Thymosin beta arg10, a major variant of thymosin beta 10 in rabbit tissues

Two homologous peptides, designated thymosin beta 4 and thymosin beta 10, respectively, have been shown to be widely distributed in mammalian cells and tissues (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker (1983) Arch. Biochem. Biophys. 221, 570-576; S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker, (1983) Arch. Biochem. Biophys. 225, 407-413). In the rabbit, thymosin beta 4 is replaced by a variant, thymosin beta ala4, that contains alanine in place of serine at the blocked NH2-terminus. It is reported that in rabbit tissues thymosin beta 10 is also replaced by a variant, designated thymosin beta arg10, that contains an additional amino acid, arginine, inserted following lysine-38. The rabbit tissues analyzed also differ from those of other mammals in the relative quantities of thymosin beta ala4 and beta arg10, which are nearly equal, compared to tissues from other mammals where the quantities of thymosin beta 10 are only one-third to one-tenth those of thymosin beta 4.     

Thymosin beta 4Xen: a new thymosin beta 4-like peptide in oocytes of Xenopus laevis

Two new thymosin beta 4-like peptides have been detected in ovaries of Xenopus laevis and Rana esculenta. Previously, it was reported that thymosin beta 4 can be found in various species, from mammals to amphibians, e.g., in X. laevis [S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker (1983) Arch. Biochem. Biophys. 221, 570-576]. However, oocytes and spleen from R. esculenta contain no thymosin beta 4 but a similar peptide without methionine. The peptide from R. esculenta elutes from a reversed-phase column about 5 min later than thymosin beta 4. The peptide from X. laevis, referred to as thymosin beta 4Xen, can hardly be distinguished from thymosin beta 4 by its retention time on HPLC, by amino acid analysis, its isoelectric point, or tryptic fingerprinting. Amino acid analyses of the tryptic fragments, however, have revealed that thymosin beta 4 and beta 4Xen are different. The amino acid sequence of thymosin beta 4Xen is reported. Thymosin beta 4 and beta 4Xen differ in the amino acid residues at positions 15, 40, and 41. At position 15 serine is replaced by alanine and at 41-42 the sequence is Thr-Ser instead of Ala-Gly. Depending on their size, defolliculated oocytes contain between 2.7 and 52.6 ng thymosin beta 4Xen which is comparable to the amount of histones in oocytes.     

Purification and crystallization of a retinoic acid-binding protein from rat epididymis. Identity with the major androgen-dependent epididymal proteins

The retinoic acid binding activity in the lumen of the rat epididymis (Ong, D., and Chytil, F. (1988) Arch. Biochem. Biophys. 267, 474-478) has been purified to homogeneity. The protein exists in two forms, one form having an additional three amino acids at the amino terminus. The amino acid sequence of the protein was determined to 20 amino acids and proved to be identical to that of the major androgen-dependent proteins from rat epididymis as deduced from the cDNA sequence. These proteins are thought to play a role in sperm maturation, perhaps, it can be suggested now, by delivering retinoic acid to the sperm. The retinoic acid-binding protein has sequence homology to the serum retinol-binding protein and is predicted to have the same overall fold of the polypeptide chain. The epididymal retinoic acid-binding protein has been crystallized from 39 to 43% saturated ammonium sulfate, 10 mm Tris, pH 8.0. The crystals are space group P2(1), with a = 39.4, b = 58.9, c = 65.4 a, beta = 105 degrees 16 min.     

Cloning and sequencing of cDNA encoding the mature form of phosphoribulokinase from spinach

Phosphoribulokinase (PRK) is a key enzyme in the Calvin cycle of autotrophic organisms. We have constructed a spinach leaf cDNA library in the phage expression vector, lambda gt11, and used a rabbit polyclonal antibody raised against spinach PRK to identify PRK clones. Analyses of the nucleotide sequences of two antibody-positive clones, 1.47 and 1.35 kb in length, showed that they encode a protein which contains the N-terminal amino acid (aa) sequence [Porter et al., Arch. Biochem. Biophys. 245 (1986) 14-23] of mature spinach PRK. The codon for the N-terminal serine of the mature protein occurs 170 bp from the 5' end of the open reading frame (ORF), suggesting that PRK is synthesized with a rather long transit peptide which is removed from the mature enzyme. The ORF, ending with an amber (TAG) codon at position 1054, predicts a mature enzyme of 351 aa with a calculated Mr of 39232.     

A pregnancy-specific beta 1-glycoprotein, a CEA gene family member, expressed in a human promyelocytic leukemia cell line, HL-60: structures of protein, mRNA and gene

Both genomic and cDNA clones encoding a precursor for a pregnancy-specific beta 1-glycoprotein (PS beta G) belonging to the CEA family, expressed in a human promyelocytic leukemia cell line, HL-60, have been isolated and the entire primary structure of the precursor is deduced. The 335-AA precursor has a 34-AA signal peptide followed by domains of N, IIA, IIB and C, which are encoded by separate exons. The genomic sequence contains extra exons IA and IB between exons N and IIA. Apparently, exon IA is excluded from the mRNA by alternative splicing while IB is a pseudo-exon having a stop codon formed by a deletion of dinucleotide in the middle of the sequence. This provides another mechanism to render exon IB abortive and is different from that we reported for another PS beta G (Biochem. Biophys. Res. Comm. (1988) 156, 68-77).     

The primary structure of the yeast hexokinase PII gene (HXK2) which is responsible for glucose repression

The nucleotide sequence of the Saccharomyces cerevisiae gene encoding the glycolytic isoenzyme hexokinase PII (HXK2), which is responsible for triggering glucose repression, has been determined. The reading frame was identified by comparison with the N-terminal undecameric amino acid (aa) sequence, determined previously [Schmidt and Colowick, Arch. Biochem. Biophys. 158 (1973) 458-470]. The codon sequence was not random, with 82.1% of the aa specified by only 25 codons. The structural gene sequence corresponded to 1455 bp, coding for 485 aa residues, corresponding to the Mr of 53 800 for the HXK2 monomer. Five initiation regions spanning 162 bp and three termination sites spanning 29 bp were detected. Sequences with similarities to a 5'-TATAAA-3' sequence were located 24-39 bp upstream of each initiation region. The most pronounced initiation region corresponded to the 5'-TATAAA-3' sequence at position -152. Two of the minor initiation sites were inside the coding sequence in front of two ATG codons.     

Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments.

We present here a study on the epitopic structure and the immunochemical characteristics of thymosin beta10 (Tbeta10), a 43 aminoacid peptide involved in important cellular mechanisms, by using the epitope mapping Multipin method. Octapeptides overlapping by one amino acid so as to represent the whole sequence of Tbeta10 were synthesized on polystyrene pins and screened, using an ELISA method, with a polyclonal antiserum raised against intact recombinant Tbeta10. The octapeptides were also tested with anti-peptide oligoclonal antisera raised against the synthetic fragments Tbeta10[1-16] and Tbeta10[31-43], with polyclonal antisera raised against natural thymosin gamma4 (Tbeta4) or thymosin beta9 (Tbeta9), and with anti-peptide oligoclonal antisera raised against various fragments of Tbeta4 (i.e. Tbeta4[1-11], Tbeta4[30-43] and Tbeta4[16-38]). Four distinct epitopic fragments were revealed, namely the sequences 1-13, 19-30, 29-40 and 36-43. Among them, the sequence 36-43 appears to offer unique immunochemical characteristics to the Tbeta10 molecule.     

Cloning and characterization of a testis-specific thymosin beta 10 cDNA. Expression in post-meiotic male germ cells.

Thymosin beta 10 is one of a small family of proteins closely related in sequence to thymosin beta 4, recently identified as an actin-sequestering protein. A single molecular weight species of thymosin beta 10 mRNA is present in a number of rat tissues. In adult rat testis, an additional thymosin beta 10 mRNA of higher molecular weight was identified. Nucleotide sequencing of cDNA clones complementary to the testis-specific thymosin mRNA indicated that this mRNA differed from the ubiquitous thymosin beta 10 mRNA only in its 5'-untranslated region, beginning 14 nucleotides upstream of the translation initiation codon. These results, together with primer extension experiments, suggest that the two thymosin beta 10 mRNAs are transcribed from the same gene through a combination of differential promoter utilization and alternative splicing. The novel thymosin beta 10 mRNA could be detected only in RNA isolated from sexually mature rat testis. Both mRNAs were present in pachytene spermatocytes; only the testis-specific mRNA was detected in postmeiotic haploid spermatids. Immunoblot analysis using specific antibodies showed that the thymosin beta 10 protein synthesized in adult testis was identical in size to that synthesized in brain. Immunohistochemical analysis showed that the protein was present in differentiating spermatids, suggesting that the testis-specific thymosin beta 10 mRNA is translated in haploid male germ cells.     

Primary structure from amino acid and cDNA sequences of two Cu,Zn superoxide dismutase variants from Xenopus laevis

A mixture of two different amino acid sequences was discovered in Cu,Zn superoxide dismutase purified from the amphibian Xenopus laevis. No N-terminal post-translational modification was found. The high number of substitutions in the sequence suggested that protein heterogeneity was a product of gene duplication. This was confirmed by isolation of two different cDNA clones. Nucleotide sequence analysis allowed the primary structure of the two peptide chains to be unambiguously assigned. The observed changes (19 in 150 residues) are distributed along the peptide chain to give similar protein net charges although substitutions of the same polarity and/or charge were the exception rather than the rule. The degree of diversity between the two Xenopus variants is comparable to that between mammalian sequences and shows that the putative increase of the rate of mutation for Cu,Zn superoxide dismutase at later evolution stages (Y. M. Lee et al., 1985, Arch. Biochem. Biophys. 241, 577-589; G. J. Steffens et al., 1986, Biol. Chem. Hoppe-Seyler 367, 1017-1024) is observed in amphibians. This is the first time complete sequences for Cu,Zn superoxide dismutase variants from the same organism have been found to be products of divergent genes and not simply allelic mutations.     

The source of oxygen in the reaction catalysed by collagen lysyl hydroxylase.

The synthetic peptides (Pro-Pro-Gly)5 and (Ile-Lys-Gly)5-Phe were hydroxylated with collagen prolyl hydroxylase and lysyl hydroxylase in an 18O2 atmosphere. The oxygen atoms in the hydroxy groups of hydroxyproline and hydroxylysine were 87% and 6.5% respectively derived from the atmospheric 18O2. The results are consistent with those reported previously for proline hydroxylation in vivo [Fujimoto & Tamiya (1962) Biochem. J. 84, 333-335; Prockop, Kaplan & Udenfriend (1962) Biochem. Biophys. Res. Commun. 9, 192-196; Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501; Prockop, Kaplan & Udenfriend (1963) Arch. Biochem. Biophys. 101, 499-503] and in vitro [Cardinale, Rhoads & Udenfriend (1971) Biochem. Biophys. Res. Commun. 43, 537-543] and for lysine hydroxylation in vivo [Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501]. In view of the similarities of these two oxygenase-type hydroxylation reactions the participation of intermediates is proposed, the oxygen atoms of which are exchangeable with those of water. The atmospheric oxygen atoms incorporated into the intermediate must be equilibrated with water oxygen atoms in the slower lysyl hydroxylase reaction.     

Rat brain hexokinase: location of the substrate nucleotide binding site in a structural domain at the C-terminus of the enzyme

8-Azido-ATP serves as a substrate for rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Irradiation of hexokinase in the presence of this photoactivatable ATP analog results in inactivation of the enzyme. ATP and hexose 6-phosphates (Glc-6-P, 1,5-anhydroglucitol-6-P) previously shown to competitively inhibit nucleotide binding protect the enzyme from photoinactivation; other hexose 6-phosphates do not. Hexoses (Glc, Man) previously shown to enhance nucleotide binding also protect against photoinactivation; other hexoses do not. These effects of hexoses and hexose 6-phosphates can be interpreted in terms of the conformational changes previously shown to result from the binding of these ligands and to influence the characteristics of the nucleotide binding site (M. Baijal and J. E. Wilson (1982) Arch. Biochem. Biophys. 218, 513-524). Limited tryptic cleavage of the enzyme produces three major fragments having molecular weights of about 10K, 40K, and 50K, and thought to represent major structural domains within the enzyme (P. G. Polakis and J. E. Wilson (1984) Arch. Biochem. Biophys. 234, 341-352). Tryptic cleavage of the enzyme, photoinactivated in the presence of 14C-labeled azido-ATP, discloses prominent labeling of the 10K and 40K domains, which are known to originate from the N- and C-terminal regions, respectively. Labeling of the 40K domain is influenced by ligands in a manner that corresponds to the effectiveness of these ligands in protecting against photoinactivation whereas labeling of the 10K domain is not affected by these same ligands. It is concluded that the 40K domain includes the binding site for nucleotide substrates. More refined two-dimensional peptide mapping techniques demonstrate that the predominant site of labeling is a peptide segment, molecular weight approximately 20K, that is located in the central and/or C-terminal region of the 40K domain. Labeling of the 10K domain is attributed to nonspecific interaction of azido-ATP with the hydrophobic sequence shown to be located at the N-terminus of brain hexokinase (P. G. Polakis and J. E. Wilson (1985) Arch. Biochem. Biophys. 236, 328-337).     

Structure and immunological properties of thymosin beta 9 Met, a new analog of thymosin beta 4 isolated from porcine thymus

During the course for the studies of thymosin beta 4 and prothymosin alpha from porcine thymus, a new analog of thymosin beta 4 has been identified. This peptide consists of 41 amino acid residues. The amino terminus is blocked by an acetyl group as revealed by fast atom bombardment mass spectrometric analysis. Amino acid sequence studies disclosed that this peptide is identical to bovine thymosin beta 9 except that leucine at position 6 in beta 9 is substituted by methionine. Thus, this new peptide has been termed thymosin beta 9 Met. The recoveries of beta 9 Met, beta 4, and prothymosin alpha in porcine tissues have been determined (in micrograms/g tissue) as follows: thymus (43, 85, 133); spleen (68, 203, 37); liver (10, 31, 27); heart (1.5, 10, 0); kidney (5, 51, 37); brain (0.8, 31, 5). Biologically, thymosin beta 9 Met was found to be more active than beta 4 in enhancing gamma-interferon production in cord blood lymphocytes. However, beta 4 appeared to stimulate higher amounts of interleukin 2 and tumor necrotic factor. The significance for the coexistence of two homologous peptides with similar functions in the thymus and a number of other organs is not clear, and deserves further investigation.     

Isolation of a fourth cysteinyl-containing peptide of the alpha-subunit of the F1 ATPase from Escherichia coli necessitates revision of the DNA sequence

The rapid determination of cysteinyl residues by Creighton's method [(1980) Nature 284, 487-489] led to the discovery of a discrepancy between protein and DNA sequence data in the alpha-subunit of the F1 ATPase from Escherichia coli [(1984) Arch. Biochem. Biophys. 229, 320-328]. We have isolated a cysteinyl-containing decapeptide from the alpha-subunit with a protein sequence (AGCAMGEYFR) which is only partially recognizable from DNA data. Re-sequencing of DNA in the region coding for the peptide has resulted in two corrections: insertion of a cytosine before position 715 and deletion of a thymine at position 731 of the uncA gene.     

Primary structure of thymosin beta 12, a new member of the beta-thymosin family isolated from perch liver.

A new polypeptide termed thymosin beta 12 has been isolated from perch liver and its primary structure elucidated. This polypeptide contains 43 amino acid residues with a molecular weight of 4822 Da. The content of thymosin beta 12 from perch liver has been determined as 43 micrograms/g of tissue. The amino-terminal end of this polypeptide is blocked by an acetyl group as deciphered by fast-atom bombardment mass spectrometric analysis. Sequence analysis reveals that thymosin beta 12 is 79% homologous to thymosin beta 4, an immunomodulator which was originally isolated from calf thymus. Thymosin beta 12 also shows 84% sequence homology to thymosin beta 11, a beta 4 analog which replaces beta 4 in two species of bony fish, oscar and rainbow trout. The evolutionary implication of such results will be discussed. The isolation of a new beta 4-related peptide from perch liver which differs from beta 11 indicates that beta-thymosin peptides are widely distributed in lower vertebrate classes.     

cDNA cloning, nucleotide sequence and expression of the gene for arylsulfatase in the sea urchin (Hemicentrotus pulcherrimus) embryo

Arylsulfatase is known to be synthesized in large amounts at the early gastrula stage of sea urchin development. We determined the amino acid sequence of a portion of the purified sea urchin embryonic arylsulfatase, and then isolated a cDNA clone for arylsulfatase by screening a sea urchin plutei lambda gt10 cDNA library with an oligodeoxynucleotide probe synthesized according to the determined amino acid sequence. The longest cDNA clones were selected and the nucleotide sequence determined. The cDNA is 2422 nucleotides long and encodes 551 amino acids. The deduced amino acid sequence has not sequence similarity with any of the peptides registered in NBRF peptide databank. Northern blot analysis revealed that the arylsulfatase cDNA hybridizes to a 2.9-kb mRNA. This mRNA exists in the unfertilized egg in small amounts, but markedly increases after the blastula stage preceding the increase of the arylsulfatase activity.     

cDNA clones completing the nucleotide and derived amino acid sequence of the alpha 1 chain of basement membrane (type IV) collagen from mouse

Six cDNA clones add 3549 nucleotides to the DNA sequence of the alpha 1 chain of basement membrane (type IV) collagen. Thus the complete nucleotide and derived amino acid sequence of the alpha 1 type IV collagen with 5007 nucleotides coding for 1669 amino acids with a calculated Mr of 160,827 is known. The six cDNA clones cover the putative N-terminal signal peptide, the 7 S region and two thirds of the helical region extending into the previously published murine nucleotide sequence [(1986) Gene 43, 301]. The protein sequence for 289 amino acids of the helical region adjacent to the 7 S region has not previously been published for any species.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.