The circumsporozoite (CS) protein of P. falciparum contains an immunodominant epitope, NADP, that is repeated 37 times in the native molecule. The presence of proline in the coat proteins of the Plasmodium parasite at various developmental stages and strains is a frequent occurrence. In this study we evaluate the influence of substitution of proline residues by glycine on the immunogenic behavior of two tandemly repeated peptides linked via glutaraldehyde to a protein carrier: The (NANP)4 P. falciparum circumsporozoite peptide and its glycine-substitute analog, (NANG)4. The results obtained show that the (NANP)4 induces antibodies which recognize the peptide free in solution, bound on a solid phase, and linked to a carrier protein. It has been previously reported that such antibodies recognize the antigenic sites of the peptide in the native protein on the surface of the sporozoite. Antibodies raised against (NANG)4 in the same experimental conditions as (NANP)4, cannot recognize the peptide free in solution or bound to the solid phase. However, these antibodies can react with the peptide when it is linked to a protein carrier. The coupling of a glycine-containing analog to a carrier results in a significant shift in its conformation, allowing it to be recognized by the antibodies.

Abstract: A peptide corresponding to position 32‐47 in tyrosine hydroxylase was synthesized (TH‐16) and polyclonal antibodies against this peptide were raised in rabbits (anti‐TH‐16). The effects of anti‐TH‐16 on modulation of tyrosine hydroxylase activity were investigated. Anti‐TH‐16 enhanced the enzymatic activity in a concentration‐dependent manner, and the antigen TH‐16 inhibited the stimulatory activity of the antiserum in a concentration‐dependent manner. The activated enzyme had a lower Kmapp for the cofactor 2‐amino‐4‐hydroxy‐6‐methyl‐5,6,7,8‐tetrahydropterin and a higher Vmax app than the nonactivated enzyme. Anti‐TH‐16 was characterized further by its ability to immunoprecipitate the enzyme activity by labeling tyrosine hydroxylase after Western blotting and by immunohistochemical labeling of cate‐cholaminergic neurons. Anti‐TH‐16 did not block activation of tyrosine hydroxylase by phosphorylation catalyzed by cyclic AMP‐dependent protein kinase. Exposure of the enzyme to anti‐TH‐16 and subsequent phosphorylation of the enzyme resulted in a greater activation of the enzyme than the sum of activation produced by these two treatments separately. However, the activation was less than additive when the enzyme was first phosphorylated and subsequently exposed to anti‐TH‐16. The present study demonstrates the utility of anti‐TH‐16 in investigating the molecular aspects of the enzyme activation.

Decay-accelerating factor (DAF) from human red cell membranes was purified by a two-step procedure involving anion exchange and immunoaffinity chromatography. The DAF preparations were purified to homogeneity as judged by silver staining. In several experiments, the final product yields were ∼23% of the total DAF present in the initial membrane extracts. The purified DAF retained its ability to inhibit the classical pathway C3-convertase and to reincorporate into cell membranes. An amino-terminal sequence was obtained by gas-phase sequencing. Rabbit antibodies to a synthetic peptide representing part of this sequence reacted with purified reduced membrane DAF by Western blotting and by a solid-phase immunoradiometric assay.

Four synthetic peptides that copy fragments of two bacterial antigens (Streptococcus pyogenes M protein and diphtheria toxin), one viral antigen (hepatitis B surface antigen), and one parasitic antigen (circumsporozoite protein of Plasmodium knowlesi) were covalently bound within the same construct. This totally synthetic polyvalent administered to mice with Freund complete adjuvant or in saline with murabutide (an adjuvant-active muramyl peptide) elicited high levels of antibodies which, in certain cases, were shown to be biologically active. The results indicated that these antibodies recognized specifically the four peptides. None of the epitopes were immunodominant. It was also demonstrated that the association of several peptides enhanced their respective immunogenicities as compared with those of their homopolymers. Finally, this study shows that a totally synthetic vaccine administered in saline with a synthetic adjuvant can be immunogenic in the absence of a protein carrier.

Ten polypeptides that stimulated the release of corticotropin from superfused rat pituitary cells and that are structurally related to porcine corticotropin-releasing factor were isolated from porcine hypothalami. The purification was carried out by gel filtration followed by reversed-phase HPLC using trifluoroacetic acid or heptafluorobutyric acid as the ion-pairing agent in water/acetonitrile solvent systems. The purified peptides were homogeneous by chromatography and by sequence analysis. One major polypeptide was characterized. Its structure is -H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Gl u-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2 [Patthy, M., Horvath, J., Mason-Garcia, M., Szoke, B., Schlesinger, D. H. & Schally, A. V. (1985) Proc. Natl. Acad. Sci. USA 82, 8762-8766]. This 41-amino acid sequence is thought to represent porcine corticotropin-releasing factor. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptides, amino acid analysis, and carboxypeptidase Y digestion, the other nine polypeptides were found to be structurally similar to this 41-amino acid sequence. Modifications of this structure include deamidation of glutamine at position 26 or 29, oxidation of methionine at positions 21 and/or 38, a blocked N terminus, and deletion of phenylalanine amide at the C terminus. Eight of these nine modified peptides retained significant corticotropin-releasing factor activity as shown by the stimulation of corticotropin release from superfused rat and pig pituitary cells. Some of these peptides may be present in pig hypothalami, while the others could have been produced during the isolation.

Rat brain and adrenal gland were analyzed by hybridization histochemistry using an RNA probe complementary to mRNA for tyrosine 3-hydroxylase (TyrOHase; tyrosine 3-monooxygenase, EC 1.14.16.2), by immunohistochemistry using TyrOHase antiserum, and by retrograde tracing using the fluorescent compound Fast blue. Cell bodies in the ventral mesencephalon contained mRNA for TyrOHase, and these cells were also TyrOHase immunoreactive. After injection of Fast blue into the striatum, such double-labeled cells in addition contained the retrograde tracer, showing that these cells send axonal projections to the injection site. These results show that hybridization histochemistry can be used to identify transmitter-specific neuron populations and that their projections can be established.

A synthetic peptide whose sequence was derived from the circumsporozoite protein of Plasmodium knowlesi coupled to bovine gamma globulin has been shown to be immunogenic when administered with Freund complete adjuvant. The present experiments were designed to test the immunogenicity of the peptide when attached to a tetanus toxoid carrier and administered with alum or murabutide, both acceptable clinical adjuvants. In both cases, the use of an adjuvant increased the levels of circulating anti-peptide antibodies over those observed when no adjuvant was used. However, when the antisera were tested for reactivity with the native protein, animals of the group receiving the conjugate associated with murabutide always had titers greatly exceeding those observed in animals that received the conjugate with alum. Moreover, the sera of the murabutide-treated group were shown to be more active in eliciting shedding of the circumsporozoite protein than were sera of animals of the Freund complete adjuvant-treated group. The use of tetanus toxoid in secondary immunizations could be eliminated when the mice primed with peptide-tetanus toxoid and murabutide were boosted with a polymer of the peptide. The results indicate that the synthetic malarial peptide-tetanus toxoid conjugate is capable of stimulating high levels of biologically active antibodies only when administered with murabutide.

A polypeptide was isolated from acid extracts of porcine hypothalami on the basis of its high ability to stimulate the release of corticotropin from superfused rat pituitary cells. After an initial separation by gel filtration on Sephadex G-25, further purification was carried out by reversed-phase HPLC. The isolated material was homogeneous chromatographically and by N-terminal sequencing. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptide and on carboxypeptidase Y digestion, the primary structure of this 41-residue polypeptide was determined to be Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2. Porcine corticotropin-releasing factor (CRF) shares a common amino acid sequence (residues 1-39) with rat and human CRF and differs from these only in positions 40 and 41. However, isoleucine was also present at position 40 in porcine CRF, but in a smaller percentage than asparagine. The sequence of porcine CRF shows 83% homology with ovine CRF. Porcine CRF markedly stimulated the release of corticotropin from superfused rat and pig pituitary cells. The biological activity and close structural relationship to CRFs of other species indicate that the peptide isolated represents porcine CRF.

Antibodies against a synthetic peptide representing the repetitive epitope of the circumsporozoite protein (CS) of Plasmodium knowlesi have properties similar to those of antibodies against the native protein. Either antibody reacts with the synthetic peptide, cross-links the CS protein on the membrane of the parasite giving the CSP reaction, and neutralizes the infectivity of sporozoites. The synthetic peptide and sporozoite extracts were equally effective when used in an immunoradiometric assay as antigens to detect antibodies to CS proteins. It is likely that the corresponding synthetic repeats from the human malaria parasites could be used to measure levels of anti-sporozoite antibodies in endemic areas, or to evaluate the humoral response to anti-sporozoite vaccines. The authors are grateful to Dr. Robert Gwadz, NIH, for supplying Anopheles mosquitoes and P. knowlesi sporozoites used in this study.

Previous studies have shown that the immunodominant region of the circumsporozoite surface (CS) protein of Plasmodium knowlesi is contained within a tandemly repeated dodecapeptide: Gln-Ala-Gln-Gly-Asp-Gly-Ala-Asn-Ala-Gly-Gln-Pro. We show here that the CS protein epitopes reacting with six monoclonal antibodies raised against the intact parasite are represented in a synthetic tandem repeat of this dodecapeptide. The specificity of four of these antibodies was studied further by preparing synthetic peptides corresponding to overlapping regions of the repeats and measuring their ability to inhibit the specific interaction between the antibodies and CS proteins. We find that three antibodies have very similar patterns of reactivity with this series of peptides and that they define an epitope of eight amino acids (Gly-Asp-Gly-Ala-Asn-Ala-Gly-Gln) within the dodecapeptide. The remaining antibody probably recognizes a configurational epitope formed by a tandem repeat of the dodecapeptide.

Complementary DNA clones that code for the immunogenic region of the Plasmodium knowlesi circumsporozoite protein were shown to contain a tandemly repeating 36-base pair unit. A synthetic dodecapeptide corresponding to the predicted reading frame of the repeating nucleotide unit behaved, in an immunoradiometric assay, identically with the native P. knowlesi circumsporozoite protein. The repeating 36-base pair unit occurred 12 times within the gene and accounts for at least one-third of the amino acid sequence of the surface antigen protein.

Organic solvents can affect the efficiency of peptide sequencing by the Edman degradation method by altering peptide extractive losses during manual sequence analysis. We present a modified phenylisothiocyanate procedure for the degradation of one to five peptides simultaneously with high repetitive yield (90–95%) with an average time per cycle of 75 min. Improvement in average yield per cycle (repetitive yield) varies with the choice of solvent and nature of peptide under investigation. The degree of extraction of a particular thiazolinone similarly can be improved by the selection of appropriate solvents.

A modified procedure for the purification of E. coli galactose-1-phosphate uridyl transferase (E.C. 2.7.6.12) was developed which reproducibly gives pure enzyme. The purified enzyme was shown to be a dimeric protein with a subunit molecular weight of 41,000 and its amino acid composition and content of free sulfhydryl groups were determined. The N-terminal and C-terminal amino acid sequences were found to be NH2-thr-gln-phe-asn-pro-val-asp and -ser(val leu)-ala-COOH respectively. This N-terminal sequence allowed the identification of the start of the transferase gene in the DNA sequence determined by GRINDLEY. Furthermore it appears to define a nine base intercistronic region between the epimerase and transferase genes.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCharacterization of a major fibroblast cell surface glycoproteinKenneth M. Yamada, David H. Schlesinger, Dorothy W. Kennedy, and Ira PastanCite this: Biochemistry 1977, 16, 25, 5552–5559Publication Date (Print):December 13, 1977Publication History Published online1 May 2002Published inissue 13 December 1977https://pubs.acs.org/doi/10.1021/bi00644a025https://doi.org/10.1021/bi00644a025research-articleACS PublicationsRequest reuse permissionsArticle Views70Altmetric-Citations138LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

FEBS LettersVolume 83, Issue 1 p. 45-47 Full-length articleFree Access The NH2-terminal sequences of galactokinase from Escherichia coli and Saccharomyces cerevisiae David H. Schlesinger, David H. Schlesinger Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 USA Department of Biochemistry, Cornell University, Ithaca, New York 14853, USASearch for more papers by this authorMark A. Schell, Mark A. Schell Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 USA Department of Biochemistry, Cornell University, Ithaca, New York 14853, USASearch for more papers by this authorDavid B. Wilson, David B. Wilson Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 USASearch for more papers by this author David H. Schlesinger, David H. Schlesinger Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 USA Department of Biochemistry, Cornell University, Ithaca, New York 14853, USASearch for more papers by this authorMark A. Schell, Mark A. Schell Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 USA Department of Biochemistry, Cornell University, Ithaca, New York 14853, USASearch for more papers by this authorDavid B. Wilson, David B. Wilson Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 USASearch for more papers by this author First published: November 01, 1977 https://doi.org/10.1016/0014-5793(77)80638-8Citations: 9AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume83, Issue1November 01, 1977Pages 45-47 ReferencesRelatedInformation

The structure of a growth-modulating tripeptide from human serum and plasma has been determined to be H-glycyl-histidyl-lysine-OH.