Structural and antigenic studies of an idiotype-bearing murine antibody to the arsonate hapten.

Mice of strain A/J responded to repeated intraperitoneal injection of Limulus hemocyanin derivatized with arsanilic acid by producing large quantities (approximately 5 mg/mL of ascites fluid) of IgG antibodies specific for this hapten. The antibodies possessed a characteristic idiotypic determinant and exhibited restricted heterogeneity as demonstrated by isoelectric focusing and primary N-terminal amino acid sequence analysis of isolated light and heavy polypeptide chains. Both light- and heavy-chain sequences were comparable to those of myeloma proteins in lack of heterogeneity. The N terminus of the light chain exhibited V KI sequence and only one position in the first 30 residues showed more than one amino acid. No variability was observed in the first 10 N-terminal residues of the heavy chain. Rabbit antiserum to the idiotype blocked binding of hapten by the purified antibody. The presence of both light- and heavy-chain antigenic determinants was required for optimal formation of the idiotypic determinant.     

Complete primary structure of prostatropin, a prostate epithelial cell growth factor

Bovine brain prostatropin is a potent and essential mitogen for prostate epithelial cell growth. The major form of prostatropin contains 154 amino acid residues in a single amino terminally blocked chain corresponding to a molecular weight of 17,400. The amino acid sequence of the 150 carboxy-terminal residues of prostatropin was derived by Edman degradation of overlapping peptides primarily generated by cleavage at lysyl and glutamyl residues. Analysis of the amino-terminal tetradecapeptide by fast atom bombardment mass spectrometry identified the blocking group as an acetyl moiety, and tandem mass spectrometry provided the sequence of the first 12 residues. Prostatropin residues 15-154 contain the sequence of bovine brain polypeptides recently described as acidic fibroblast growth factor and class I heparin-binding growth factor. The sequence of the first 25 residues of prostatropin is acetyl-Ala-(Gly, Glu)-Glu-Thr-Thr-Thr-Phe-Thr-Ala-Leu-Thr-Glu-Lys-Phe-Asn-Leu-Pro-Leu-Gly -Asn-Tyr-Lys-Lys-Pro. Reduced and carboxymethylated prostatropin exhibits mitogenic activity, suggesting that disulfide bonds among cysteine residues 30, 61, and 97 are not functionally essential. These results demonstrate by rigorous structural analysis that the brain-derived polypeptide previously described only as a mesenchymal and neuroectodermal cell mitogen is also an epithelial cell growth factor that may be involved in support of prostate hyperplasia and adenocarcinoma.     

A novel form of the polypeptide PHI isolated in high yield from bovine upper intestine. Relationships to other peptides of the glucagon-secretin family

A novel form of the polypeptide termed PHI (peptide HI with N-terminal histidine and C-terminal isoleucine amide) has been isolated from bovine upper intestine. This bovine peptide was obtained in a 40 times higher yield than the corresponding polypeptide isolated from porcine intestine. Bovine PHI is, like porcine PHI, composed of 27 amino acid residues. The complete amino acid sequence of the bovine peptide is His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Tyr-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser- Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence differs from porcine PHI at position 10 and from human PHI at positions 10, 12 and 27. The amino acid residue exchange between porcine and bovine PHI makes the latter more similar to the vasoactive intestinal polypeptide (VIP), gastric inhibitory polypeptide (GIP), glucagon and the growth-hormone-releasing factor (GRF).     

Complete amino acid sequence of a new murine T-cell growth factor P40

A new murine T-cell growth factor, designated P40, which supports growth of helper T-cells in the absence of interleukin-2, interleukin-4 and antigen has been isolated from helper T-cell lines in sufficient quantities (100 micrograms) to permit its complete amino acid sequence determination. This was achieved by a combination of sensitive peptide mapping using microbore reversed-phase high performance liquid chromatography and automated microsequence analysis. Attempts to obtain N-terminal sequence data on P40 were unsuccessful due to N-terminal blockage of the native molecule. The nature of this N-terminal blocking was established using a combination of amino acid analysis, fast-atom-bombardment mass spectrometry and peptide synthesis. The P40 molecule, a single polypeptide chain comprising 126 amino acid residues, is structurally distinct from other known T-cell growth factors. No similarity was revealed when the amino acid sequence of P40 was compared with other proteins whose biochemical structure is known. The protein sequence data reported here predict four N-linked glycosylation sites in the P40 molecule.     

Amino acid sequence of bovine protein Z: a vitamin K-dependent serine protease homolog

The amino acid sequence of protein Z has been determined from sequence analysis performed on fragments obtained by chemical and enzymatic degradations. The polypeptide consists of a single chain containing 396 amino acid residues (Mr 43 677). Comparison with the vitamin K-dependent plasma proteins reveals an extensive homology. The N-terminal part, containing 13 gamma-carboxyglutamic acid and one beta-hydroxyaspartic acid residue, is extensively homologous to and of similar length to the light chain of factor X. The remainder of protein Z is homologous to the serine proteases and of similar size to the heavy chain of factor Xa, but of the active site residues only aspartic acid-102 is present. Histidine-57 and serine-195 are replaced in protein Z by threonine and alanine, respectively. The physiological function of protein Z is still uncertain.     

Characterization of the effects of human placental HGF on rat hepatocytes.

Hepatocyte growth factor (HGF) also known as hepatopoietin A (HPTA) (Michalopoulos, FASEB J., 4:176-187, 1990) is a heparin-binding growth factor whose characterization and tissue distribution have been reported elsewhere. This growth factor was recently cloned and its amino acid sequence determined under the name of hepatocyte growth factor (HGF) (Miyazawa et al., Biochem. Biophys. Res. Commun., 163:967-973, 1989; Zarnegar et al., Biochem. Biophys. Res. Commun., 163:1370-1376, 1989; Nakamura et al., Nature, 342:440-443, 1989). Human placenta is one of the tissues that contains significant amounts of HGF. We isolated HGF from human placenta and characterized its biologic effect on rat hepatocytes. Human placenta HGF was isolated in high purity as a single chain molecule. Single chain HGF stimulated DNA synthesis in primary rat hepatocyte cultures in serum-free medium. The maximal effect was seen at 5-10 ng/ml. The maximal response occurred at 25-48 hours after plating of the hepatocytes. Protein synthesis was also stimulated by HGF in primary rat hepatocyte cultures. There were peak responses at 19-24 and 37-42 hours after plating of the hepatocytes. TGF beta 1 inhibited more than 95% of HGF-induced DNA synthesis but only 25% of HGF-induced protein synthesis. HGF interacted in an additive manner with EGF, a well-known hepatocyte mitogen. There was not an additive interaction between HGF and aFGF. Regenerating liver hepatocytes obtained from rats which underwent two-thirds partial hepatectomies (PHX) also responded to HGF in a dose-dependent manner as the hepatocytes from normal liver.     

Isolation and characterization of a cDNA coding for human myeloperoxidase

A cDNA encoding the carboxyl-terminal fragment of the human myeloperoxidase heavy chain was isolated and characterized. It was then used to determine the locations of the myeloperoxidase light and heavy chains in the polypeptide precursor. A cDNA library from poly(A)+ RNA from human leukemia HL-60 cells was constructed in pBR322 and screened by differential hybridization with enriched and depleted cDNA probes and then by hybridization with an oligonucleotide probe. A cDNA clone containing 1278 bp with an open reading frame of 474 bp and a 3' noncoding region of 804 bp was isolated. The amino acid sequence deduced from the nucleotide sequence consisted of 158 residues including a sequence of 14 amino acids known to be present in the heavy chain of the molecule. The cDNA also included a stop codon of TAG followed by a noncoding sequence that included a potential recognition site for polyadenylylation and a poly(A) tail. RNA transfer blot analysis with the cDNA probe indicated that myeloperoxidase mRNA was approximately 3.3 kb in length. In vitro translation of the mRNA selected by cDNA hybridization revealed preferential synthesis of a 74,000-Da polypeptide precursor that could be precipitated with anti-myeloperoxidase IgG. Antibodies specific for the heavy and light chains of myeloperoxidase were isolated from antiserum by affinity chromatography employing Sepharose columns covalently bound to the heavy or light chains. Antibodies specific for the light chain or the heavy chain readily precipitated the 74,000-Da precursor polypeptide. These results indicated that myeloperoxidase is synthesized as a single chain which undergoes processing into a light and heavy chain. Furthermore, the heavy chain of myeloperoxidase originates from the carboxyl terminus of the precursor polypeptide.     

Molecular cloning and nucleotide sequences of the complementary DNAs to chicken skeletal muscle myosin two alkali light chain mRNAs.

We report here the molecular cloning and sequence analysis of DNAs complementary to mRNAs for myosin alkali light chain of chicken embryo and adult leg skeletal muscle. pSMA2-1 contained an 818 base-pair insert that includes the entire coding region and 5' and 3' untranslated regions of A2 mRNA. pSMA1-1 contained a 848 base-pair insert that included the 3' untranslated region and almost all of the coding region except for the N-terminal 13 amino acid residues of the A1 light chain. The 741 nucleotide sequences of A1 and A2 mRNAs corresponding to C-terminal 141 amino acid residues and 3' untranslated regions were identical. The 5' terminal nucleotide sequences corresponding to N-terminal 35 amino acid residues of A1 chain were quite different from the sequences corresponding to N-terminal 8 amino acid residues and of the 5' untranslated region of A2 mRNA. These findings are discussed in relation to the structures of the genes for A1 and A2 mRNA.     

Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase.

The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa.     

Amino acid sequence of porcine spleen cathepsin D light chain

The complete amino acid sequence of the light chain of cathepsin D from porcine spleen has been determined. The light chain consists of a single polypeptide chain with 97 amino acid residues. The sequence is: (formula; see text) The molecular weight of the light chain was calculated from this sequence to be 10,548 (without carbohydrates). A single disulfide bond links two half-cystine residues between positions 46 and 53. A cysteine residue is located at position 27. The light chain sequence is extensively homologous to the NH2-terminal sequence of other aspartyl proteases. It shows a 59% identity with the sequence of mouse submaxillary gland renin and a 49% identity with that of porcine pepsin. A single glycosylation site is located at residue 70 of the cathepsin D light chain. This site corresponds to position 67 of pepsin by homology. The active site aspartyl residue, corresponding to Asp-32 of pepsin, is located at residue 33 in the cathepsin D light chain.     

Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r.

The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r.     

Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis

A cDNA clone, pFL18, carrying a putative full-length fibroin light chain (L-chain) sequence was isolated and its nucleotide sequence was determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 262 amino acid residues. The sequence was concluded to be that of the L-chain with its signal peptide because corresponding amino acid sequences for the seven tryptic and the four chymotryptic peptides from the purified L-chain were all included and an N-terminal region having typical properties of a signal peptide was present. The N terminus of the mature form of L-chain was identified as N-acetyl serine by analyzing the acyl-dansylhydrazide derived from the N-acyl-amino acid which had been released from the N-terminal blocked chymotryptic peptide by the acylamino acid-releasing enzyme. It was suggested that a signal peptide had cleaved between Pro18 and Ser19, yielding a mature L-chain polypeptide consisting of 244 amino acid residues. The molecular weight of the L-chain was calculated to be 25,800 including the N-acetyl group. The L-chain contained three Cys residues, two of which were suggested to form an intramolecular disulfide linkage, leaving the third one at the most C-terminal position and in a relatively hydrophilic region as the most probable site of disulfide linkage with the fibroin heavy chain.     

A novel 17 kD heparin-binding growth factor (HBGF-8) in bovine uterus: purification and N-terminal amino acid sequence

We have purified to near homogeneity a novel 17 kD growth factor from bovine uterus which we designated heparin-binding growth factor-8 (HBGF-8). The growth factor binds tightly to cation exchange resins and to Heparin-Sepharose and is stable to acetone precipitation and labile in acid. Based upon total activity in acetone extracts of bovine uterus stimulating 3H-thymidine incorporation into DNA of serum-starved NIH 313 cells, a 6940 fold purification was achieved with an overall yield of HBGF-8 activity of 0.4%, using extraction of acetone powders and chromatographic separations at neutral pH. Approximately 18 micrograms protein was obtained from 1.2 kg wet weight of tissue. HBGF-8 was clearly separated from 17.5 kD bovine uterus basic fibroblast growth factor (bFGF) by purification and its N-terminal amino acid sequence analyzed. A polypeptide with a unique 25 N-terminal amino acid sequence was found. HBGF-8 was as active as acidic fibroblast growth factor (aFGF) and slightly less active than bFGF in the mouse NIH 3T3 fibroblast mitogenic assay system with an intrinsic specific activity of 5000 dpm/ng under standard assay conditions.     

Human low-molecular-weight urinary urokinase. Partial characterization and preliminary sequence data of the two polypeptide chains

Low-molecular-weight urokinase (molecular weight 33100) was separated by analytical and preparative isoelectric focusing into five major subforms with isoelectric points between 8.7 and 9.6. These subforms are very similar in molecular weight, specific activity, amino acid composition and content of amino sugar and their N-terminal sequence constellation is identical. Low-molecular-weight urokinase consists of two polypeptide chains connected by a single disulfide bridge. The N-terminal region of the heavy chain (calculated Mr 30700) exhibits homology within the first 46 residues analyzed, with the known primary structure of other serine proteases. The mini chain (Mr 2426), whose complete sequence was determined, consists of 21 residues which show homology with the primary structure of the C-terminal region of the plasmin heavy chain. Based on sequence data and homology criteria with serine proteases a single-chain urokinase precursor is postulated having a peptide bond constellation between heavy and light chain region compatible with the requirements for serine protease activation.     

Amino acid sequence of rat liver cathepsin L

The complete amino acid sequences of the heavy and light chains of rat liver cathepsin L (EC 3.4.22.15) were determined at the protein level. The heavy and light chains consisted of 175 and 44 amino acid residues, respectively, and their Mr values without glycosyl groups calculated from these sequences were 18941 and 5056, respectively. The amino acid sequence was also determined from the N-terminal sequences of the heavy and light chains, and the sequences of cleavage fragments of the heavy chain with lysylendopeptidase and cyanogen bromide. The fragments were aligned by comparison with the amino acid sequence deduced from the sequence of cDNA of rat preprocathepsin L. The sequence of rat liver cathepsin L determined at the protein level was identical with that deduced from the cDNA sequence except that in the heavy chain, residues 176-177 (Asp-Ser) were not present at the C-terminus and alanine was replaced by proline at residue 125. Asn-108 in the heavy chain is modified with carbohydrate.     

Cloning and characterization of the growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3) in the rat.

We report for the first time the complete amino acid sequence for the growth hormone dependent insulin-like growth factor binding protein (IGFBP-3) in the rat. A human IGFBP-3 clone was generated using the polymerase chain reaction (PCR) and used to screen a rat liver cDNA library. cDNA clones of the rat IGFBP-3 were isolated and the full amino acid sequence deduced. The sequence begins with a putative, 26 amino acid signal peptide followed by a 265 amino acid binding protein. The amino acid sequence is over 80% homologous with the equivalent human IGFBP-3 form and shows complete conservation of 18 cysteine residues that are clustered at the amino and carboxy ends of the protein. IGFBP-3 is the binding subunit of the major circulating IGFBP in the rat, and hence the availability of precise structural data and cDNA probes provides an important opportunity for a detailed study of the control of IGFBP-3 synthesis at the level of gene expression.     

Amino acid sequence of bovine brain derived class 1 heparin-binding growth factor

The major class 1 heparin-binding growth factor from bovine brain is a single-chain polypeptide of 140 amino acids with a molecular weight of 15 877. It has the amino acid sequence Phe1-Asn-Leu-Pro-Leu-Gly-Asn-Tyr-Lys-Lys-Pro-Lys-Leu-Leu- Tyr15-Cys-Ser- Asn-Gly-Gly-Tyr-Phe-Leu-Arg-Ile-Leu-Pro-Asp-Gly-Thr30-Val-Asp-Gly- Thr-Lys-Asp-Arg- Ser-Asp-Gln-His-Ile-Gln-Leu-Gln45-Leu-Cys-Ala-Glu-Ser-Ile-Gly- Glu-Val-Tyr-Ile- Lys-Ser-Thr-Glu60-Thr-Gly-Gln-Phe-Leu-Ala-Met-Asp-Thr-Asp-Gly- Leu-Leu-Tyr-Gly75- Ser-Gln-Thr-Pro-Asn-Glu-Glu-Cys-Leu-Phe-Leu-Glu-Arg-Leu- Glu90-Glu-Asn-His-Tyr- Asn-Thr-Tyr-Ile-Ser-Lys-Lys-His-Ala-Glu-Lys105-His-Trp-Phe-Val -Gly-Leu-Lys-Lys- Asn-Gly-Arg-Ser-Lys-Leu-Gly120-Pro-Arg-Thr-His-Phe-Gly-Gln-Lys -Ala-Ile-Leu-Phe-Leu-Pro-Leu135-Pro-Val-Ser-Ser-Asp140-OH. The mitogen is homologous to the class 2 heparin-binding growth factor pituitary fibroblast growth factor with about 50% of the amino acids being identical between the two mitogens.