Bovine Subunit Research Articles

Purification and properties of the low-molecular-weight α-crystallin from normal goat lens: Comparison with bovine lens

Low-molecular-weight α-crystallin ( α L-crystallin) isolated from decapsulated lens of goat ( Capra hiscus) has been purified to an apparently homogeneous population. Goat α L-crystallin closely resembles its bovine counterpart in size, shape, exposition of sulfhydryl groups, subunit composition and the nature of its UV-absorption profile. Like bovine α L-crystallin, dissociated subunits of goat α L-crystallin assemble upon reassociation into a particle of almost half the size of the native one. However, subunits of goat α L-crystallin are found to contain more aromatic amino acid residues than those of bovine subunits leading to a higher value of extinction coefficient ( E 1 cm 1%) at 280 nm for the goat protein.

ATP synthase of yeast mitochondria. Characterization of subunit d and sequence analysis of the structural gene ATP7.

The subunit analogous to the d-subunit of ATP synthase from bovine heart mitochondria was isolated from the purified yeast enzyme. Partial protein sequences were determined by direct methods. From this information, two oligonucleotide probes were constructed and used for screening a DNA genomic bank of Saccharomyces cerevisiae. The sequence of yeast subunit d was deduced from the DNA sequence of ATP7 gene. Mature yeast subunit d is 173 amino acids long. Its NH2-terminal serine is blocked by an N-acetyl group, and the protein has no processed NH2-terminal sequence other than the removal of the initiator methionine. The protein is predominantly hydrophilic. The amino acid sequence is 22% identical and 44% homologous to bovine subunit d. A null mutant was constructed. The mutant strain was unable to grow on glycerol medium. The mutant mitochondria had no detectable oligomycin-sensitive ATPase activity, and the catalytic sector F1 was loosely bound to the membranous part. The mutant mitochondria did not contain subunit d, and the mitochondrially encoded hydrophobic subunit 6 was not present.

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Complete and partial glycophospholipid anchors are found on a fusion protein consisting of luteinizing hormone beta subunit followed by a carboxyl-terminal domain of Thy-1.

The Thy-1 antigen is anchored to the cell surface by a carboxyl-terminal glycophospholipid moiety. To investigate the extent of anchor addition which occurs when such proteins cannot move efficiently to the cell surface, we have expressed a recombinant fusion protein composed of 107 amino-terminal amino acids of bovine luteinizing hormone beta subunit and 46 COOH-terminal amino acids of murine Thy-1 (Thy-1.2 allele). Although the limited amount of fusion protein transported to the cell surface is glycophospholipid-anchored, most of the protein accumulates in an intracellular, endoglycosidase H-sensitive form. The intracellular protein has an unusual structure that contains ethanolamine but does not bind detergent, suggesting either that anchor addition proceeds via a hydrophilic partial intermediate, or that anchor-degradative enzymes exist along the secretory path.

Binding of terbium and of an elastase inhibitor to bovine pancreatic subunit III, an inactive protease E.

Using the Tb3+ luminescence technique, we showed that bovine subunit III, a defective pancreatic serine endopeptidase-like protease, possessed a single metal ion binding site able to bind Tb3+ with a high affinity comparable to that of porcine elastase. The topology of the metal ion binding site in subunit III is predicted from sequence homologies and modeling experiments based on the known crystallographic three-dimensional structures of the equivalent sites in porcine elastase 1 and bovine beta-trypsin. Moreover, the Tb3+ luminescence technique in parallel to a 19F NMR investigation, allowed us to measure the binding of a very potent specific inhibitor of porcine elastase (trifluoroacetyl-L-lysyl-alanyl p-trifluoromethylphenylanilide) to bovine subunit III. These results confirm that, although devoid of any specific activity, subunit III might possess a conformation close to that of an active enzyme and further support the analogy between subunit III and an elastase-like family.

Identification of a procarboxypeptidase A-truncated protease E binary complex in human pancreatic juice

The characterization, in human pancreatic juice, of a binary complex associating procarboxypeptidase A with a 32 kDa inactive glycoprotein (G32) is reported in this paper. Free G32 was isolated after dissociation of the binary complex. N-terminal sequence analysis revealed a complete homology between this protein and human protease E (HPE 1), except for the two strongly hydrophobic N-terminal residues (Val-Val) which are missing in G32. This protein might be a truncated protease E highly analogous to the subunit III of the ruminant procarboxypeptidase A-S6 ternary complex. The analogy with bovine subunit III is further supported by interspecies reassociation experiments showing that bovine procarboxypeptidase A can specifically bind human G32.

Cytochrome Oxidase Subunit V Gene of Neurospora crassa: DNA Sequences, Chromosomal Mapping, and Evidence that the cya-4 Locus Specifies the Structural Gene for Subunit V

The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA.

Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V.

The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA.

Sequence of cDNAs encoding subunit Vb of human and bovine cytochrome c oxidase

We have isolated a full-length human fetal muscle cDNA clone specifying the nuclear-encoded subunit Vb of the human mitochondrial respiratory chain enzyme, cytochrome c oxidase (COX; EC 1.9.3.1), and a partial-length brain cDNA clone specifying the analogous bovine subunit. The two cDNAs are 85% identical at the nucleotide level. Similar to other proteins imported into mitochondria, the deduced human COX Vb protein contains a presequence, 31 amino acids long, rich in basic residues. We find no evidence for tissue-specific transcripts for subunit Vb of human COX, as Northern analysis of total RNA from human muscle, liver, and brain showed a single, identically sized transcript in each cell type, while partial-length cDNA clones isolated from human muscle and endothelial cell cDNA libraries were identical in sequence to the fetal muscle cDNA.

ATP synthase from bovine mitochondria: The characterization and sequence analysis of two membrane-associated sub-units and of the corresponding cDNAs

ATP synthase from bovine mitochondria is a complex of 13 different polypeptides, whereas the Escherichia coli enzyme is simpler and contains eight subunits only. Two of the bovine subunits, b and d, which had not been characterized, have been isolated from the purified enzyme. Subunits with sizes corresponding to bovine subunits b and d are evident in preparations of the enzyme from mitochondria of other species. Partial protein sequences have been determined by direct methods. On the basis of some of this information, two oligonucleotide mixtures, 17 and 18 bases in length, have been synthesized and used as hybridization probes in the isolation of clones of the cognate cDNAs. The sequences of the two proteins have been deduced from their DNA sequences. Subunit b is 214 amino acid residues in length and has a free N terminus. Subunit d is 160 amino acid residues long. Its N-terminal alanine is blocked by an N-acetyl group, as demonstrated by fast atom bombardment mass spectrometry of N-terminal peptides. The sequence near the N terminus of the b subunit is made predominantly of hydrophobic residues, whereas the remainder of the protein is mainly hydrophilic. This N-terminal hydrophobic region may be folded into an β-helical structure spanning the lipid bilayer. In its distribution of hydrophobic residues, this protein resembles the b subunits of ATP synthase complexes in bacteria and chloroplasts. The b subunit in E. coli forms an important structural link between the extramembrane sector of the enzyme F 1, and the intrinsic membrane domain, F 0. It is proposed that the bovine mitochondrial subunit b serves a similar function. If this is so, the mitochondrial enzyme, as the chloroplast ATP synthase, contains equivalent subunits to all eight of those that constitute the E. coli enzyme. Subunit d has no extensive hydrophobic sequences, and is not apparently related to any subunit described in the simpler ATP synthases in bacteria and chloroplasts.

Subunit III of bovine procarboxypeptidase A: its relationship to human pancreatic elastase 1.

This paper is a continuation of our study of various animal pancreatic enzymes which are related to human pancreatic elastase 1 (Sziegoleit, A. & Linder, D. (1986) Biol. Chem. Hoppe-Seyler, 367, 527-531). The isolation and immunological analysis of the related protein from bovine pancreas disclosed that the third subunit of the procarboxypeptidase A complex is the antibody-binding component. The similarity of this subunit to elastase 1 is affirmed by comparison of their primary structures. While the complete amino-acid sequence of bovine subunit III recently has been published (Venot, N., Sciaky, M., Puigserver, A., Desnuelle, P. & Laurent, G. (1986) Eur. J. Biochem. 157, 91-99), we here present the amino-acid sequence of the carboxy-terminal tryptic peptide of human pancreatic elastase 1 showing a high degree of homology.

Biochemical and immunological studies of bovine and porcine neurofilament triplet proteins by peptide mapping after cyanogen bromide cleavage

1. 1. Peptide mapping of the three bovine and porcine neurofilament protein subunits (“L”, “M” and “H”) with apparent mol. wts of 70, 160 and 210 kDa were performed with CNBr, leading to the cleavage of methionyl bonds. 2. 2. We have obtained two characteristic large fragments with molecular weights of 85 kDa for the “M” bovine subunit and 135 kDa for the “H” subunit of bovine neurofilament. 3. 3. A comparison of the electrophoretic patterns of CNBr generated polypeptides of “L” subunit from beef and pig showed that they are highly related structures. 4. 4. The peptide mappings of CNBr peptides of “M” and “H” subunits from beef and pig were significantly different. 5. 5. Antibodies were raised against the 85 kDa and 135 kDa CNBr fragments. Immunoblotting results with anti-85 kDa and anti-135 kDa of beef are in favour of large differences of structure between the “M” subunits from pig and beef. 6. 6. The “H” proteins were very similar and they also showed that the C-terminal part of bovine “H” and “M” proteins share common antigenic determinants.

Existence of ternary complexes of procarboxypeptidase A in the pancreas of some ruminant species.

The existence of procarboxypeptidase A, in the form of a non-covalent ternary complex containing the apparently inactive serine protease (subunit III), has so far been observed only in the ox pancreas. Evidence, obtained in the present study, shows that a ternary complex of procarboxypeptidase A, with a subunit III highly homologous with that of the bovine complex, is also present in two other ruminant species, sheep and goat. The biological significance of these complex forms of procarboxypeptidase A and the consistently high biosynthesis level of the apparently inactive subunit III in all three ruminant species is still unknown. Yet the synthesis of subunit III is not related to the animal diet since in the horse, which is a non-ruminant herbivorous animal, the procarboxypeptidase A is monomeric. Reassociation assays between either bovine subunits II or III and monomeric as well as binary forms of procarboxypeptidase A from various species show that, unlike subunit II, the recognition site for subunit III is highly conserved in all the procarboxypeptidases A and that bovine subunit II is different from porcine chymotrypsinogen C with regard to association.

Analysis of several bovine lutropin beta subunit cDNAs reveals heterogeneity in nucleotide sequence.

Several, independent lutropin beta subunit cDNA clones have been isolated from a lambda gt-11 pituitary cDNA library. The complete nucleotide sequence of four clones was determined. A number of single nucleotide differences were detected including three changes within the coding sequence. One of these nucleotide changes resulted in a change in the predicted amino acid sequence, while two did not. These changes may reflect heterogeneity in the animals from which pituitaries were obtained, or they may reflect errors which occurred during the construction or cloning of the cDNA. Two other differences in nucleotide sequence suggest differences in the processing of precursor mRNAs. One difference involves the deletion of three nucleotides from the coding sequence of one of the clones. This probably reflects the use of alternate splicing sites at intron-exon boundaries. The other likely processing difference is reflected by the fact that one of the clones contains a much longer 3' untranslated region than the other clones. This difference probably arose due to a single nucleotide difference leading to selection of an alternate polyadenylation site.

O-phosphoserine content of intermediate filament subunits

Purified subunits of intermediate filaments obtained from a variety of tissues and cell types contain O-phosphoserine and, in some cases, smaller amounts of O-phosphothreonine. The O-phosphoserine content was estimated by reaction of performic acid oxidized subunits with methylamine in NaOH. Decamin of BHK-21 and CHO fibroblasts contained about 1 mol/mol. Avian and mammalian desmin consists of two subunits, an acidic (alpha) subunit which contained 2 mol/mol and a more basic (beta) nonphosphorylated subunit. The principal (Mr approximately 60 000) subunit of squid brain neurofilaments contained 5 mol/mol. Most mouse and bovine keratin subunits contained 3--6 mol/mol, although certain bovine subunits of higher molecular weight contained none. The O-phosphoserine contents of keratin subunits purified from the viable and stratum corneum layers were the same. The O-phosphoserine was located in non-alpha-helical regions of the subunits which presumably project out from the alpha-helical wall of the intermediate filaments. Most subunits could be partially dephosphorylated in vitro with alkaline phosphatase. It was found that the capacity of such partially dephosphorylated subunits for assembly into native-type filaments in vitro was independent of their phosphate content.

Alignment of the amino terminal amino acid sequence of human cytochrome c oxidase subunits I and II with the sequence of their putative mRNAs.

Thirteen of the first fifteen amino acids from the NH2-terminus of the primary sequence of human cytochrome c oxidase subunit I and eleven of the first twelve amino acids of subunit II have been identified by microsequencing procedures. These sequences have been compared with the recently determined 5'-end proximal sequences of the HeLa cell mitochondrial mRNAs and unambiguously aligned with two of them. This alignment has allowed the identification of the putative mRNA for subunit I, and has shown that the initiator codon for this subunit is only three nucleotides away from the 5'-end of its mRNA; furthermore, the results have substantiated the idea that the translation of human cytochrome c oxidase subunit II starts directly at the 5'-end of its putative mRNA, as had been previously inferred on the basis of the sequence homology of human mitochondrial DNA with the primary sequences of the bovine subunit.

Heterogeneity of S-100 protein: Comparison of bovine and guinea-pig S-100 proteins

1. 1. Two populations of S-100 proteins (I & II) were obtained from bovine and guinea-pig brains after DEAE-cellulose chromatography. 2. 2. While S-100 I was predominant in bovine (70%), S-100 II was present in major amounts in guinea-pig (92%) brain. Both S-100 populations were active against anti-S-100 serum. 3. 3. SDS-urea gradient slab-gel electrophoresis revealed four subunits of 7.8, 6.8, 6.1 and 5.2 K in bovine while the 5.2 K subunit was absent in guinea-pig. 4. 4. Protein distribution among the subunits revealed the predominance of 5.2 and 6.1 K in bovine and guinea-pig respectively. 5. 5. The immunoreactivity of the subunits was quantified by rocket immunoelectrophoresis using bovine anti-S100 serum. 6. 6. In general, the guinea-pig subunits were 2–3 times more reactive than the most active bovine subunit (5.2 K) even though the intact S-100 protein from the two species were almost equally active.

Proton nuclear magnetic resonance studies on bovine lutropin, its subunits, and on the alpha subunit of pregnant mare serum gonadotropin. Assignment of histidine resonances in the alpha subunit.

The pK values of the 3 histidine residues in the common alpha subunits of bovine and equine glycoprotein hormones have been determined from titration curves generated from their C-2 proton nuclear magnetic resonances at different pH values. Assignment of resonances to specific histidines is based on a comparison between the two species, which have 1 histidine residue in different positions in their sequences, and of the bovine alpha subunit after removal of its histidine 94 by treatment with carboxypeptidases. In both species, those histidines closest to the COOH terminus titrate with near normal pK values of 6.2. The histidine residue found in the bovine subunit at position 87 titrates with an approximate pK value of 5.4. Histidine 83, adjacent to an oligosaccharide moiety in both species, does not titrate over a pH range of 4.0 to 8.0 and thus appears inaccessible to solvent. Similarly, in bovine lutropin-beta, 1 of 3 histidine residues does not titrate between pH 5.0 and 7.0. In the intact hormone, 2 "nontitratable" histidine residues are found. Changes in the characteristics of the signals, however, preclude unambiguous assignment of these two resonances to the nontitrating histidines in the isolated subunits. It appears that changes in the environment of at least some histidines occur when the subunits combine to yield intact hormone.

The Subunits of Human Pituitary Thyroid-stimulating Hormone: ISOLATION, PROPERTIES, AND COMPOSITION

Abstract The subunits, α and β, of human thyroid-stimulating hormone (TSH) from the pituitary have been obtained by the same procedures utilized for the preparation of bovine TSH subunits, namely, dissociation in 1 m propionic acid and separation by gel filtration. The amino acid composition of human TSH-α indicates that this subunit is identical or nearly so with the α subunits of human luteinizing hormone and human chorionic gonadotropin. The NH2-terminal residue is valine and it appears that human TSH-α is shortened at its amino end by 7 residues relative to bovine TSH-α; a similar relationship has been shown by others for human LH-α relative to the ovine or bovine subunit. The β subunit of human TSH differs markedly in amino acid composition from those of the two human gonadotropins, particularly with respect to tyrosine, isoleucine, valine, proline, arginine, and lysine. The carbohydrate analyses for the 2 subunits are given. A major difference between human and bovine TSH is the presence of sialic acid in the former, nearly all in the α subunit. Immunological comparisons by double diffusion show that antisera to the intact hormone or its α subunit cross-react with homologous TSH and with the human gonadotropins but that the antiserum against the β subunit is specific for thyroid-stimulating hormone. The human subunits were shown, by gel electrophoresis and bioassay, to form intra- and interspecies recombinants with complementary subunits from other glycoprotein hormones.

Immunologic and biologic behavior of hCG and bovine LH subunit hybrids.

Bovine LH subunits were combined with hCG subunits. The hybrids were characterized biologically and immunologically using the ovarian ascorbic acid depletion assay and specific homologous radioimmunoassays for hCG and its subunits. Hybrids comprised of complementary subunits formed biologically active preparations. The immunologic behavior of the hCG subunits was unaltered by combination with complementary bovine LH subunits. One would expect the biologically active hybrids to have similar conformations, assuming the latter to be a requisite for biologic activity. Consequently, it would appear that our radioimmunoassay systems were not dependent on the conformation necessary for biologic activity since the immunologic and biologic behaviors of the hybrids were disparate. (Endocrinology 92: 411, 1973)