrophilic state to a hydrophobic state as judged by fragment A binding to micelles of a mild detergent (Brij 96). At low pH fragment A also rapidly and tightly binds to and penetrates model membranes. Binding is reversed when pH is neutralized. The transition pH, the apparent midpoint of the change between the hydrophilic state and the membrane-penetrating hydrophobic state, occurs at about pH 3.5 in the presence of Brij 96 micelles, pH 4 in the presence of small unilamellar vesicles (SUV) composed of zwitterionic phosphatidylcholine, and pH 5 in the presence of SUV composed of 25 mol % anionic phosphatidylglycerol and 75% phosphatidylcholine. The effects of high temperature provide an important clue as to the nature of the changes at low pH. At neutral pH and high temperature, i.e. in the thermally denatured state, a conformational change similar to that observed at low pH occurs, although fragment A does not become hydrophobic. In addition, the effects of low pH and high temperature on the stability of the native state are cumulative. This indicates that the changes in fragment A both at high temperature and at low pH involve denaturation, although there appears to be only partial unfolding under these conditions. Based on the results of this study, the role of fragment A in diphtheria toxin membrane penetration and translocation is evaluated.

Chromatography, High Pressure Liquid↗
PubMed1988

Low pH-induced release of diphtheria toxin A-fragment in Vero cells. Biochemical evidence for transfer to the cytosol.

When Vero cells with surface-bound 125I-labeled, nicked diphtheria toxin were exposed to pH 4.5, two polypeptides of Mr 20,000 and 25,000 became protected against externally applied Pronase E. The 20-kDa polypeptide appears to be the toxin A-fragment, whereas the 25-kDa polypeptide must be derived from the B-fragment. Permeabilization of the cells with saponin allowed efflux of the 20-kDa fragment to occur, whereas most of the 25-kDa polypeptide remained associated with the cells. A number of compounds and conditions which protect cells against diphtheria toxin prevented the protection against Pronase E. Protection of the 25-kDa polypeptide occurred even when the transmembrane proton gradient (delta pH) was dissipated by acidification of the cytosol, whereas protection and release of the A-fragment were prevented under these conditions. Electrical depolarization and ATP depletion of the cells did not inhibit protection and release of the A-fragment. The data indicate that delta pH is required for the transfer of the A-fragment to the cytosol, whereas the insertion of part of the B-fragment into the membrane occurs at low pH, even in the absence of a delta pH.

Adenosine Diphosphate Ribose↗
PubMed1987

Structure activity relationship studies with C-terminal fragments of vasopressin and oxytocin on avoidance behaviors of rats.

The potency of various C-terminal fragments of the neurohypophyseal hormone [Arg8]vasopressin [AVP-(1-9)] was determined using different avoidance behavioral test procedures in rats. Passive avoidance behavior was facilitated by these peptides. The fragments [Cyt6]AVP-(5-8) and [Cyt6]AVP-(5-9) were the most potent peptides tested after postlearning injection (consolidation) and preretention treatment (retrieval), respectively, after s.c. treatment. Comparable results were found when the peptides were injected into the lateral brain ventricle, but after this route of administration the peptides were about 3 to 4 order of magnitude more potent as compared to the s.c. route. Pentylenetetrazol-induced retrograde amnesia was reversed by AVP-(1-9) and various C-terminal fragments. In this respect [Cyt6] AVP-(5-9) and [pGlu4,Cyt6]-AVP-(4-9) appeared to be the most potent peptides. Injection of AVP-(1-9) and various C-terminal fragments after the acquisition of pole-jumping avoidance behavior induced resistance to extinction of the behavior. The fragment [pGlu4,Cyt6]AVP-(4-8) was the most potent peptide for this effect. Passive avoidance behavior was attenuated by s.c. administered oxytocin [OXT-(1-9)] and various C-terminal fragments. [pGlu4,Cyt6]-OXT-(4-9), [pGlu4,Cyt6]OXT-(4-8) and [Cyt6]OXT-(5-8) were the most potent of the peptides tested after postlearning administration, whereas [Cyt6]OXT-(5-9) was the most potent sequence after preretention injection. In all experiments the effects of the peptides were dose-dependent and of a long term nature. The results show that C-terminal fragments of the neurohypophyseal hormones potently modulate various aspects of memory processes, as assessed with different avoidance behaviors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Proteolysis of smooth muscle myosin light chain kinase. Formation of inactive and calmodulin-independent fragments.

Proteolysis by trypsin of gizzard myosin light chain kinase in the absence of Ca2+-calmodulin causes a biphasic effect on kinase activity. During the initial phase of proteolysis, Ca2+-calmodulin-dependent kinase activity is reduced over a thousand-fold. Further proteolysis, in the second phase, causes an increase in activity that is independent of Ca2+-calmodulin. Loss of activity is associated with the formation of a 64,000-dalton fragment. Calmodulin-independent activity is associated with the formation of a 61,000-dalton fragment. Procedures for the isolation of each fragment are outlined. Tryptic hydrolysis of the isolated 64,000-dalton peptide generates the 61,000-dalton peptide and increases calmodulin-independent activity. Km values for ATP and light chains for the native kinase and two fragments are the same, i.e. approximately 100 and 5 microM, respectively. Neither fragment binds to F-actin. Amino acid analyses of both fragments are given. Synthetic peptides corresponding to the calmodulin-binding regions of the smooth and skeletal muscle kinases are potent inhibitors of the 61,000-dalton fragment. These data demonstrate the existence of an inhibitory region that is suggested to be located between the active site and the calmodulin-binding site. Whether it is distinct from or at the N-terminal end of the calmodulin-binding site cannot be determined from these data.

Amino Acid Sequence↗
PubMed1986

[The role of A-cells in affecting the immunostimulating effect of F(ab')2-fragments].

The immunoregulatory effect of F(ab')2 fragments on normal rabbit IgG and that preincubated with A-cells from spleen have been compared. Both products were tested for their ability to enhance primary immune response of rabbit spleen cells to SRBC. It was demonstrated that low molecular mass product appeared after F(ab')2 fragments incubation with A-cells at 37 degrees C and possessed immunostimulating activity similar to that of initial F(ab')2 fragments. In addition, it was shown that F(ab')2 reduction to monovalent Fab' fragment with the following alkylation of SH-group abolished the ability of Fab' fragment to enhance the immune response. It may signify that half cystein Fab' fragment residue is essential for processing of the fragment in A-cells and (or) for immune response enhancement.

Alkylation↗
PubMed1979

Studies with fragments of a highly active analogue of luteinizing hormone releasing hormone.

The minimal structural requirements for gonadotrophin releasing activity were studied with fragments of a highly active analogue of luteinizing hormone releasing hormone (LH-RH), [D-Ser(But)6]LH-RH(1-9)nonapeptide-ethylamide (Hoe 766). All fragments are related to the C-terminal structure of LH-RH and have increased enzyme stability. Ovulation in phenobarbitone-blocked rats was induced with a median effective dose/rat, of 1.9 microgram of the (3-9)-heptapeptide, Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-ethylamide and 6.8, 18.0 and 38.3 microgram for the (4-9), (5-9) and (6-9) fragments respectively. The (3-9)- heptapeptide and (4-9)-hexapeptide induced release of LH and FSH in phenobarbitone-blocked rats with a ratio similar to that of LH-RH. Degradation of LH-RH by enzyme preparations of liver, kidney and hypothalamic or anterior pituitary tissue was not modified by addition of the (3-9)-heptapeptide fragment. The organ distribution of the 125I-labelled (3-9)-heptapeptide fragments was similar to LH-RH, but not to Hoe 766. The peptide accumulated in liver and kidney, but was eliminated from the anterior pituitary gland 15 min after i.v. injection, whereas Hoe 766 showed progressive accumulation in the pituitary gland (tissue:plasma ratio = 6.6 after 60 min). In contrast to C-terminal fragments of LH-RH, the corresponding fragments of nonapeptide analogues retained significant biological activity, and the minimal structural requirements for LH release may be related to the C-terminal sequence of LH-RH.

Animals↗
PubMed1987

Cytotoxicity of diphtheria toxin A fragment to toxin-resistant murine cells delivered by pH-sensitive immunoliposomes.

pH-sensitive immunoliposomes composed of dioleoylphosphatidylethanolamine and oleic acid (8:2 molar ratio) mediated the delivery of the cytotoxic fragment A of diphtheria toxin to the cytoplasm of target L-929 cells. Free fragment A, fragment A encapsulated in antibody-free liposomes, or fragment A encapsulated in pH-insensitive immunoliposomes was not effective in the inhibition of the cellular protein synthesis. pH-sensitive immunoliposomes containing diphtheria fragment A were not toxic to nontarget diphtheria-resistant A31 cells or to nontarget diphtheria-sensitive Vero cells. Pretreatment of target L-929 cells with the weak bases NH4Cl or chloroquine, agents which raise the endosome/lysosome pH, blocked the cytotoxic effect of the pH-sensitive immunoliposomes containing fragment A. Excess free antibody or excess empty pH-sensitive immunoliposomes also blocked the cytotoxic effect. Since it is known that fragment A alone cannot cross lipid membranes, the results of this study indicate that pH-sensitive immunoliposomes are able to release the toxin into the cytoplasm, probably by fusing with the endosome membrane following a receptor-mediated endocytosis of the immunoliposome.

Animals↗
PubMed1979

Affinity isolation and characterization of immunoglobulin G Fc fragment-binding glycoprotein from human blood platelets.

Immune complexes bind to several eukaryotic cell types including human blood platelets through the Fc fragment of immunoglobulin (Ig) G. Utilizing immobilized Fc fragment of IgG enabled us to isolate from human blood platelets a glycoprotein of an apparent Mr = 255,000 which, upon reduction, dissociated into sub-units of an apparent Mr = 50,000. This Fc fragment-binding glycoprotein has an isoelectric point between pH 6.3 and 6.9 and is composed of 34% hydrophobic, 25% acidic, and 14% basic amino acids. The Fc fragment-binding glycoprotein was also isolated from human platelet membrane preparations and was unaffected by prior treatment of platelets with thrombin. Isolated Fc fragment-binding glycoprotein formed an in vitro complex with aggregated immunoglobulin G. These results suggest that the isolated Fc fragment-binding component may prove useful in studies concerning the functional role of glycoproteins as cellular receptors for the Fc fragment of IgG.

Antigen-Antibody Complex↗
PubMed1979

Purification and characterization of rabbit anti-mouse renin specific Fab fragments.

Antibodies, raised against pure renin from the submaxillary gland of mice, were used to obtain renin specific Fab fragments. The purification steps were DEAE-chromatography, followed by papain digestion with separation of the undigested IgG preparation from the Fab/Fc fragments on a Sephadex G-100 column. Finally the Fab fragments were subjected to affinity chromatography on a CH-Sepharose 4B column with submaxillary renin attached. The purified Fab fragments revealed only a single band in SDS-polyacrylamide gel electrophoresis and a single precipitation line in cross immunoelectrophoresis. The association constants for the reaction of renin with the purified Fab fragments compared to the divalent antibodies were of the same magnitude, 0.7 x 10(11) l/mol and 1.0 x 10(11) l/mol, respectively. Comparison of the affinity of the Fab fragments for the antigenic determinants and the enzymatic inhibition of renin were determined to be approximately the same. Thus, the pure specific immunoreactive Fab fragment of antirenin, with an inhibitor constant of 1.5 x 10(-11), is the most potent inhibitor of mouse renin, so far.

Animals↗