Bovine serum transferrin phenotypes AA,D1D1, D2D2, EE: Their carbohydrate compositions and electrophoretic multiplicity

Samples of homozygous bovine serum transferrins have been prepared and their purity has been ascertained by immunological techniques and electrophoretic analysis in SDS. Measurements of carbohydrate composition show that no significant differences exist among the phenotypic variants AA, D₁D₁, D₂D₂, and EE. Chromatography of transferrin AA on DEAE-cellulose separated four subfractions, each of which corresponded well with one band obtained by polyacrylamide gel electrophoresis. Carbohydrate analyses of the individual subfractions did not show significant differences in sialic acid, hexose, or hexosamine contents. After desialylation with neuraminidase, each subfraction was converted to a major band and a minor band on gel electrophoresis. From the relative band positions of the desialylated transferrins, it was concluded that possession of sialyl residues by bovine transferrin is not the primary cause of electrophoretic multiplicity. Rather, sialic acid masks an underlying heterogeneity which most likely resides within the polypeptide chain. Further characterization of this heterogeneity will best be undertaken with the isolated asialotransferrin subfractions.This research was supported by Grants MT-4074 and MA-5554 from the Medical Research Council of Canada and a Senior Fellowship (to M. W. C. H.) from the Ontario Heart Foundation.     

The preparation and partial characterization of N-terminal and C-terminal iron-binding fragments from rabbit serum transferrin.

Two iron-binding fragments of Mr 36 000 and 33 000 corresponding to the N-terminal domain of rabbit serum transferrin were prepared. One iron-binding fragment of Mr 39 000 corresponding to the C-terminal domain was prepared. The N-terminal amino acid sequence of rabbit serum transferrin is: Val-Thr-Glu-Lys-Thr-Val-Asn-Trp-?-Ala-Val-Ser. One glycan unit is presented in rabbit serum transferrin and it is located in the C-terminal domain.     

The N- and C-terminal amino acid sequences of the heavy chain from a pathological human immunoglobulin IgG

The heavy chain of a pathological human immunoglobulin IgG and also the Fd fragment have been isolated. No free alpha-amino group was present on either and the N-terminal sequence of both has been identified as pyrrolid-2-one-5-carbonylvalylthreonine. Splitting at the four methionine residues of the heavy chain with cyanogen bromide gave five fractions. The fraction from the C-terminal end of the chain was isolated in high yield and the amino acid sequence was: His-Glu-Ala-Leu-His-Asp(NH(2))-His-Tyr-Thr-Glu(NH(2))-Lys-Ser-Leu-Ser-Leu-Ser-Pro-Gly These results give strong support to the view that the heavy chain of immunoglobulin is a single peptide chain.     

N-terminal amino acid sequences of precursor and mature forms of alpha-1-antitrypsin

alpha-1-Antitrypsin is found in hepatocytes as a high-mannose glycoprotein (Mr 49 000), extracellularly as a complex-type glycoprotein (Mr 54 000). Deglycosylation of both forms with peptide: N-glycosidase led to proteins of identical app. Mr (41 000). The sequence of 26 N-terminal amino acids of rat alpha 1-antitrypsin was determined. A high content of polar amino acids was found. The partially characterized presequence of in vitro synthesized alpha 1-antitrypsin showed a cluster of hydrophobic amino acids. A pre-peptide of 24 amino acids is proposed. There is no evidence for the existence of a propeptide.     

Involvement of the N- and C-terminal fragments of bovine pancreatic deoxyribonuclease in active protein folding

The three-dimensional structure of bovine pancreatic (bp) DNase revealed that its N- and C-termini form an antiparallel beta-sheet structure. The involvement of this beta-sheet structure in the active protein folding of bpDNase was thus investigated via a series of deletion and substitution variants. Several substitution variants of N-terminal Leu1 and C-terminal Leu259, and one variant with only the last Thr260 deleted, remained fully active. However, the other deletion variants, in which 2-10 amino acid residues were removed from the C- or N-terminus, all lost the DNase activity. The results indicated that the backbone hydrogen bonding in the antiparallel beta-sheet, rather than the side-chain interactions, is crucial for the correct protein folding. When the deletion variants were complemented with synthetic peptides of the deleted N- or C-terminal sequences, the DNase activity was generated. The highest DNase activity was generated when the C-terminal 10-residue-deleted brDNase(Delta251-260) was admixed with the C-terminal 10-residue peptide (peptide C10) in a molar ratio of 1:400. The noncovalent binding between brDNase(Delta251-260) and peptide C10 exhibited a dissociation constant of 48 microM. Circular dichroism spectra showed that the deletion variants were partially folded with mainly helical structures and that admixture with corresponding peptides facilitated their folding into the nativelike beta-sheet-rich structure. Thermal denaturation profiles also revealed that the transition temperature for brDNase(Delta251-260) was increased from 55 to 63 degrees C after incubation with peptide C10. The folding activation process for the deletion variant occurred in two stages, and Ca(2+) was required.     

N-terminal amino acid sequences of the hemopexins from chicken, rat and rabbit

The N-terminal amino acid sequences of the hemopexins purified from the plasma of rat, rabbit and chicken were compared with each other and with that of human hemopexin. Although the N-terminal sequences differ among these species, residues 2, 3 and 14 are identical in all four hemopexins. Ten of the first 28 residues are identical in all but the chicken protein. When introducing gaps into the sequence, a much greater homology is observed between the human and rat or rabbit hemopexins (60%) than when the sequences were compared directly (40%).     

N-terminal amino acid sequences and amino acid compositions of the Spirochaeta aurantia flagellar filament polypeptides.

The amino-terminal sequences and amino acid compositions of the three major and two minor polypeptides constituting the filaments of Spirochaeta aurantia periplasmic flagella were determined. The amino-terminal sequence of the major 37.5-kDa outer layer polypeptide is identical to the sequence downstream of the proposed signal peptide of the protein encoded by the S. aurantia flaA gene. However, the amino acid composition of the 37.5-kDa polypeptide is not in agreement with that inferred from the sequence of flaA. The 34- and 31.5-kDa major filament core polypeptides and the 33- and 32-kDa minor core polypeptides show a striking similarity to each other, and the amino-terminal sequences of these core polypeptides show extensive identity with homologous proteins from members of other genera of spirochetes. An additional 36-kDa minor polypeptide that occurs occasionally in preparations of S. aurantia periplasmic flagella appears to be mixed with the 37.5-kDa outer layer polypeptide or a degradation product of this polypeptide.     

The catalytic chain of human complement subcomponent C1r. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments.

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the 'charge-relay' system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the 'histidine loop', a disulphide bond that is present in all other known serine proteinases.     

cDNA and amino acid sequences of bovine tissue factor.

A cDNA encoding bovine tissue factor has been isolated from a lambda gt11 bovine adrenal cDNA library. The cDNA insert was 1877 base pairs with an open reading frame of 876 base pairs that encoded a presequence of 35 amino acids and a mature tissue factor of 257 amino acids. Bovine tissue factor had three potential N-glycosylation sites, four extracellular cysteine residues, a cytoplasmic cysteine residue, and one tripeptide tryptophan-lysine-serine motif. Identities of the amino acid sequences of the mature forms between the bovine tissue factor and each of human, mouse, and rabbit tissue factors were 70.4%, 57.2%, and 74.1%, respectively.     

Purification and N-terminal amino acid sequences of Chlamydia trachomatis histone analogs.

DNA-binding proteins specific to Chlamydia trachomatis elementary bodies have been described and recently characterized as procaryotic histone analogs. I have developed an affinity purification procedure for the 18-kDa histone analog, Hc1, based on its affinity for polyanions. The availability of highly purified Hc1 has allowed for determination of its N-terminal amino acid sequence and should prove useful in studies of its biological function. The variable C. trachomatis histone analog not obtained by this procedure was electrophoresed onto Immobilon paper for sequencing. The N terminus of the variable histone was conserved among C. trachomatis serotypes L2, D, and B and was distinct from that of Hc1.     

Comparison of bovine serum transferrin A and D2. I. Amino acid residue differences

A comparison is made of single components of the homozygous variants A and D2 of bovine serum transferrin by tryptic, chymotryptic and cyanogen bromide digestion. It is concluded that there are three substitutions A:D2-Glu:Asp, Lys:Arg and Asp:Gly. In the light of the recent work of Brock et al. (1980) it is concluded that all three substitutions occur in the C-terminal sequence of the chain. By homology with the sequence of human serum transferrin (MacGillivray et al., 1982) the Lys:Arg and Asp:Gly substitutions probably occur at residues 527 and 446, respectively, from the N-terminus. The Asp:Gly substitution is considered more likely than our earlier conclusion (Maeda, McKenzie & Shaw, 1977) that there is a deletion in the chain of D2 (A:D2, Asp:--). The location of the Glu:Asp substitution is not known.     

Structural proteins of Western equine encephalitis virus: amino acid compositions and N-terminal sequences

The structural proteins of Western equine encephalitis virus, a member of the alphavirus group, have been characterized by the determination of their amino acid compositions and by N-terminal sequence analysis. More than 60 residues of the N-terminal sequences of each of the envelope glycoproteins have been determined. A comparison of these sequences with the previously determined sequences of two related alphaviruses. Sindbis virus and Semliki Forest virus, strongly supports the view that all three viruses have evolved from a common ancestor and provides information on the pattern of this evolution. The analysis of the capsid proteins of Western equine encephalitis virus shows that the nucleocapsid of this virus can accommodate a considerable degree of variability in its protein component and that at least some regions of alphavirus capsid proteins show more extensive differences between different viruses than do the envelope glycoproteins.