Isolation and partial characterization of two different subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase

The molybdenum-iron protein of Azotobacter vinelandii nitrogenase was separated into two subunits of equal concentration by ion exchange chromatography on sulfopropyl (SP) Sephadex at pH 5.4 in 7 M urea. Better than 90% yield of each subunit was obtained on a preparative scale if the reduced carboxymethylated molybdenum-iron protein was incubated at 45 degrees C for 45 min prior to chromatography. Without the heating step low yields of the subunits were obtained. Although the amino acid compositions of the two subunits were very similar, the NH2-terminal sequences were completely different as determined by automated sequential Edman degradation. The sequence for the alpha subunit was NH2-Ser-Gln-Gln-Val-Asp-Lys-Ile-Lys-Ala-Ser-Tyr-Pro-Leu-Phe-Leu-Asp-Gln-Asp-Tyr- and for the beta subunit the sequence was NH2-Thr-Gly-Met-Ser-Arg-Glu-Glu-Val-Glu-Ser-Leu-Ile-Gln-Glu-Val-Leu-Glu-Val-Tyr-. Likewise the COOH-terminal sequences for the two subunits, as determined with carboxypeptidase Y, were tota-ly different. The sequence for the alpha subunit was -Leu-Arg-Val-COOH and that for the beta subunit was -Ile-(Phe, Glu)-Ala-Phe-COOH. Radioautographs of tryptic peptide maps were prepared for the molybdenum-iron protein and the two subunits which had been labeled at the cysteinyl residues with iodo[2-14C]acetic acid. These maps indicated that the two subunits had no cysteinyl peptides in common and that the cysteinyl residues were clustered in both subunits.     

Isolation, properties, and the complete amino acid sequence of a second form of 60-kDa glycoprotein esterase. Orientation of the 60-kDa proteins in the microsomal membrane

A second form (form 2) of glycosylated esterase was isolated from liver microsomal membranes and characterized. The subunit molecular weight of form 2 is identical to that of the 60-kDa protein previously reported (Ozols, J. (1987) J. Biol. Chem. 262, 15316-15321). The NH2 terminus of the form 2 enzyme is blocked. Digestion of form 2 with pyroglutamyl aminopeptidase, followed by electroblotting and sequence analysis of the blotted protein, indicated that a pyroglutamyl residue was located at the NH2 terminus. Sequence analysis of the deblocked protein as well as characterization of the peptides obtained from enzymatic and chemical cleavages of the intact protein led to the elucidation of its complete amino acid sequence. The protein is a single polypeptide consisting of 532 residues. Carbohydrate is attached at asparaginyl residue 249. The sequence of form 2 esterase is 50% identical to the sequence of form 1 enzyme. The amino acid sequence of the first 26 residues of form 1 enzyme from human liver microsomes shows that 23 residues are identical to that of rabbit form 1, but only 8 residues that are identical to form 2. Treatment of the forms 1 and 2 isozymes with N-glycosidase F or endo-N-acetylglucosaminidase H resulted in a decrease of their subunit molecular weights, indicating that the carbohydrate attached is of the high mannose type. To determine whether the 60-kDa proteins are located on the cytoplasmic or luminal side of the endoplasmic membrane, microsomes were treated with proteolytic enzymes and the two 60-kDa isozymes were isolated and characterized. Sequence analysis of both proteins indicated that their NH2 termini were unaffected by proteolysis. Form 1 isozyme isolated from trypsin-treated microsomes, however, lacked the COOH-terminal heptapeptide (residues 533-539). These results, in addition to the finding of an N-linked carbohydrate, suggest that the two 60-kDa proteins are oriented on the luminal side of the endoplasmic membrane.     

Cloning and sequencing of the gene encoding cytochrome c553 from Desulfovibrio vulgaris Hildenborough.

The gene encoding cytochrome c553 from Desulfovibrio vulgaris Hildenborough was cloned by using two synthetic deoxyoligonucleotide probes. The amino acid sequence derived from the sequence of the gene differs from that reported by Bruschi and LeGall (Biochim. Biophys. Acta 271:48-60, 1972). Renewed protein sequencing confirmed the correctness of the DNA-derived sequence. The gene sequence indicates cytochrome c553 to be synthesized as a precursor protein with an NH2-terminal signal sequence of 24 residues.     

Isolation and nucleotide sequence of the extracellular acid protease gene (ACP) from the yeast Candida tropicalis

The extracellular acid protease of Candida tropicalis was purified from the supernatant fraction of culture medium containing bovine serum albumin as nitrogen source and the NH2-terminal amino acid (aa) sequence of the protein was determined. The gene for the acid protease (ACP) was isolated using a pool of synthetic oligonucleotides as a probe and a segment of the deduced aa sequence was found to be in agreement with the NH2-terminal aa sequence of the protein. The deduced aa sequence of ACP is similar to the aa sequence of proteases of the pepsin family. The nucleotide sequence of the 5' portion of this gene revealed a coding sequence for a 60 residue propeptide containing two Lys-Arg amino acid pairs that have been identified as sites for peptidase processing of several exported peptides and proteins. The final Lys-Arg site occurs at the junction with the mature extracellular form of the acid protease.     

Structural analysis of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Sequences of proteolytic fragments, cysteine-containing peptides, and a NADPH-protected cysteine peptide

Detergent-solubilized NADPH-cytochrome P-450 reductase was purified from porcine hepatic microsomes and compared to the rabbit enzyme isolated under identical conditions. The porcine enzyme had an equivalent specific activity toward cytochrome c compared to the rabbit enzyme. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the porcine enzyme exhibited a major band at Mr = 80,000 and two additional bands at Mr = 20,000 and 60,000. The 20-kDa fragment was shown to be the COOH-terminal portion of the protein which contains a hydrophobic sequence of 28 residues homologous to the pyrophosphate-binding portion of the FAD-binding protein p-hydroxybenzoate hydroxylase. The 60-kDa fragment corresponded to the NH2-terminal portion of the protein since this peptide and the intact protein have blocked NH2 terminal. The trypsin-solubilized porcine enzyme has an NH2-terminal sequence which is homologous to the equivalent trypsin-solubilized enzymes from rat and rabbit (80% sequence homology). Eight cysteine-containing peptides were isolated from a tryptic digest of the S-carboxymethylated pig enzyme. Significant sequence homology was not found between these peptides and other flavoproteins, except for one peptide (Glu-Val-Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg) which exhibited partial homology with the known NADPH-binding site of glutathione reductase. When the NADPH-protected enzyme was first S-alkylated with unlabeled iodoacetate, NADPH depleted, and further alkylated with 14C-labeled iodoacetate, the above radiolabeled peptide was isolated from a tryptic digest. The equivalent peptide was also isolated by a similar procedure from rabbit liver cytochrome P-450 reductase.     

Human tumor necrosis factor. Production, purification, and characterization

Human tumor necrosis factor (TNF) was purified to homogeneity from serum-free tissue culture supernatants of the HL-60 promyelocytic leukemia cell line induced by 4 beta-phorbol 12-myristate 13-acetate. The purification scheme consisted of controlled-pore glass and DEAE-cellulose chromatography, Mono Q-fast-protein liquid chromatography, and reverse-phase high performance liquid chromatography. The purified protein was homogeneous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. The specific activity of purified tumor necrosis factor is approximately 10(8) units/mg. The protein has a molecular weight of approximately 17,000, an isoelectric point of 5.3, and contains two cysteines involved in a disulfide bridge. Approximately 50% homology between TNF and another cytolytic lymphokine, lymphotoxin, exists when the NH2-terminal 34 residues of TNF and internal sequence generated by tryptic, Staphylococcus aureus V8 protease, and chymotryptic digests of TNF are aligned with the complete amino acid sequence of lymphotoxin.     

The primary structure of rat ribosomal protein L35

The amino acid sequence of the rat 60S ribosomal subunit protein L35 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Ribosomal protein L35 has 122 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 14,412. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 15-17 copies of the L35 gene. The mRNA for the protein is about 570 nucleotides in length. Rat L35 is related to the archaebacterial ribosomal proteins Halobacterium marismortui L33 and Halobacterium halobium L29E; it is also related to Escherichia coli L29 and to other members of the prokaryotic ribosomal protein L29 family. The protein contains a possible internal duplication of 11 residues.     

Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae.

A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced. The DNA sequence encodes a precursor protein containing 387 amino acid residues. The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids. The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established. In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases. Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells.     

Purification and amino-terminal sequence of the bovine cardiac sodium-calcium exchanger: evidence for the presence of a signal sequence

The Na(+)-Ca2+ exchange carrier was purified from bovine cardiac tissue by a new procedure which relies principally upon anion-exchange chromatography. The purified protein exhibited two major bands on sodium dodecyl sulfate gels, at 120 and 160 kDa. The relative intensities of the two bands could be altered by variations in the procedures used for preparing the samples for electrophoresis, suggesting that they represent two different conformational states of the same protein. The NH2-terminal amino acid sequences of the 120- and 160-kDa bands were identical and agreed closely with a region of the deduced amino acid sequence of the recently cloned canine cardiac exchanger. The NH2-terminal sequence was preceded in the deduced sequence by a 32-residue segment that exhibited the characteristics of a signal sequence; the initial amino acid in the NH2-terminal sequence followed immediately after the predicted cleavage site for the signal sequence. The Na(+)-Ca2+ exchanger appears to be unique among membrane transport carriers in encoding a cleaved signal sequence. The characteristics of the sequences flanking the first putative transmembrane segment of the mature exchanger suggest that the signal sequence is necessary to ensure the correct topological orientation of the exchanger in the membrane.     

The structure of the mutant dihydrofolate reductase from Streptococcus faecium. Partial sequence and order of the limited tryptic and cyanogen bromide peptides.

The major form of dihydrofolate reductase from a methotrexate-resistant mutant (strain A) of Streptococcus faecium var. Durans has been purified on a large scale. Amino acid analysis of this form of the enzyme (isoenzyme 2) reveals an absence of cystine or cysteine, and sedimentation studies indicate a molecular weight of 20,800. The NH2-terminal sequence was determined by Edman degradation of the intact protein and the COOH terminus by selective tritiation and by carboxypeptidase treatment. After the action of trypsin on the citraconylated protein, seven of the expected nine peptides were purified from the digest, and after cyanogen bromide treatment of the unmodified protein, all seven of the anticipated peptides were isolated. The amino acid composition of all of these peptides has been established as well as their complete or partial sequences. From the results it was possible to order these peptides within the sequence and to establish the sequence of the NH2-terminal 60 residues and the COOH-terminal 11 residues.     

Analysis of antibody diversity: V-D-J mRNA nucleotide sequence of four anti-GAT monoclonal antibodies. A paucigene system using alternate D-J recombinations to generate functionally similar hypervariable regions

The nucleotide sequence of four anti-(Glu60-Ala30-Tyr10)n (GAT) monoclonal gamma 1 heavy chain mRNAs was determined from codon 10 to 120. This sequence overlaps with the NH2-terminal amino acid sequence, allowing elucidation of the complete protein sequence encompassing regions VH, D and JH. These sequences, which are highly conserved, indicate that anti-GAT antibodies expressing the same public idiotypic specificities represent a paucigene system, which uses at least two D-J combinations leading to functionally similar hypervariable regions involved in the recognition of the dominant Glu-Tyr determinant. D regions are encoded by D genes which are closely related either to the D-SP2 or the D.FL16 germ line gene cores.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.     

The primary structure of rat ribosomal protein L38.

The amino acid sequence of the rat 60S ribosomal protein L38 was deduced from the sequence of nucleotides in three recombinant cDNAs. Ribosomal protein L38 has 69 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 8,081. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 11-13 copies of the L38 gene. The mRNA for the protein is about 450 nucleotides in length.     

A 25-kDa NH2-terminal fragment carries all the antibacterial activities of the human neutrophil 60-kDa bactericidal/permeability-increasing protein

We have isolated, after limited proteolysis of the bactericidal/permeability-increasing protein (BPI) of human neutrophils, a 25-kDa fragment that possesses the bactericidal and envelope-altering activities of the 60-kDa parent protein. On a molar basis, the fragment is as potent as holo-human BPI against rough Escherichia coli, is more potent than holo-BPI against more resistant smooth E. coli, and retains the specificity of BPI toward Gram-negative bacteria. NH2-terminal amino acid sequence analysis shows that the fragment is derived from the NH2 terminus of the BPI molecule. These findings suggest that all of the molecular determinants of the antibacterial properties of BPI reside within the NH2-terminal 25-kDa segment, implying a novel structural/functional organization for a cytotoxic protein.     

Frog brain expresses a 60 KDa Ca2+ binding protein similar to mammalian calreticulin

The present report was undertaken in an effort to characterize the nature of Ca2+ binding protein(s) in the central nervous system of less evolved vertebrates. In particular we investigated whether the brain microsomal fraction of Rana esculenta expresses calsequestrin, calreticulin and/or other related Ca2+ binding protein(s). We found that a 60 KDa protein having an NH2-terminal amino acid sequence similar to mammalian calreticulin is the major microsomal Ca2(+)-binding protein.     

Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2)

Clusterin, a glycoprotein originally isolated from ram rete testis fluid, is a dimer composed of monomers with non-identical NH2-terminal amino acid sequences. In view of its possible role in cell-cell interactions in the seminiferous epithelium, we sought to identify such a protein in the rat. Using the bioassay developed for the ovine protein, rat clusterin was purified to apparent homogeneity by HPLC from primary Sertoli cell-enriched culture media. This protein is also a heterodimer consisting of monomers of Mr 43,000 (alpha) and Mr 35,000 (beta). NH2-Terminal amino acid sequence analysis indicated that the alpha subunit has a sequence of NH2-SLMPLSHYGPLSFHNMFQPFFDMIHQAQQA and the beta subunit, NH2-EQEFSDNELQELSTQGSRYVNKEIQNAVQG. These two subunits show marked similarity with the corresponding subunits of ram clusterin isolated from rete testis fluid. Using an antibody against the alpha subunit of rat clusterin, a cDNA clone was isolated from a rat testicular lambda gt11 cDNA library. Analyses of the amino acid sequence derived from the isolated rat clusterin cDNA and of the NH2-terminal amino acid sequences indicate that rat clusterin is identical to a Sertoli cell glycoprotein previously designated sulfated glycoprotein-2.