Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3'-kinase binding.

Progress has been made in our understanding of the mechanism by which the binding of vascular endothelial growth factor (VEGF) to cognate receptors induces a range of biological responses, but it is far from complete. Identification of receptor autophosphorylation sites will allow us to determine how activated VEGF receptors are coupled to specific downstream signalling proteins. In the present study, we have expressed human VEGF receptors in insect cells using the baculovirus expression system, identified a major autophosphorylation site on the VEGF receptor fms-like tyrosine kinase-1 (Flt-1) by HPLC-electrospray ionization (ESI)-MS, and characterized in vitro interactions between Flt-1 and phosphatidylinositol 3'-kinase (PI3-kinase). Infection of High 5 insect cells with Flt-1 recombinant virus resulted in the expression of a 170 kDa glycoprotein, which bound VEGF with a K(d) of 2 x 10(-10) M in intact insect cells. The overexpressed recombinant Flt-1 receptors exhibited tyrosine kinase activity and were constitutively phosphorylated. Analysis of Flt-1 tryptic peptides by HPLC-ESI-MS with selective phosphate ion monitoring identified a hexapeptide (YVNAFK; where single-letter amino-acid code has been used) containing a phosphotyrosine (pTyr) residue at position 1213. Using synthetic phosphopeptides, this pTyr residue was found to be directly involved in the binding of PI3-kinase in vitro even though it did not fall within a consensus pYM/VXM PI3-kinase binding motif. These results suggest that phosphorylated Flt-1 associates with PI3-kinase at pTyr(1213) to mediate the activation of this pathway in VEGF signalling.

Amino acid analysis.

Amino acid analysis (AAA) is one of the best methods to quantify peptides and proteins. Two general approaches to quantitative AAA exist, namely, classical postcolumn derivatization following ion-exchange chromatography and precolumn derivatization followed by reversed-phase HPLC (RP-HPLC). Excellent instrumentation and several specific methodologies are available for both approaches, and both have advantages and disadvantages. This unit focuses on picomole-level AAA of peptides and proteins using the most popular precolumn-derivatization method, namely, phenylthiocarbamyl amino acid analysis (PTC-AAA). It is directed primarily toward those interested in establishing the technology with a modest budget. PTC derivatization and analysis conditions are described, and support and alternate protocols describe additional techniques necessary or useful for most any AAA method--e.g., sample preparation, hydrolysis, instrument calibration, data interpretation, and analysis of difficult or unusual residues such as cysteine, tryptophan, phosphoamino acids, and hydroxyproline.

Cloning and functional expression of a human heparanase gene.

We have cloned a gene (HSE1) from a human placental cDNA library that encodes a novel protein exhibiting heparanase activity. The cDNA was identified through peptide sequences derived from purified heparanase isolated from human SK-HEP-1 hepatoma cells. HSE1 contains an open reading frame encoding a predicted polypeptide of 543 amino acids and possesses a putative signal sequence at its amino terminus. Northern blot analysis suggested strong expression of HSE1 in placenta and spleen. Transient transfection of HSE1 in COS7 cells resulted in the expression of a protein with an apparent molecular mass of 67-72 kDa. HSE1 protein was detectable in conditioned media but was also associated with the membrane fraction following cell lysis. The HSE1 gene product was shown to exhibit heparanase activity by specifically cleaving a labeled heparan sulfate substrate in a similar manner as purified native protein.

Identification of proteins electroblotted to polyvinylidene difluoride membrane by combined amino acid analysis and bioinformatics: An ABRF multicenter study.

The ABRF amino acid analysis study evaluated the general utility of amino acid analysis (AAA) for identification of proteins after denaturing gel electrophoresis and electroblotting to polyvinylidene difluoride (PVDF) membrane.Thirty-eight participating laboratories analyzed a known control (ovalbumin, 5 microg applied to the gel) and either lysozyme or bovine serum albumin as unknown samples (1-, 5-, and 10-microg amounts applied to the gel). Analyses of the unknowns yielded average compositional errors of approximately 30%, 19%, and 18%, respectively, from the low, intermediate, and higher sample amounts; the ovalbumin control exhibited an approximately 17% average error. Compositional data were submitted to the ExPASy and PROPSEARCH Internet sites for protein identification.Without search parameter adjustments or restrictions, both computer programs provided identification of about 20%, 66%, and 74% of the data from the 1-, 5-, and 10-microg gel samples, respectively. Deleting problematic data (Gly, Met, and Pro) did not always facilitate protein identification. Incorporating control results into the ExPASy search increased identifications 2% to 10%, and restricting search parameters by species, isoelectric pH, and molecular weight increased identifications by more than 80%. Average amounts analyzed for correct identifications were approximately 0.4 microg, 1.8 microg, and 2.9 microg for the 1-, 5-, and 10-microg gel samples, respectively.The results support the efficacy of AAA in the low microgram and nanogram range for the identification of PVDF-immobilized proteins from two-dimensional gels.

Cellular retinaldehyde-binding protein ligand interactions. Gln-210 and Lys-221 are in the retinoid binding pocket.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the retinal pigment epithelium (RPE) and Müller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa. Ligand interactions determine the physiological role of CRALBP in the RPE where the protein is thought to function as a substrate carrier for 11-cis-retinol dehydrogenase in the synthesis of 11-cis-retinal for visual pigment regeneration. However, CRALBP is also present in optic nerve and brain where its natural ligand and function are not yet known. We have characterized the interactions of retinoids with native bovine CRALBP, human recombinant CRALBP (rCRALBP) and five mutant rCRALBPs. Efforts to trap and/or identify a Schiff base in the dark, under a variety of reducing, denaturing, and pH conditions were unsuccessful, suggesting the lack of covalent interactions between CRALBP and retinoid. Buried and solvent-exposed lysine residues were identified in bovine CRALBP by reductive methylation of the holoprotein followed by denaturation and reaction with [3H]acetic anhydride. Radioactive lysine residues were identified by Edman degradation and electrospray mass spectrometry following proteolysis and purification of modified peptides. Human rCRALBP mutants K152A, K221A, and K294A were prepared to investigate possible retinoid interactions with buried or partially buried lysines. Two other rCRALBP mutants, I162V and Q210R, were also prepared to identify substitutions altering the retinoid binding properties of a random mutant. The structures of all the mutants were verified by amino acid and mass spectral analyses and retinoid binding properties evaluated by UV-visible and fluorescence spectroscopy. All of the mutants bound 11-cis-retinal essentially like the wild type protein, indicating that the proteins were not grossly misfolded. Three of the mutants bound 9-cis-retinal like the wild type protein; however, Q210R and K221A bound less than stoichiometric amounts of the 9-cis-isomer and exhibited lower affinity for this retinoid relative to wild type rCRALBP. Residues Gln-210 and Lys-221 are located within a region of CRALBP exhibiting sequence homology with the ligand binding cavity of yeast phosphatidylinositol-transfer protein. The data implicate Gln-210 and Lys-221 as components of the CRALBP retinoid binding cavity and are discussed in the context of ligand interactions in structurally or functionally related proteins with known crystallographic structures.

Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.

Changes in biological activity and folding of guanylate cyclase-activating protein 1 as a function of calcium.

Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca2+-binding protein, activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca2+-binding proteins from the calmodulin superfamily, the Ca2+-free form of GCAP1 stimulates the effector enzyme. In this study, we analyzed the Ca2+-dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca2+-sensitive modulation of GC1 activity. The change from a Ca2+-bound to a Ca2+-free form of GCAP1 increased susceptibility of Ca2+-free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca2+-bound form, only the N-terminus (myristoylated Gly2-Lys9) and C-terminus (171-205 fragment) of GCAP1 are removed by trypsin, while in the Ca2+-free form, GCAP1 is readily degraded to small fragments. Successive inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca2+-dependent inactivation of GCAP1. GCAP1(E75D,E111D,E155D) mutant did not bind Ca2+ and stimulated GC1 in a [Ca2+]-independent manner. GCAP1 and GCAP2, but not S-100beta, a high [Ca2+]free activator of GC1, competed with the triple mutant at high [Ca2+]free, inhibiting GC1 with similar IC50's. These competition results are consistent with comparable affinities between GC1 and GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca2+ binding and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein from the activator to the inhibitor of GC1.

Inhibition of human cytomegalovirus UL80 protease by specific intramolecular disulfide bond formation.

A symmetrically substituted disulfide compound, CL13933, was identified as a potent inhibitor of human cytomegalovirus UL80 protease. Two types of inhibited protease were observed, depending on inhibitor concentration. At high concentrations, CL13933 formed a covalent adduct with the protease on Cys residues. At lower concentrations, this compound induced specific intramolecular disulfide formation between Cys84 and Cys87, and between Cys138 and Cys161. In contrast, Cys202 did not form disulfide bonds. Inhibition was reversed upon reduction of the protease. Each of the five cysteines of the UL80 protease was individually mutated to Ala. Each of the mutant proteases retained enzymatic activity, but mutants C138A and C161A were resistant to inhibition by CL13933, suggesting that disulfide bond formation between Cys138 and Cys161 is responsible for inhibition. This disulfide is apparently not induced by air oxidation. Examination of the CL13933 loading patterns of wild type and the five mutant proteases by mass spectrometry revealed that residues Cys87, Cys138, and Cys161 react with CL13933, and that the disulfide pair partner of each (Cys84, Cys161, and Cys138, respectively) is able to displace the compound via thiol-disulfide exchange. The possible significance of these reactive thiols in the protease is discussed.

Proximity mapping of the Tet repressor-tetracycline-Fe2+ complex by hydrogen peroxide mediated protein cleavage.

We demonstrate in a quantitative in vitro induction assay that tetracycline-Fe2+ is a more than 1000-fold stronger inducer of Tet repressor compared to tetracycline-Mg2+. Oxidative cleavage of the Tet repressor-tetracycline-Fe2+ complex with H2O2 and ascorbate results in an Fe(2+)-dependent specific fragmentation of the protein. The maximal yield of about 15% and a reaction time of less than 30 s are only observed in the presence of the drug, whereas about 1% cleavage is obtained after 30 min in the presence of Fe2+ without tetracycline. Cleavage is not inhibited by several radical scavengers, suggesting a highly localized reactivity of the redox-active oxo intermediates in the proximity of the Fe(2+)-tc chelater where they are generated. The products can be separated by HPLC only after denaturation, indicating that the complex is not disrupted by cleavage. Residues at which the cleavage takes place are identified using the masses of the fragments determined by electrospray mass spectrometry and their N-terminal sequences. The major cleavage site maps to residues 104 and 105 of Tet repressor. Less efficient cleavages occur at residues 56 and 136, and the least efficiently cleaved sites are around residues 144 and 147. The cleavage efficiencies correlate to the distances and orientations of the respective peptide bonds to Mg2+ in the crystal structure of the Tet repressor-tetracycline-Mg2+ complex. We discuss potential reaction mechanisms leading to protein cleavage.

Biological activity and phosphorylation sites of the bacterially expressed cytosolic domain of the KDR VEGF-receptor.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor which binds to two structurally similar receptor tyrosine kinases, KDR and FLT1. Towards the goal of clarifying the signal transduction pathways by which VEGF activates endothelial cells, we expressed in bacteria an enzymatically active form of the cytosolic domain of the KDR receptor. The expressed protein undergoes autophosphorylation in both bacterial cells and in its purified form. Using peptide mapping and sequencing of peptides, we identified four tyrosine residues that are phosphorylated corresponding to residues 951, 996, 1054, and 1059 of the KDR protein. The location of the phosphorylated residues in the bacterially expressed protein, and/or the consensus sequences around these sites, suggest they may be identical to the phosphorylated sites of KDR in mammalian cells.

Refolding and characterization of human recombinant heparin-binding neurite-promoting factor.

Heparin-binding neurite-promoting factor (HBNF) is a highly basic, cysteine-rich 136-residue protein, and a member of a new class of heparin-binding proteins. It exhibits a neurite-outgrowth promoting activity and its expression is both temporally and spacially regulated during fetal and postnatal development. A high interspecies sequence conservation suggests important, presently unknown, biological functions. HBNF is structurally and most likely functionally related to the product of a developmentally regulated gene, MK (midkine). To elucidate biological roles of these proteins, recombinant forms of the proteins were produced. Expression of human recombinant HBNF and MK in Escherichia coli lead to the formation of insoluble aggregated protein that accounted for about 25% of the total cellular protein. Homogeneous, monomeric forms of each protein were recovered from inclusion bodies by reduction with dithiothreitol and solubilization in 8 M urea. Refolding of the reduced and denatured protein occurred upon dialysis at pH 7.4. Human recombinant (hr) HBNF and hrMK prepared in this manner were further purified by heparin affinity chromatography. Chromatographic evidence demonstrates that refolding and concomitant disulfide bond formation in hrHBNF proceeds in high yield with minimal formation of stable nonnative disulfides. Studies on the redox status of the 10 cysteine residues of bovine brain HBNF and the refolded recombinant protein indicate that all cysteines are engaged in disulfide bond formation. The disulfide arrangements for the recombinant protein were found to be identical to those in the native protein isolated from bovine brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and partial amino acid sequence of a mu opioid receptor from rat brain.

A rat brain opioid receptor protein was isolated by binding [epsilon-biotinyl-Lys32] beta-endorphin to membranes, solubilizing the receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine and purifying on immobilized streptavidin and wheat germ agglutinin. The purified glycoprotein had a molecular mass of 60-70 kDa. Recovery of this protein was blocked by the nonselective opioid antagonist naloxone and the highly mu-selective agonist [D-Ala2,N-methyl-Phe4,Glyol5]-enkephalin but not by the highly delta-selective agonist [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin when these compounds were added as competitors at the binding step. The 60-70-kDa receptor protein co-purified through the streptavidin column with 40-kDa protein recognized by anti-Gi alpha antibodies. GTP and Na+ influenced dissociation of the solubilized R.125I-L complex and elution of the receptor and G protein from streptavidin in fashions consistent with the pharmacology of mu-opioid receptors. A 23-amino acid residue sequence from the purified receptor differs at 4 positions from a similar sequence in the murine delta-opioid receptor and is encoded within a novel rat brain cDNA isolated by polymerase chain reaction with oligonucleotide primers related to the murine delta-opioid receptor gene.

Enzymatic generation of the amino terminus of the beta-amyloid peptide.

The major pathological change in Alzheimer's disease is the deposition of 39-42-amino acid beta-amyloid peptide (BAP) in the brain. Since BAP begins at the aspartate residue (Asp1, or codon 672 of the amyloid precursor protein (APP)770 transcript), the ability of several proteases to cleave the peptide bond methionine-Asp1 (M/D) was evaluated by using peptides and recombinant APP molecules as substrates. Cathepsin G and chymotrypsin cleave the synthetic peptide HSEVKMDAEF at M/D under acidic conditions, whereas cleavage at lysine-methionine (K/M) predominates when the pH is alkaline. Trypsin and cathepsins B, D, and L are unable to cleave the synthetic peptide at M/D. Peptide SEVNLDAEF, representing the mutation found in early onset Alzheimer's disease families from Sweden, is cleaved by cathepsin G and chymotrypsin at leucine-aspartate (L/D). Incubation of cathepsin G with soluble protease nexin-2 obtained from recombinant APP (APP-REP) derivatives resulted in proteolytic cleavage at or near the amino terminus of BAP. Cathepsin G-mediated cleavage was also observed in the domain representing the amino terminus of BAP when mature plasma membrane-associated APP-REP molecules were used as substrates. Our results strongly suggest the involvement of a chymotrypsin-like serine protease in the generation of the amino terminus of BAP beginning at Asp1.

Genomic organization of the human HBNF gene and characterization of an HBNF variant protein as a splice mutant.

The organization of the human heparin-binding neurite outgrowth promoting factor (HBNF) gene is presented. Based on Southern analysis and the isolation of genomic DNA clones from a lambda phage library, the minimum size of the gene is 42 kb. Sequences comprising the HBNF mRNA are contained in five exons which account for the 1650 nt mRNA size observed by northern analysis. From the structure of the gene it is predicted that a variant human HBNF cDNA with a three basepair deletion is a result of alternative splicing at the acceptor site of exon 5. Evidence is presented that indicates the existence of a variant HBNF protein, des-Ala119-HBNF, in bovine brain which has a corresponding amino acid deletion. This alternate form comprises approximately 20% of the total HBNF protein present in bovine brain.

Comparison of the disulfide bond arrangements of human recombinant and bovine brain heparin binding neurite-promoting factors.

Heparin binding neurite-promoting factor (HBNF) is a highly basic 136 amino acid protein containing 10 cysteine residues. We have determined the redox status and the disulfide arrangement of the cysteine residues in HBNF from bovine brain and refolded human recombinant protein produced in E. coli. Our data indicate that all 10 cysteines are involved in disulfide bond formation. The disulfide linkages of human recombinant and bovine brain HBNF, as determined after proteolytic digestions of the non-reduced proteins by peptide mapping and sequence analysis are: Cys15-Cys44, Cys23-Cys53, Cys30-Cys57, Cys67-Cys99 and Cys77-Cys109. Thus, recombinant HBNF has the same disulfide arrangement as the native brain-derived protein.

Expression and analysis of the human cytomegalovirus UL80-encoded protease: identification of autoproteolytic sites.

The 45-kDa assembly protein of human cytomegalovirus is encoded by the C-terminal portion of the UL80 open reading frame (ORF). For herpes simplex virus, packaging of DNA is accompanied by cleavage of its assembly protein precursor at a site near its C terminus, by a protease encoded by the N-terminal region of the same ORF (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). By analogy with herpes simplex virus, we investigated whether a protease is contained within the N-terminal portion of the human cytomegalovirus UL80 ORF. The entire UL80 ORF was expressed in Escherichia coli, under the control of the phage T7 promoter. UL80 should encode a protein of 85 kDa. Instead, the wild-type construct produces a set of proteins with molecular masses of 50, 30, 16, 13, and 5 kDa. In contrast, when mutant UL80 is deleted of the first 14 amino acids, it produces only an 85-kDa protein. These results suggest that the UL80 polyprotein undergoes autoproteolysis. We demonstrate by deletional analysis and by N-terminal sequencing that the 30-kDa protein is the protease and that it originates from the N terminus of UL80. The UL80 polyprotein is cleaved at the following three sites: (i) at the C terminus of the assembly protein domain, (ii) between the 30- and 50-kDa proteins, and (iii) within the 30-kDa protease itself, which yields the 16- and 13-kDa proteins and may be a mechanism to inactivate the protease.

Partial amino acid sequence of a somatostatin receptor isolated from GH4C1 pituitary cells.

A somatostatin receptor isolated from GH4C1 rat pituitary tumor-derived cells was cleaved with cyanogen bromide or cyanogen bromide+trypsin to obtain sequenceable fragments. Five unique amino acid sequences ranging from 6 to 27 amino acid residues were obtained. The sequence was identical to sequence recently reported for one of two somatostatin receptors cloned from human pancreas [Yamada et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 251-255] except for a single valine to isoleucine substitution. This is the first report of amino acid sequence from a purified somatostatin receptor.

Molecular characterization of interleukin 12.

Interleukin 12 (IL-12), formerly known as cytotoxic lymphocyte maturation factor and natural killer cell stimulatory factor, is a cytokine secreted by a human B lymphoblastoid (NC-37) cell line when induced in culture with phorbol ester and calcium ionophore. This factor has been purified to homogeneity and shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells. In addition, purified IL-12 stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. The protein is a heterodimer composed of a 40- and a 35-kDa subunit. Amino acid sequence analysis confirmed predicted sequences from the cloned cDNAs of each subunit. Chemical and enzymatic deglycosylation of the heterodimer demonstrated that the 40- and 35-kDa subunits contain 10 and 20% carbohydrate, respectively. Structural analysis of IL-12 using site-specific chemical modification revealed that intact disulfide bonds are essential for bioactivity. The 40-kDa subunit of IL-12 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by immunoblotting as being present in NC-37 cell supernatant solutions in relatively large amounts uncomplexed to the 35-kDa subunit. Previously it had been shown that the 40-kDa subunit alone does not cause the proliferation of activated human T lymphocytes or enhance the cytolytic activity of human natural killer cells. However, results obtained by site-specific chemical modification suggesting that a tryptophan residue is at or near the active site of IL-12 may imply a direct role of the subunit in interacting with the IL-12 receptor. These data may support the recent proposal (D.P. Gearing and D. Cosman (1991) Cell 66, 9-10) that IL-12 consists of a complex of cytokine and soluble receptor.

The role of arginine residues in interleukin 1 receptor binding.

Interleukin 1 (IL-1) is a family of polypeptide cytokines that plays an essential role in modulating immune and inflammatory responses. IL-1 activity is mediated by either of two distinct proteins, IL-1 alpha or IL-1 beta, both of which bind to the same receptor found on T-lymphocytes, fibroblasts and endothelial cells (Type 1 receptor). The effect of specific chemical modification of recombinant IL-1 alpha and IL-1 beta on receptor binding was examined. Modification of the proteins with phenylglyoxal, an arginine-specific reagent, resulted in the loss of Type 1 IL-1 receptor binding activity. The stoichiometry of this modification revealed that a single arginine in either IL-1 alpha or IL-1 beta is responsible for the loss of activity. Cyanogen bromide cleavage of phenylglyoxal modified IL-1 alpha and IL-1 beta, followed by sequencing of the peptides, revealed that arginine-12 in IL-1 alpha and arginine-4 in IL-1 beta, which occupy the same topology in the respective crystallographic structures, are the target of phenylglyoxal. These results suggest that an arginine residue plays an important role in ligand-receptor interaction.

Isolation and partial sequence of goat spleen prothymosin alpha.

Goat prothymosin alpha, a highly acidic polypeptide of pI3.5, 109 amino acid residues, has been isolated from lymphoid and non-lymphoid tissues of young female goats. Unlike rat, murine and porcine prothymosins alpha, goat prothymosin alpha appears at a higher concentration in the spleen compared with the thymus. The sequence of segments of the polypeptide involving known mutations has been determined, by automatic sequencing of its tryptic peptide fragments. The acidic amino acid-rich segment in the middle of the molecule, including residues 49-83, has not been sequenced. Goat prothymosin alpha closely resembles bovine prothymosin alpha, with only one substitution, proline for alanine at position 85. It also resembles human prothymosin alpha, with only three substitutions. It differs more significantly from rat and murine prothymosins alpha, by two deletions and three substitutions. The results show the highly conserved nature of the molecule, with substitutions at given positions only.