Proteolysis of rabbit immunoglobulin M by papain.
Ontology highlight
ABSTRACT:
SUBMITTER: McIlroy BM
PROVIDER: S-EPMC1168411 | biostudies-other | 1974 Nov
REPOSITORIES: biostudies-other
Similar Datasets
Project description:About two-thirds of the hexose of human and rabbit immunoglobulin G (IgG) was located in the Fc fragment and one-third in the ;hinge' region of the gamma (heavy) polypeptide chain at the junction of the Fab and Fc fragments. In contrast, bovine IgG contained more hexose in the ;hinge' region than in the Fc fragment. The initial cleavage of susceptible IgG molecules into Fab and Fc fragments by papain under the conditions given by Porter (1959) had reached completion after digestion for 2hr., though bovine IgG was digested somewhat more slowly than human or rabbit IgG. The release of ;hinge' peptides from human and rabbit IgG had also reached completion by 2hr., but was slower from bovine IgG and continued for several hours longer. Since bovine IgG molecules contained on the average a greater amount of hexose in the ;hinge' region, carbohydrate on this part of the gamma-chain may influence not only the initial rate of enzymic hydrolysis into Fab and Fc fragments, but also, and to a greater extent, the rate of further limited hydrolysis of the N-terminal regions of the Fc fragment. The presence of carbohydrate in the ;hinge' region does not appear to account for the resistance of some IgG molecules to papain digestion and of some Fc fragments to N-terminal degradation.
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Project description:Four types of Fc fragments of different sizes were isolated by papain treatment of rabbit immunoglobulin G under various conditions and by subsequent chromatographic procedures. 1. Brief digestion at neutral pH without reduction produced a molecule in which the Fab and Fc fragments were still linked by a pair of labile disulphide bridges, and the Fc fragment released by cleaving these bonds, called 1Fc fragment, contained a portion of the ;hinge' region including an interchain disulphide bridge. Both complement-binding and guinea-pig skin-binding activities were retained by this fragment, which had mol. wt. 48000. 2. Prolonged digestion at neutral pH of immunoglobulin G whose labile inter-heavy-chain disulphide bridges had been reduced removed the ;hinge' region, giving mFc fragments (mol. wt. 46000), which lacked the capacity to bind guinea-pig skin but retained the antigenic as well as the complement-binding activities of 1Fc fragment completely. 3. Digestion at pH5.0 yielded a smaller fragment, sFc (mol. wt. 40000), which was no longer able to bind complement. Though the antigenic structure was intact, sFc fragment was curiously unable to precipitate with antibodies to the N-terminal determinants. 4. Fragment stFc (mol. wt. 25000), representing the C-terminal portion of Fc fragment, was formed from all the larger fragments by digestion at pH4.5. Only the C-terminal antigenic determinants were retained by stFc fragment.
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Project description:Rabbits have been used extensively as a model system for the elucidation of the mechanism of immunoglobulin diversification and for the production of antibodies. We employed Next Generation Sequencing to analyze Ig germline V and J gene usage, CDR3 length and amino acid composition, and gene conversion frequencies within the functional (transcribed) IgG repertoire of the New Zealand white rabbit (Oryctolagus cuniculus). Several previously unannotated rabbit heavy chain variable (VH) and light chain variable (VL) germline elements were deduced bioinformatically using multidimensional scaling and k-means clustering methods. We estimated the gene conversion frequency in the rabbit at 23% of IgG sequences with a mean gene conversion tract length of 59±36 bp. Sequencing and gene conversion analysis of the chicken, human, and mouse repertoires revealed that gene conversion occurs much more extensively in the chicken (frequency 70%, tract length 79±57 bp), was observed to a small, yet statistically significant extent in humans, but was virtually absent in mice.
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Project description:Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins.
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Project description:Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ?1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.
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Project description:1. Proteolysis of immunoglobulin G with long-acting thyroid-stimulating activity with papain and pepsin shortened the duration of thyroid stimulation in mice prepared for the McKenzie (1958) bioassay. The time-course appeared to be related to molecular size. 2. The activity after proteolysis was recovered solely in the electrophoretically slow F(ab)-fragment. 3. There was no immunological cross-reaction between pituitary thyroid-stimulating hormone and the active fragments of the long-acting thyroid stimulator despite their similar behaviour in the bioassay. 4. The implications of these findings are discussed.
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Project description:Creatine kinase from rabbit muscle is inactivated by limited proteolysis with proteinase K from Tritirachium album. Gel-filtration and cross-linking studies showed that the limited proteolysis did not affect the molecular weight of the enzyme under non-denaturing conditions, but did cause changes in the reactivity of the reactive thiol group on each subunit and in the ability of the enzyme to form a 'transition-state analogue' complex in the presence of magnesium acetate plus ADP plus creatinine plus NaNO3.
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Project description:Rabbit muscle phosphoglycerate mutase is inactivated by proteolysis with thermolysin. Inactivation is correlated with the breakage of one (or a few) bond(s) near one end of the polypeptide chain. There is no change in the overall conformation, quaternary structure or binding to Cibacron Blue on proteolysis. The possible analogy with the existence of a flexible tail in the yeast enzyme is discussed.
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Project description:The Ig heavy chain variable region (V(H)) genes encode the antigen-binding regions of antibodies. The rabbit genome contains more than 100 V(H) genes, but only one (V(H)1) is preferentially used in the VDJ gene rearrangement. Three highly divergent alleles occur at this V(H)1 locus in most rabbit populations. These three V(H) alleles are also present in snowshoe hare populations, indicating that the polymorphism of the V(H)1 alleles is trans-specific. Here we report the results of a phylogenetic analysis of rabbit Ig germ-line V(H) genes (alleles) together with V(H) genes from humans and mice. We have found that all rabbit V(H) genes belong to one mammalian V(H) group (group C), which also includes various human and mouse V(H) genes. Using the rate of nucleotide substitution obtained from human and mouse V(H) sequences, we have estimated that the V(H)1 polymorphism in the rabbit lineage has been maintained for about 50 million years. This extremely long persistence of V(H)1 polymorphism is apparently caused by overdominant selection, though the real mechanism is unclear.
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Project description:By using sodium dodecyl sulphage/polyacrylamide-gel electrophoresis it was shown that rabbit muscle creatine kinase, both in a homogenate and purified, appears to be composed of a mixture of two peptides (mol.wts. 42100 and 40300) differing in length by about 15 amino acids. It is found that low concentrations of proteinase K from the fungus Tritirachium album can cleave about 38 amino acids from each chain of creatine kinase, leaving two large fragments (mol.wts 37700 and 35500). Scission of the whole enzyme was found to be concomitant with complete loss of enzyme activity. MgADP in the presence of absence of creatine slowed the rate of proteolysis by about 50%, but the transition-state analogue complex creatine-NO3--MgADP appeared to protect completely. The time course for the proteolytic inactivation in the presence of this complex, but not in its absence, was biphasic.
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