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Biomedical subjects

F W Putnam

Publications and source records attributed to F W Putnam.

At least 127 records · Page 7Linked to original sources
PubMed1979

Primary structure of a human IgA1 immunoglobulin. I. Isolation, composition, and amino acid sequence of the chymotryptic peptides.

As the initial phase of the determination of the complete covalent structure of a human immunoglobulin A, 52 chymotryptic peptides, ranging in length from 2 to 37 residues, were isolated and characterized from the reduced and carboxymethylated alpha1 heavy chain of the myeloma IgA protein Bur. The peptides were subjected to sequence analysis by the dansylation technique, manual and automatic Edman degradation, and carboxypeptidase digestion. The results, in conjunction with the data on the tryptic and thermolysin peptides and the cyanogen bromide fragments reported in the accompanying papers, established the complete primary structure of a human IgA chain.

Amino Acid Sequence↗
PubMed1979

Primary structure of a human IgA1 immunoglobulin. IV. Streptococcal IgA1 protease, digestion, Fab and Fc fragments, and the complete amino acid sequence of the alpha 1 heavy chain.

In order to establish the complete amino acid sequence of the human IgA alpha1 chain Bur, IgA1 protease from Streptococcus sanguis was employed to generate Fabalpha and Fcalpha fragments in the final stage of this investigation. Cyanogen bromide cleavage of the Fabalpha fragment followed by reduction and aminoethylation produced the Fd' fragment (residues 84 to 227); this contains part of the variable region (VR), the whole first constant domain (Calpha1), and part of the hinge region of this heavy chain. The tryptic peptides of the Fd' fragment were isolated, characterized, and sequenced. The results together with the data in the preceding papers on chymotryptic, tryptic, and thermolysin peptides permitted the complete amino acid sequence of the human IgA alpha1 chain to be established.

Amino Acid Sequence↗
PubMed1979

Primary structure of a human IgA1 immunoglobulin. II. Isolation, composition, and amino acid sequence of the tryptic peptides of the whole alpha1 chain and its cyanogen bromide fragments.

As part of the strategy for determining the covalent structure of a human IgA1 molecule (Bur), a tryptic digest was prepared of the reduced and carboxymethylated alpha1 heavy chain. In addition to the main experiment, tryptic peptides were prepared from the succinylated aminoethylated alpha1 chain and from fragments obtained by CNBr scission of the alpha1 chain. Complete recovery of the peptides was impeded by the large size of some of the tryptic peptides and of the principal CNBr fragment, and difficulty in separating other glycopeptides. Twenty-eight tryptic peptides of the reduced and carboxymethylated alpha1 chain were purified and sequenced, accounting for more than 300 residues. Additional information was obtained by sequence analysis of trypudies described in this series of papers contributed to the complete sequence analysis of the alpha1 chain.

Amino Acid Sequence↗
PubMed1979

Primary structure of a human IgA1 immunoglobulin. III. Isolation, composition, and amino acid sequence of the thermolysin peptides.

As part of the strategy for determination of the complete covalent structure of a human IgA immunoglobulin, 66 peptides were isolated from a thermolysin digest of reduced and carboxymethylated IgA alpha1 chain Bur and were purified. They range in length from 2 to 24 residues. Some of the peptides have been characterized and sequenced in order to supply needed information that was not obtained from the chymotryptic and tryptic peptides. These thermolysin peptides provide much necessary data to produce a rigorous proof for the primary structure of the human alpha1 chain. The remaining peptides from the thermolysin digest whose amino acid composition and NH2-terminal residues were sufficient to identify them unequivocally have also been assigned in the structure. They supply additional information that helps remove ambiguity in the structure, and they provide useful data about the profile of the peptide bonds that are susceptible to thermolysin digestion.

Amino Acid Sequence↗
PubMed1979

Structural studies of human IgD: isolation by a two-step purification procedure and characterization by chemical and enzymatic fragmentation.

A myeloma IgD immunoglobulin (designated WAH) that was present in high concentration in plasma ( approximately 3.5 g/dl) was purified in >90% yield by a two-step procedure of ammonium sulfate precipitation plus AcA 34 gel filtration. Although the plasma had been stored for 2 years without the addition of a proteolytic inhibitor, no "spontaneous" degradation was apparent and the isolated IgD remained structurally intact. However, the purified IgD showed extreme susceptibility in vitro to various proteolytic enzymes; e.g., Fab(delta) (M(r) approximately 47,000) and Fc(delta) (M(r) approximately 80,000) fragments were generated quantitatively after only 10 min of incubation with papain in the absence of cysteine. By combining limited enzymatic digestion, reductive cleavage, and cyanogen bromide fragmentation, several series of well defined fragments corresponding to the different regions and domains of the IgD molecule were generated. These fragments are useful for physical, chemical, and immunological studies, as well as for the sequence determination of the IgD delta chain. A model of the IgD molecule was derived from such studies and from overlapping of the series of fragments. The possible existence of an extra constant domain in the delta chain appears unlikely in view of our finding of an extended hinge region of about 50 residues which can be cleaved off the amino terminus of the papain Fc(delta) by brief treatment with trypsin. In addition to a distinct stretch of carbohydrate attachment sites, the delta-chain hinge region contains a segment unusually rich in electrical charge. This charged segment is responsible for the lability of IgD to spontaneous degradation and may be related to its biological role as a B lymphocyte receptor.

Cyanogen Bromide↗
PubMed1979

Structure of the A2m(1) allotype of human IgA--a recombinant molecule.

The complete amino-acid sequence of the constant (C) region of the alpha2 heavy chain of a human IgA2 protein of the A2m(1) allotype has been determined. Excluding the hinge region and the carbohydrate content, this alpha2 allotype differs from the alpha1 chain in only 14 amino-acid positions; all of these are identical to the A2m(2) allotype of the alpha2 chain and confer subclass (or isotypic) character on the alpha2 chains. However, the A2m(2) allotype differs in six positions where A2m(1) and alpha1 are identical; the first two are just before the hinge and the other four are in the last (C(H)3) domain. The A2m allotypic character of alpha2 chains is attributed to several conformational factors in the sequence at positions 211-221, just before the hinge. The isoallotypic determinant shared by alpha1 chains and the A2m(1) allotype of alpha2 resides in the identity of their C(H)3 domains. Thus, the A2m(1) allotype appears to be a hybrid chain that is identical with alpha1 in the C(H)3 domain and identical with the A2m(2) alpha2 chain in the C(H)1 and C(H)2 domains and in the hinge, except for the allotypic determinants arising from four structural differences from residues 211-221. The genetic origin of isotypes, allotypes, and isoallotypes of the alpha chain has involved several events of homologous crossing over and neutral point mutations accumulated late in the evolutionary development of IgA immunoglobulins. Since the crossing over appears to occur between C(H)2 and C(H)3, heavy chain domains may be coded for by independent units in embryonic DNA that are analogous to the variable (V) and C segments of light-chain genes.

Amino Acid Sequence↗
PubMed1979

Complete amino acid sequence of a histidine-rich proteolytic fragment of human ceruloplasmin.

The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in alpha helices, about a third in beta-sheet structure, and the remainder in beta turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication.

Amino Acid Sequence↗
PubMed1978

Complete amino acid sequence of the alpha 2 heavy chain of a human IgA2 immunoglobulin of the A2m (2) allotype.

The complete amino acid sequence of the alpha 2 heavy chain of a human IgA2 immunoglobulin of the A2m(2) allotype has been determined and is compared to the sequence of the alpha 1 chain of the human IgA1 subclass. The characteristic differences between the alpha 1 and alpha 2 chains are greatest in the hinge region and in the location and number of the oligosaccharides. Apart from the duplication in the hinge region of alpha 1 and the deletion in alpha 2, there are 23 amino acid exchanges in the constant (C) regions of the two chains. Accepted mutations are related to the surface accessibility of the residues and the proximity of carbohydrate. The results indicate that human IgA and IgG subclasses arose late in evolution and reflect similar mutationa pressures.

Amino Acid Sequence↗
PubMed1978

Cold pepsin digestion: a novel method to produce the Fv fragment from human immunoglobulin M.

A strategy for the proteolytic fragmentation of human IgM has been developed. This method is called "cold pepsin digestion" because of its unique feature of achieving restricted peptic cleavages at 4 degrees and pH 4.0. Cold pepsin digestion has been applied successfully to produce an Fv fragment from 14 human IgM proteins. The Fv fragment consists of the heavy chain variable domain (VH) and the light chain variable domain (VL) held together by strong noncovalent interaction. Thus, each Fv fragment contains one intact antigen-binding site and represents the minimal active fragment derivable from an antibody molecule. A series of other structurally and functionally important fragments were also isolated and characterized. Two basic digestion pathways were recognized; these mainly reflect the relative accessibility of five sets of major interdomain cleavage sites.

Binding Sites, Antibody↗
PubMed1977

Chemical evidence that proteolytic cleavage causes the heterogeneity present in human ceruloplasmin preparations.

Nine samples of human ceruloplasmin [iron(II):oxygen oxidoreductase; EC 1.16.3.1] prepared by different procedures have been examined for heterogeneity; gel electrophoresis showed that seven contained a number of components with molecular weights ranging from 20,000 to 130,000, and two contained largely a single component of molecular weight 130,000. Digestion of a single-component preparation with plasmin produced fragments with molecular weights similar to those found in the multicomponent preparations. Amino-terminal analysis, peptide mapping, and amino acid analysis showed that plasmin digestion generated a fragment of 20,000 molecular weight, which corresponded to a component present in a multicomponent ceruloplasmin preparation. The 20,000 molecular weight fragment appears to correspond to the so-called alpha-subunit or L-chain of human ceruloplasmin. Chemical evidence is thus provided that ceruloplasmin is a single-chain protein and that the so-called subunits are fragments. The 20,000 molecular weight fragment contains a single cysteine; amino acid sequence studies have shown that the sequence in the vicinity of this residue is similar to that around the single cysteine residue in plant plastocyanins and bacterial azurins, which are small, blue, copper-containing proteins.

Amino Acid Sequence↗
PubMed1977

Location and structural significance of the oligosaccharides in human Ig-A1 and IgA2 immunoglobulins.

The location, number, and kinds of oligosaccharides in human IgA1 and IgA2 immunoglobulins have been determined by amino acid sequence analysis of the alpha heavy chains. Both A2m allotypes of the alpha2 chain of IgA2 have two GlcN oligosaccharides that are absent in the alpha1 chain, but they lack GalN. The A2m(2) allotype has a fifth GlcN oligosaccharide. The alpha chains of IgA proteins also have subclass-specific and allotype-specific differences in amino acid sequence. Although other classes of human immunoglobulins differ in the number and kind of oligosaccharides, the sites are often homologous and are related to the immunoglobulin domain structure. Evolutionary preservation of the tripeptide acceptor sequence for GlcN probably indicates both a structural and biological role for carbohydrate.

Amino Acid Sequence↗
PubMed1976

Complete covalent structure of a human IgA1 immunoglobulin.

The complete covalent structure has been determined for a human myeloma IgA1 immunoglobulin. This protein has unique features in the amino acid sequence and disulfide bridge structure of the variable (V) and constant (C) regions of both the alpha heavy and the lambda light chains, and in the number and loci of oligosaccharides. Whereas C region domains of heavy chains have evolved independently over eons, recent isotypic variations have occured in lambda light chains and possibly in alpha heavy chains.

Amino Acid Sequence↗
PubMed1976

Structure, function, and evolutionary relationships of Fc domains of human immunoglobulins A, G, M, and E.

Human immunoglobulins, A, G, M, and E have strong homology in amino acid sequence (about 30 percent) distributed nonuniformly throughout the Fc region. Immunoglobulins M are A are least alike in the first Fc domain and most alike in the second. Individual domains of heavy chains have evolved with different mutation rates but with conservation of essential structural features. No relation of primary structure to complement binding ability is apparent.

Amino Acid Sequence↗