Lack of biological activity of preproendothelin [110-130] in several endothelin assays

A 21 amino acid peptide containing the prepropendothelin sequence from amino acids 110 to 130 and two intrachain disulfide bonds was synthesized and tested for biological activity in the following endothelin assays: 1.) a competition binding assay using [125I]ET-1 and dog heart membranes, 2.) three RIA's using 125I-ET-1, -2 and -3 and the respective anti-ET rabbit antisera; and 3.) a contractile activity bioassay using hamster aortic rings. The synthetic peptide which has been referred to as the "endothelin-like" peptide occurs 36 amino acids C-terminal to endothelin in the prepro-protein sequence. It contains only 40% sequence homology to the three endothelin isoforms, but has the same sequence and cyclization pattern of cysteines at positions 1, 3, 11 and 15. Despite the overall similarity in secondary structure to the three isoforms of endothelin and sarafotoxin S6b, preproendothelin [110-130] had no activity in any of the assays when tested at concentrations of 10(-10)M to 10(-5)M.     

Importance of N- and C-terminal regions of gastrin-Gly for preferential binding to high and low affinity gastrin-Gly receptors

G17-Gly has been shown to stimulate the growth of DLD-1 human colon cancer cells in a biphasic manner via high and low affinity receptors. In the current study, the existence of heterogeneous receptor populations for G17-Gly on the HT-29 human colon cancer cell line was investigated. The effect of either N- or C-terminal peptide truncation on receptor binding and cell growth stimulation was also explored. [Leu15]G17-Gly bound to both high (nM) and low (microM) affinity sites on HT-29 cells. The peptide stimulated cell growth in a dose-dependent and biphasic manner with maximal stimulation at 10(-9) M peptide concentration, suggesting that, as in the case of DLD-1 cells, it is the high affinity receptor which is responsible for the growth-promoting effects. In contrast, G17(1-12) stimulated the growth of HT-29 cells in a sigmoidal fashion with an EC50 of 4.6x10(-9) M. Sequential N-terminal truncation of [Leu15]G17-Gly results in decreased binding to the high affinity G17-Gly receptor on DLD-1 cells. [Leu15]G17(11-17)Gly bound to the low affinity G17-Gly receptor with an affinity similar to that of the full sequence peptide but was unable to displace the radioligand from high affinity sites. G17(1-6)-NH2 was unable to displace [3H]G17-Gly from either site. These results suggest that the important residues for binding to the low affinity receptor are in the C-terminal region of the peptide while those required for interaction with the high affinity receptor lie further towards the N-terminus.     

Identification of membrane-embedded domains of lipophilin from human myelin

The organization of lipophilin in the intact human myelin membrane has been studied by labeling with the carbene photogenerated from 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). This hydrophobic probe labels mostly lipophilin (the main intrinsic protein of myelin) and the lipids within the bilayer. The domains of lipophilin which are embedded within the membrane have been identified by proteolytic fragmentation of the [125I]TID-labeled myelin, extraction with organic solvents, and separation by chromatography. Four labeled peptides were purified in this way. Polyacrylamide gel electrophoresis, amino acid compositions, automated sequencing, and carboxy-terminal analyses identified a 15K molecular weight peptide, T1 (residues 1-143), as representing the amino-terminal fragment, a 10K peptide, T2 (residues 1-97), representing a smaller amino-terminal fragment, a 5K peptide, T4 (residues 53-97), which represented the COOH-terminal half of peptide T2, and a 7K peptide, T3 (residues 205-268), which represented a sequence near the COOH terminus of lipophilin. The specific radioactivities of the peptides were determined; peptides T1 and T2 had similar specific activities, which were twice the specific activities of peptides T3 and T4. The data provide direct chemical evidence that human lipophilin has membrane-embedded domains between residues 1-97, 53-97, and 205-268, in agreement with some of the predictions of other investigators based on the sequence of bovine myelin lipophilin (proteolipid apoprotein) and a hydrophobicity diagram.     

Quantitative Immunoprecipitation Studies with Anti-γ-Aminobutyric AcidA Receptor γ2 1–15 Cys Antibodies

Antibodies raised against the synthetic peptide NH2-QKSDDDYEDYASNKTC-COOH (gamma 2 1-15 Cys), which corresponds to the N-terminal amino acid sequence with a C-terminal cysteine of the human gamma 2 subunit of the gamma-aminobutyric acidA (GABAA) receptor, were used to study the quantitative immunoprecipitation of agonist benzodiazepine binding sites from bovine brain. Anti-gamma 2 1-15 Cys antibodies were found to immunoprecipitate specifically in parallel [3H]flunitrazepam- and [3H]muscimol-reversible binding sites in a dose-dependent manner. The maximum percentages of [3H]flunitrazepam binding sites immunoprecipitated from detergent extracts of bovine cerebral cortex, cerebellum, and hippocampus were 68, 77, and 83%, respectively. Immunoprecipitation studies with anti-alpha 1 324-341 antibodies carried out in parallel with anti-gamma 2 1-15 Cys antibodies provided evidence for the promiscuity of the gamma 2 subunit within native GABAA receptors. These results substantiate the association of the gamma 2 polypeptide with native GABAA receptors.     

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.     

Interaction of synthetic sarafotoxin with rat vascular endothelin receptors

The effects of synthetic analogs of sarafotoxin (STX) S6b, a snake venom peptide with a high sequence homology to the endothelium-derived vasoconstrictor endothelin (ET), on ET receptor binding activity and cytosolic free Ca2+ concentration [( Ca2+]i) were studied in cultured rat vascular smooth muscle cells. Binding studies revealed that [Cys1-15, Cys3-11] STX competed with 125I-ET for the binding to its vascular receptors with lower affinity than that of ET, but was far more effective than [Cys1-11, Cys3-15]STX in inhibiting the binding. [Cys1-15, Cys3-11]STX had a less potent effect on increasing [Ca2+]i than ET, whereas [Cys1-11, Cys3-15]STX was inactive. These data suggest that there may exist heterogenous subpopulations of the vascular ET/STX receptors, and that the proper double cyclic structure of STX is essential for interacting with its putative receptors to induce the [Ca2+]i response.     

Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe-1,Val1,Asn2, Gln3,His4,Ser8, His9,Glu12,Tyr15,Leu16]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has greater than 1,000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln3,Ala4]IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr15,Leu16]IGF-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. This peptide is also equipotent to hIGF-I at the types 1 and 2 IGF receptors. The peptide in which these four-point mutations are combined, [Gln3,Ala4,Tyr15,Leu16]IGF-I, has 600-fold reduced affinity for the serum binding proteins. This peptide has 10-fold increased potency for the insulin receptor, but is equipotent to hIGF-I at the types 1 and 2 IGF receptors. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, these peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I.     

Bradykinin and its Gly6 analogue are substrates of cyclophilin: a fluorine-19 magnetization transfer study

Fluorine-19 magnetization transfer experiments have been used to determine the rates of cis/trans isomerization about the X-Pro7 peptide bond in [p-fluoro-Phe8]bradykinin (cis/trans ratio approximately 0.1) and its Gly6 analogue (cis/trans ratio approximately 0.4). The measurements were carried out both prior to and after the addition of cyclophilin, which has recently been shown to have peptidyl-proline cis/trans isomerase activity and is the apparent target enzyme of the immunosuppressive agent cyclosporin A. Magnetization transfer measurements over the temperature range 40-75 degrees C in the absence of enzyme give activation energies of 22.8 and 23.0 kcal/mol for [p-fluoro-Phe8]bradykinin and its Gly6 analogue, respectively. The values for the uncatalyzed cis----trans rate constant, kc, are determined by extrapolation to be 4.8 x 10(-2) and 2.1 x 10(-2) s-1 for the two peptides at 25 degrees C. The enzyme-catalyzed enhancement of the cis/trans interconversion rate was proportional to added cyclophilin concentration and was strongly sequence specific, with bradykinin a much better substrate than [Gly6]bradykinin. At a peptide concentration of 2.2 mM, the catalytic activity expressed as kc per micromolar cyclophilin was determined to be 1.2 s-1/microM for [p-fluoro-Phe8]bradykinin and 0.13 s-1/microM for the Gly6 analogue. The increased cis----trans interconversion rates were strongly inhibited by cyclosporin A and the 6-(methylalanine) derivative, which bind to cyclophilin, but not by the 1-(tetrahydrofurfuryl) derivative of cyclosporin that binds weakly.     

Binding of human tumor necrosis factor alpha to multimeric complementary peptides.

A peptide with binding properties for tumor necrosis factor (TNF alpha) sequence 144-157 has been designed, using a computer-assisted method able to create peptide sequences hydropathically complementary to a given sequence. The complementary peptide was synthesized in a multimeric form starting from an octadentate polylysine core, to facilitate its immobilization and to provide interaction multivalency. Once immobilized on a solid support to prepare an affinity column, it recognized the target TNF144-157 peptide selectively from crude peptide mixtures containing TNF fragments encompassing the entire TNF alpha sequence. Similar selectivity and specificity were shown for full-length recombinant TNF alpha, allowing its purification from crude Escherichia coli extracts. The octameric complementary peptide preserved its recognition properties for TNF alpha and biotinylated TNF alpha even after coating on microtiter plates. Competitive binding occurred with unlabeled TNF alpha in the range between 0.01 and 10 micrograms/ml, in the presence of detergent such as 0.05% Tween 20 and in the presence of 1% normal goat serum. The effect of complementary peptide multimerization was evidenced by its enhanced binding affinity for TNF alpha, which exists in solution as a trimer, while the target TNF[144-157] peptide was recognized with much lower strength. The dissociation constant for interaction with TNF alpha was close to 10 nM, allowing its easy detection by solid phase assays in concentrations as low as 10 pmol/ml.     

The streptococcal flavoprotein NADH oxidase. I. Evidence linking NADH oxidase and NADH peroxidase cysteinyl redox centers

The FAD-containing NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the four-electron reduction of O2----2H2O, has been purified by an improved procedure for analyses of its structural and redox properties. The enzyme is apparently a dimer of two identical subunits, each containing 1 mol of FAD. Dithionite reduction of the enzyme proceeds in two distinct phases corresponding to approximately 0.5 and 1.1 eq/FAD, respectively. Thiol assays of the NADH oxidase, reduced anaerobically with 1 eq of NADH/FAD prior to denaturation, are consistent with the presence of a single redox-active cysteinyl residue/subunit. Analysis of the cysteinyl peptides of the oxidase, identified in tryptic digests of the enzyme labeled metabolically with [35S]cysteine, reveals a sequence which is closely related to the redox-active cysteinyl peptide sequence recently determined for the streptococcal flavoprotein NADH peroxidase. A second cysteinyl peptide sequence, when aligned with residues 3-17 of the peroxidase NH2-terminal sequence, reveals identity in 7 of 15 positions and satisfies several of the criteria described for ADP-binding structures. Additional probes of the structural and redox properties of the NADH oxidase, including visible circular dichroism spectroscopy and sensitivity to inactivation by hydrogen peroxide, provide further evidence for a fundamental structural connection between flavin-dependent NADH oxidase and peroxidase functions.     

Sequences of B-chain/domain 1-10/1-9 of insulin and insulin-like growth factor 1 determine their different folding behavior

Although insulin and insulin-like growth factor-1 (IGF-1) belong to one family, insulin folds into one thermodynamically stable structure, while IGF-1-folds into two thermodynamically stable structures (native and swap forms). We have demonstrated previously that the bifurcating folding behavior of IGF-1 is mainly controlled by its B-domain. To further elucidate which parts of the sequences determine their different folding behavior, by exchanging the N-terminal sequences of mini-IGF-1 and recombinant porcine insulin precursor (PIP), we prepared four peptide models: [1-9]PIP, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1 by means of protein engineering, and their disulfide rearrangement, V8 digestion, circular dichroic spectra, disulfide stability, and in vitro refolding were investigated. Among them only [1-9]PIP, like mini-IGF-1/IGF-1, was expressed in yeast as two isomers: isomer 1 (corresponding to swap IGF-1) and isomer 2 (corresponding to native IGF-1), which are supported by the experimental results of disulfide rearrangements, peptide mapping of V8 endoprotenase digests, circular dichroic analysis, in vitro refolding, and disulfide stability analysis. The other peptide models, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1, fold into one stable structure as PIP does, which indicates that sequence 1-4 of mini-IGF-1 is important for the folding behavior of mini-IGF-1/IGF-1 but not sufficient to lead to a bifurcating folding. The results demonstrated that the folding information, by which mini-IGF-1/IGF-1-folds into two thermodynamically structures, is encoded/written in its sequence 1-9, while sequences 1-10 of B chain in insulin/PIP play an important role in the guide of its unique disulfide pairing during the folding process.     

Design of hydropathically complementary peptides for Big Endothelin affinity purification.

Molecular recognition between Big Endothelin (Big ET) and a computer generated peptide hydropathically complementary to Big ET[16-29] sequence has been studied by analytical high performance liquid affinity chromatography (HPLAC), circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Specific binding was observed between solid support immobilized complementary peptide and Big ET[1-38], [1-32], and [16-32], but not with Big ET fragments [1-21], [16-21], [22-32], and [22-38], obtained by chymotrypsin proteolytic degradation. Selectivity in the recognition process was clearly demonstrated by the ability of complementary peptide affinity column to purify the Big ET molecule from complex peptide mixtures, even when present in very low concentrations. Similar selectivity was evidenced with the Big ET fragment [16-32], [NH2-HLDIIWVNTPEHIVPYG-COOH] containing the entire hydropathically complementary sequence. Binding was followed by marked spectroscopic changes, as monitored by circular dichroism and one- and two-dimensional nuclear magnetic resonance experiments. The NMR spectra of the complementary peptides 1:1 mixture showed variations in the chemical shifts of proton resonances in several residues, both in the main chain (amide protons) and in the side chains (aliphatic and aromatic protons). These data support the hypothesis of a multilocalized type of interaction between complementary peptides, where many residues along the peptide chains participate in co-operative stabilizing contacts in the forming complex.     

Actions of several substituted short analogues of porcine galanin on isolated rat fundus strips: a structure-activity relationship.

The activity of porcine galanin (Gal) fragments and analogues were tested in vitro using rat gastric fundus strips. The peptides contracted longitudinal smooth muscle in a concentration-dependent manner with the following order of potency: [Nle4]Gal(1-15), Gal(-15), [Cle4]Gal(1-15), [Hse6]Gal(1-15), [Va14]Gal(1-15), [Ile4]Gal(1-15), [endoTrip2a, Cle4]Gal(1-15), [desThr3, Cle4]Gal(1-15), [D-Leu4] Gal(1-15), [desLeu4]Gal(1-15). On the contrary [desTrp2, Val4]Gal (1-15) remained inactive up to 10 microM. The values of Hill's coefficients estimated from the appropriate concentration-contraction curves for all analogues except for [Val4]Gal(1-15), [Hse6]Gal(1-15), [endoTrp2a,Cle4]Gal(1-15), [desLeu4]Gal(1-15) and [D-Leu4] Gal(1-15) did not significantly differ from unity. Our results indicate that the integrity of the first four N-terminal amino acids of Gal molecule is essential for the full excitatory myogenic action of the peptide in rat gastric fundus. Similarly, substitution, addition or deletion of amino acid residues in positions two, three, four and six can considerably influence the ability of Gal analogues to interact with Gal receptors. The data acquired in the course of our structure-activity study suggest that both N- and C-terminals of Gal molecule contribute towards the affinity and activity of Gal in rat gastric smooth muscle cell receptors.     

Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells

Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an Mr 95,000 protein (identified as the insulin receptor beta subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) antisera) and an Mr 180,000 protein (which was unreactive with all anti-insulin receptor antibodies). After purification and tryptic digestion of the Mr 95,000 protein, tryptic peptides containing Tyr(P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. The partial amino acid sequence obtained by gas- and solid-phase Edman degradation was compared to the amino acid sequence of the intracellular extension of the rat insulin receptor deduced from the genomic sequence. Approximately 80% of all beta subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg from the tyrosine kinase domain, which is recovered mainly as the double phosphorylated species (predominantly in the form with Tyr(P) at residues 3 and 7 from the amino terminus; the remainder with Tyr(P) at residues 3 and 8), with 10-15% as the triple phosphorylated species. A second tryptic peptide is located near the carboxyl terminus, contains 2 tyrosines, and has the sequence, Thr-Tyr-Asp-Glu-His-Ile-Pro-Tyr-Thr-; this contains 20-30% of beta subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. Approximately 10% of beta subunit [32P]Tyr(P) resides on an unidentified tryptic peptide of Mr 4,000-5,000. The insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells thus involves at least 6 of the 13 tyrosine residues located on the beta subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin. This multisite regulatory tyrosine phosphorylation is the initial intracellular event in insulin action.     

Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine.

The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)     

A highly basic N-terminal extension of the mitochondrial matrix enzyme ornithine transcarbamylase from rat liver

We have deduced the amino acid sequence of the N-terminal leader peptide of the mitochondrial enzyme ornithine transcarbamylase from a cDNA clone obtained from a rat liver cDNA library. The sequence is remarkable in being highly basic, having 4 arginine, 3 lysine and 1 histidine with no acidic residues in a total of 32 residues. The leader sequence has no extensive hydrophobic stretches, has 72% homology with the leader peptide of human ornithine transcarbamylase [1], and in terms of its basic character resembles the N-terminal extensions on a number of fungal mitochondrial [2-5] and pea chloroplast [6] proteins. Thus the basic nature of these leader peptides may constitute the signal for mitochondrial import.     

Fully synthetic immunogens. Part III. Synthesis of hinge-peptide/gastrin conjugates and their immunological properties.

As core molecule for the multiple attachment of antigenic peptides we have selected the human IgG1 hinge fragment 225-232/225'-232'. Two types of conjugates of this double-chain bis-cystinyl hinge-peptide were prepared i) by linking its C-termini to [NIe15]-human-little-gastrin-[2,17] and ii) by elongating the resulting hinge-peptide/[NIe15]-little-gastrin-[2-17] conjugate at the two N-termini with the human big-gastrin sequence 1-14 to produce the big-gastrin-[1-14]/hinge-peptide/little-gastrin-[2-17] conjugate. For the synthesis of these peptide structures both the route via the preformed double-chain bis-cystinyl peptide and the route via suitably protected monomeric bis-cysteinyl peptides were used. For the latter approach advantage was taken of the previous observation about the preferred oxidation of the bis-cysteinyl hinge-peptide 225-232 to the dimer in parallel alignment. Both synthetic routes led to identical products. Immunization experiments in guinea pigs with the synthetic hybrids led to surprisingly strong immune responses with anti-little-gastrin antibody titers comparable to those induced by the iso-1-cytochrome c/little-gastrin-[2-17] conjugate as carrier-hapten system. These findings show that the two gastrin constructs are fully competent immunogens. Additionally, the gastrin receptor-like specificity of the antibodies indicates that both the synthetic hybrids and the cytochrome c conjugate allow for expression of a little-gastrin-specific conformational epitope similar to the bioactive structure of this hormone. The usefulness of such synthetic hybrids is further confirmed by the observation that the bivalent immunogen, containing both the little-gastrin 2-17 and the big-gastrin 1-14 sequence, is capable of inducing an immune response against both antigenic sequences, although with different efficiency. These results fully confirm our expectations.     

Detection in Vivo of Very Rapid Red Light-Induced Calcium-Sensitive Protein Phosphorylation in Etiolated Wheat (Triticum aestivum) Leaf Protoplasts

Etiolated wheat (Triticum aestivum cv Mercia) leaf protoplasts respond to brief red-light irradiation by increasing in volume over a 10-min incubation period (M.E. Bossen, H.A. Dassen, R.E. Kendrick, W.J. Vredenberg [1988] Planta 174: 94-100). When the calcium-sensitive dye Fluo-3 was incorporated into these protoplasts, red-light irradiation initiated calcium transients lasting about 2 min (P.S. Shacklock, N.D. Read, A.J. Trewavas [1992] Nature 358: 153-155). Release of calcium in the protoplasts by photolysis of incorporated 1-{2-amino-5-[1-hydroxy-1-(2-nitro-4, 5-methylenedioxyphenyl)-methyl]-phenoxy}-2-(2[prime]-amino-5[prime]-methylp henoxy)-ethane-N,N, N[prime],N[prime] -tetraccetic acid, tetrasodium salt (caged calcium) or caged inositol trisphosphate frequently induced transient increases in intracellular calcium levels, although the kinetics of these changes showed variation between experiments. Upon exposure to red light, a pronounced increase in the phosphorylation of a 70-kD and to a lesser extent a 60-kD peptide was observed, commencing within 15 s and continuing for up to 2 min. Simultaneous far-red and red irradiation attenuated the response. Upon release of incorporated caged calcium by cage photolysis, the labeling of these two peptides was greatly increased. When incorporated caged inositol trisphosphate was photolyzed, only the labeling of the 70-kD peptide was enhanced. Phosphorylation of the 70-kD peptide was also increased when extracellular calcium was elevated, but it decreased with increasing extracellular EGTA. These data thus provide direct evidence for the operation of an in vivo transduction sequence involving red light-dependent, calcium-sensitive protein phosphorylation.     

Identification of a tyrosine residue in rat guanidinoacetate methyltransferase that is photolabeled with S-adenosyl-L-methionine.

Exposure of rat guanidinoacetate methyltransferase to ultraviolet light in the presence of S-adenosyl-L-[methyl-3H]methionine ([methyl-3H]AdoMet) results in covalent linking of radioactivity to the enzyme protein. The incorporation of radioactivity shows no lag and is linear with respect to time up to 1 h. The photolabeling is saturable with [methyl-3H]AdoMet, and the binding constant of the enzyme for AdoMet determined in this experiment is similar to that obtained by equilibrium dialysis. Low concentrations of competitive inhibitors S-adenosyl-L-homocysteine and sinefungin effectively prevent the photoinduced labeling by AdoMet. Although guanidinoacetate methyltransferase is irreversibly inactivated upon ultraviolet irradiation in the absence of AdoMet, the enzyme inactivated by 1-h exposure to ultraviolet irradiation has been shown to bind AdoMet with an affinity identical to that of the native enzyme. These results indicate that photolabeling occurs at the active site. Following proteolysis of the [methyl-3H]-AdoMet-labeled enzyme with chymotrypsin, a radioactive peptide is isolated having a sequence Asp-Thr-X-Pro-Leu-Ser-Glu-Glu-Thr-Trp. The peptide corresponds to residues 134-143, with X being modified Tyr-136. The same peptide is photolabeled when [carboxy-14C]AdoMet is used. High-performance liquid chromatography of this peptide after acid hydrolysis and phenyl isothiocyanate derivatization suggests that the entire molecule of AdoMet is attached to Tyr-136.     

Merozoite surface protein 2 of Plasmodium falciparum: expression, structure, dynamics, and fibril formation of the conserved N-terminal domain

Merozoite surface protein 2 (MSP2) is a GPI-anchored protein on the surface of the merozoite stage of the malaria parasite Plasmodium falciparum. It is largely disordered in solution, but has a propensity to form amyloid-like fibrils under physiological conditions. The N-terminal conserved region (MSP2(1-25)) is part of the protease-resistant core of these fibrils. To investigate the structure and dynamics of this region, its ability to form fibrils, and the role of individual residues in these properties, we have developed a bacterial expression system that yields > or =10 mg of unlabeled or (15)N-labeled peptide per litre of culture. Two recombinant versions of MSP2(1-25), wild-type and a Y7A/Y16A mutant, have been produced. Detailed conformational analysis of the wild-type peptide and backbone (15)N relaxation data indicated that it contains beta-turn and nascent helical structures in the central and C-terminal regions. Residues 6-21 represent the most ordered region of the structure, although there is some flexibility around residues 8 and 9. The 10-residue sequence (MSP2(7-16)) (with two Tyr residues) was predicted to have a higher propensity for beta-aggregation than the 8-mer sequence (MSP2(8-15)), but there was no significant difference in conformation between MSP2(1-25) and [Y7A,Y16A]MSP2(1-25) and the rate of fibril formation was only slightly slower in the mutant. The peptide expression system described here will facilitate further mutational analyses to define the roles of individual residues in transient structural elements and fibril formation, and thus contribute to the further development of MSP2 as a malaria vaccine candidate.