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Peptide maps of Fab and Fc fragments of a locus negative and a locus positive IgG-immunoglobulin molecules
Free Communications
863
The chemistry of the antigenic components of basement membranes from vascular and avascular tissues N. A. Kefalides University of Chicago, Ill., U.S.A. Antibodies were produced against a fraction of lens capsule (ALC), Descemet's membrane (DM) and glomerular basement membrane (GBM) obtained by reduction and alkylation in 8 M urea and against the isolated collagen component of lens capsule. The reduced and alkylated fraction resembles the intact membrane in its amino acid and carbohydrate composition and is characterized by the presence of large amounts of hydroxylysine, hydroxyproline, neutral hexose, hexosamine and sialic acid. Antibodies to the reduced and alylated GBM cross-react with ALC and DM and are directed against a small molecular weight and a large molecular weight glycoprotein which have an amino acid composition unlike that of the collagen component. The collagen component has only glucose and galactose as its sugar moiety. Antibodies to the collagen component cross-react with the collagens isolated from various basement membranes. Fractionation studies suggest that the three antigenic components of basement membranes interact by hydrogen and disulfide bonds. The relative proportion of the antigenic components varies among basement membranes and it may determine their functional properties.
Peptide maps of Fab and Fc fragments of a locus negative and a locus positive IgGimmunoglobulin molecules K. L. Knight and A. Gilman-Sachs University of Illinois at the Medical Center, Chicago, lU.,
U.S.A. Ten to 30 per cent of normal rabbit IgG immunoglobulin molecules lack the allotypic specificity of the a heavy chain locus. Large quantities of these a locus negative molecules can be obtained from rabbits injected neonatally with anti a locus antisera. More than 95 per cent of the purified IgG immunoglobulin molecules from an a locus suppressed rabbit lacked the a locus specificity. Papain digestion of the a negative IgG molecules resulted in the formation of a crystalline fragment similar to the Fc fragment observed with normal rabbit IgG immunoglobulin. The Fc fragment of a negative molecules and Fc fragment of a positive molecules were digested with trypsin and the resulting peptides were compared by peptide mapping on thin layer cellulose plates. Approximately 34 peptides were observed and no difference between the peptides of the a negative and a positive molecules was found. In contrast to this, comparison of the peptide maps of the Fab fragment of the a negative and a positive molecules revealed 2-3 peptide differences. These results show that subclasses of rabbit gamma chains, as identified by their allotypic specificities in the Fd region, differ in the Fd portion with respect to their peptide maps but that the Fc portion of the molecules is identical. Allotypes to the a negative molecules have recently been identified in our laboratory and concurrent linkage and structural studies, will be helpful for understanding the genetic basis for the assembly of the variable and constant portion of the heavy chain.
Application of displacement radioimmunologic determination of fructose-l, 6diphosphatase t
H.J. Kolb and G. M. Grodsky Inst.f. Diabetesforschung, Munich, Germany Displacement radioimmunoassay was applied to quantitatively estimate rabbit liver fructose-l,6-diphosphatase (FDPase) using antiserum from guinea pig. FDPase was radioiodinated by the chloramine-T procedure modified to provide high specific activity, despite lability of enzyme in oxidative environment. The immunoassay was sensitive to
863
The chemistry of the antigenic components of basement membranes from vascular and avascular tissues N. A. Kefalides University of Chicago, Ill., U.S.A. Antibodies were produced against a fraction of lens capsule (ALC), Descemet's membrane (DM) and glomerular basement membrane (GBM) obtained by reduction and alkylation in 8 M urea and against the isolated collagen component of lens capsule. The reduced and alkylated fraction resembles the intact membrane in its amino acid and carbohydrate composition and is characterized by the presence of large amounts of hydroxylysine, hydroxyproline, neutral hexose, hexosamine and sialic acid. Antibodies to the reduced and alylated GBM cross-react with ALC and DM and are directed against a small molecular weight and a large molecular weight glycoprotein which have an amino acid composition unlike that of the collagen component. The collagen component has only glucose and galactose as its sugar moiety. Antibodies to the collagen component cross-react with the collagens isolated from various basement membranes. Fractionation studies suggest that the three antigenic components of basement membranes interact by hydrogen and disulfide bonds. The relative proportion of the antigenic components varies among basement membranes and it may determine their functional properties.
Peptide maps of Fab and Fc fragments of a locus negative and a locus positive IgGimmunoglobulin molecules K. L. Knight and A. Gilman-Sachs University of Illinois at the Medical Center, Chicago, lU.,
U.S.A. Ten to 30 per cent of normal rabbit IgG immunoglobulin molecules lack the allotypic specificity of the a heavy chain locus. Large quantities of these a locus negative molecules can be obtained from rabbits injected neonatally with anti a locus antisera. More than 95 per cent of the purified IgG immunoglobulin molecules from an a locus suppressed rabbit lacked the a locus specificity. Papain digestion of the a negative IgG molecules resulted in the formation of a crystalline fragment similar to the Fc fragment observed with normal rabbit IgG immunoglobulin. The Fc fragment of a negative molecules and Fc fragment of a positive molecules were digested with trypsin and the resulting peptides were compared by peptide mapping on thin layer cellulose plates. Approximately 34 peptides were observed and no difference between the peptides of the a negative and a positive molecules was found. In contrast to this, comparison of the peptide maps of the Fab fragment of the a negative and a positive molecules revealed 2-3 peptide differences. These results show that subclasses of rabbit gamma chains, as identified by their allotypic specificities in the Fd region, differ in the Fd portion with respect to their peptide maps but that the Fc portion of the molecules is identical. Allotypes to the a negative molecules have recently been identified in our laboratory and concurrent linkage and structural studies, will be helpful for understanding the genetic basis for the assembly of the variable and constant portion of the heavy chain.
Application of displacement radioimmunologic determination of fructose-l, 6diphosphatase t
H.J. Kolb and G. M. Grodsky Inst.f. Diabetesforschung, Munich, Germany Displacement radioimmunoassay was applied to quantitatively estimate rabbit liver fructose-l,6-diphosphatase (FDPase) using antiserum from guinea pig. FDPase was radioiodinated by the chloramine-T procedure modified to provide high specific activity, despite lability of enzyme in oxidative environment. The immunoassay was sensitive to