The cellular immune response to synthetic peptides containing sequences known to be heptenic in performic acid-oxidized ferredoxin from Clostridium pasteurianum

The NH₂-terminal heptapeptide and the COOH-terminal pentapeptide of performic acid-oxidized ferredoxin from Clostridium pasteurianum have been shown to encompass the two major haptenic regions of this molecule. These peptides were conjugated to succinylated bovine serum albumin (S-BSA) to yield an immunologically bivalent hapten-carrier conjugate (N + C-S-BSA). Peptides were also synthesized which contained the NH₂-terminal and COOH-terminal haptenic peptides linked by a bridge of five amino acids (N-5-C), these two peptides linked by 10 amino acids (N-10-C), and one containing two COOH-terminal peptides linked by 12 amino acids (C-12-C). The ability of these preparations to elicit various immunological responses was tested. In O-Fd-sensitized guinea pigs, N + C-S-BSA, N-5-C, and N-10-C elicited immediate and delayed skin reactions; N-5-C and N-10-C inhibited the migration of macrophages; N + C-S-BSA and N-10-C stimulated the proliferation of lymphocytes from these sensitized animals, however, N-5-C and C-12-C did not. In animals sensitized to N + C-S-BSA, skin reactions were elicited by O-Fd, S-BSA, and the NH₂-and COOH-terminal peptides alone. In these animals, lymphocyte proliferation was stimulated significantly by either O-FD or S-BSA. The N-5-C peptide was found to be nonimmunogenic by the schedule used here. However, the N-10-C peptide was found to be strongly immunogenic, and, in animals sensitized to N-10-C, skin reactions and MIF were elicited by N-10-C and 0-Fd, and lymphocyte proliferation was stimulated by N-10-C and O-Fd, but not by C-12-C. The implications of these results in relation to the bicellular mechanism of the immune response are discussed.     

Effect of cyclic AMP on RNA and protein synthesis in Candida albicans.

Sequence analysis by the automated Edman degradation shows that dopamine $\text{β}$-hydroxylase (dopamine $\text{β}$-monooxygenase; EC 1.14.17.1) from bovine adrenal medulla contains equal amounts of NH₂-terminal alanine and serine residues. The sequence data are in agreement with the proposal that this enzyme consists of two types of polypeptide chains which are identical in the NH₂-terminal ends, except that one of the chains lack the NH₂-terminal tripeptide Ser-Ala-Thr.     

Initiation of ovalbumin synthesis in chicken oviduct magnum: Transient labeling of the NH2-terminus by methionine

The incorporation of [³⁵S]methionine into ovalbumin, a protein containing NH₂-terminal N-acetylglycine, has been studied in chicken oviduct magnum cells. The purification of [³⁵S]methionine-labeled ovalbumin from total oviduct proteins was accomplished by dialysis of a crude extract at pH 3.6 followed by chromatography on carboxymethyl cellulose. The radioactive ovalbumin eluted from the column in three peaks (P₀, P₁, and P₂-containing 0, 1, and 2 moles of phosphate, respectively, per mole of ovalbumin). The kinetics of labeling of peaks P₀ and P₁ showed that the ratio of radioactivity in NH₂-terminal methionine to total incorporation was greater at 2 min of labeling than at later times. The transient labeling of the NH₂-terminus of ovalbumin with methionine indicates that methionine is the initiator amino acid for the synthesis of this protein, which in its mature form contains NH₂-terminal N-acetylglycine.     

NH2-terminal analysis of Wolfgram and Folch-Lees proteolipid proteins

Abstract— The NH₂-terminal amino acids of Wolfgram and Folch-Lees proteolipids of bovine and human CNS myelin were determined using the cyanate method (Starke & Smyth , 1963) followed by direct amino acid analysis of the products. Glycine predominated in every case and was recovered in amounts similar to the results described by Whikehart & Lees (1973), who used a dansylation technique followed by thin layer chromatography of the DNS-amino acids. In the present study substantial amounts of glutamic acid, serine, alanine and aspartic acid were also recovered, plus traces of other amino acids. Few differences were observed between Wolfgram and Folch-Lees proteolipids. The end group products of purified W1 proteolipid of bovine Wolfgram fraction, of diazometholysed Folch-Lees proteolipid, and of a sample of phosphatidyl serine had essentially the same composition. The similarity of these results, especially for both fractionated and unfractionated Wolfgram proteolipid, may be evidence that the observed products are derived from phosphoglycerides present in proteolipid rather than from the actual NH₂-terminals of the protein.     

Human pituitary growth hormone: immunochemical investigations of biologically active fragments

Guinea pig antisera to human growth hormone were tested for their ability to recognize the two biologically active fragments of the hormone produced by human plasmin digestion and a synthetic active fragment. A precipitin line was obtained with native human growth hormone, plasmin-treated human growth hormone, and its NH₂-terminal fragment (residues 1–134). In the microcomplement-fixation and radioimmunoassay experiments, the NH₂-terminal plasmin fragment (residues 1–134) showed a greater immunoreactivity than the COOH-terminal plasmin fragment (residues 141–191). This, in turn, was more active than the synthetic fragment (residues 95–136).     

Response of human and chick kidney adenylate cyclase to biologically active synthetic fragments of human parathyroid hormone

The 34-amino acid NH₂-terminal fragment of human parathyroid hormone synthesized according to the sequence described by Niall $\text{et al.}$ (1) is approximately 140 times more potent than the fragment synthesized according to Brewer $\text{et al}$ (2) in activating human renal cortex adenylate cyclase. The potencies of the two peptides, relative to the effect of MRC standard bovine parathyroid hormone preparation $\text{67}\text{342}$ in this system, were 5600 ± 600 (S.E.M.) units/mg and 40 ± 5 units/mg respectively. The potencies of the more active peptide and the corresponding bovine parathyroid hormone sequence were similar in this system and also in assays based upon the production of cyclic AMP by chick kidney both $\text{in vivo}$ and $\text{in vitro}$.     

Covalent modification of the solubilized rat liver vitamin K-dependent carboxylase with pyridoxal-5′-phosphate

The carboxylation of the pentapeptide substrate, Phe-Leu-Glu-Glu-Ile, by a rat microsomal vitamin K-dependent carboxylase was stimulated two- to threefold at pyridoxal-5′-P concentrations between 0.5 and 1.0 mm. This stimulation was reduced at concentrations higher than 1.0 mm. The K_m for the pentapeptide was lowered twofold in the presence of 1 mm pyridoxal-5′-P. The activation by pyridoxal-5′-P is specific, as 1 mm pyridoxal, pyridoxine, pyridoxine-5′-P, pyridoxamine, pyridoxamine-5′-P, or 4-pyridoxic acid did not stimulate the pentapeptide carboxylation rate. All six analogs, as well as formaldehyde and acetaldehyde, inhibited the carboxylation reaction in a concentration-dependent manner. The activation of the carboxylase by pyridoxal-5′-P appeared to be mediated by its direct binding to the enzyme via Schiff base formation. Sodium borohydride reduction of solubilized microsomes in the presence of pyridoxal-5′-P, followed by dialysis to remove unbound material, resulted in a carboxylase preparation with a specific activity twice that of the untreated control microsomes. The derivatized enzyme was not further stimulated by added pyridoxal-5′-P. This derivatized carboxylase could be obtained in the absence of pentapeptide and divalent cations. The stimulation of the carboxylase activity by divalent cations and pyridoxal-5′-P was mediated at separate site(s) on the enzyme. Studies of the NH₂-terminal pyridoxalated pentapeptide with both a normal and PLP-modified enzyme, in the presence and absence of PLP, demonstrated competition of the pentapeptide PLP moiety to a PLP site on the enzyme. It was concluded that pyridoxal-5′-P forms a covalent attachment to an ?-NH₂ of a lysine near the active site of the carboxylase.     

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) localizes to endoplasmic reticulum not peroxisomes, and NH2-terminal hydrophobic property determines the subcellular localization of ABC subfamily D proteins

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) is a member of ATP-binding cassette (ABC) protein subfamily D. ABC subfamily D proteins are also known as peroxisomal ABC proteins. Therefore, P70R is thought to be a peroxisomal membrane protein. However, the subcellular localization of P70R is not extensively investigated. In this study, we transiently expressed P70R in fusion with HA (P70R-HA) in CHO cells and examined subcellular localization by immunofluorescence. Surprisingly, P70R-HA was localized to the endoplasmic reticulum (ER), not to peroxisomes. To examine the ER-targeting property of P70R, we expressed various NH₂-terminal deletion constructs of P70R. Among the NH₂-terminal deletion constructs, mutant proteins starting with hydrophobic transmembrane segment (TMS) were localized to ER, but the ones containing the NH₂-terminal hydrophilic cytosolic domain were not. ABC subfamily D proteins destined for peroxisomes have NH₂-terminal hydrophilic region adjacent to TMS1. However, only P70R lacks the region and is translated with NH₂-terminal hydrophobic TMS1. Furthermore, attachment of the NH₂-terminal hydrophilic domain to the NH₂-terminus of P70R excluded P70R from the ER-targeting pathway. These data suggest that P70R resides in the ER but not the peroxisomal membranes, and the hydrophobic property of NH₂-terminal region determines the subcellular localization of ABC subfamily D proteins.     

Amino Acid Sequence Analysis of Reverse Transcriptase Subunits from Avian Myeloblastosis Virus

The NH₂-terminal amino acid sequences of the α and β chains of avian myeloblastosis αβ DNA polymerase were determined by using microsequence analysis in the subnanomole range and were found to be identical up to 17 residues. The common sequence was as follows: Thr-Val-Ala-Leu-His-Leu-Ala-Ile-Pro-Leu-Lys-Trp-Lys-Pro-Asn-His-Thr-. This result provides convincing chemical evidence that the α chain is derived from the NH₂-terminal region of the β chain by proteolytic cleavage, whereas the amino acid composition for these α and β subunits and p32 DNA endonuclease suggests that the latter is derived from the carboxyl-terminal region of the β chain.     

Prohormone Thiol Protease and Enkephalin Precursor Processing: Cleavage at Dibasic and Monobasic Sites

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys-Arg, Arg-Arg, and Lys-Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin-containing peptides, peptide E and BAM-22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM-22P at the Lys-Arg site between the two basic residues. The Arg-Arg site of both peptide E and BAM-22P was cleaved at the NH₂-terminal side of the paired basic residues to generate [Met]-enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH₂-terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys-Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH₂-terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.     

Structural evidence for a liver-specific glyceraldehyde-3-phosphate dehydrogenase.

The amino acid sequences near the amino termini of glyceraldehyde-3-phosphate dehydrogenase from bovine and porcine liver have been determined. Using classical peptide isolation techniques as well as automated Edman degradation, the NH₂-terminal 30 residues of the bovine liver enzyme were determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Asn-Ser-Gly-Lys-Val-Asp-Ile-Val-Phe-Ile. Twenty-two residues from the NH₂-terminus of the porcine liver enzyme, determined using the automated Edman degradation, were identical to the corresponding sequence from bovine liver enzyme. Both liver enzymes have Asn at position 6. The corresponding residue 6 in the muscle and yeast glyceraldehyde-3-phosphate dehydrogenases is Asp. This evidence suggests that the Asn-6 residue is specific for the liver tissues. The exchange of Asn for Asp may significantly alter the allosteric properties of muscle and liver enzymes especially the activity of the liver enzymes in gluconeogenesis.     

Some factors that influence the nonenzymatic glycation of peptides and polypeptides by glyceraldehyde

The rate of reaction of glyceraldehyde with a series of peptides was found to be dependent on their amino acid composition, sequence, and chain length. The presence of a histidine near the NH₂-terminal increased the rate of glycation, whereas the presence of a carboxyl group near the reaction site led to a decrease in reaction rate. In general, tripeptides reacted faster than dipeptides, and dipeptides reacted faster than amino acids. Sodium phosphate and 2,3-diphosphoglycerate enhanced the rate of reaction of glyceraldehyde with all the dipeptides tested. Sodium chloride inhibited the reaction in phosphate buffer, but not in HEPES buffer. The NH₂-terminal heptapeptide from the -chain of human hemoglobin A (HbA), where histidine is the second residue, reacted with glyceraldehyde faster than the NH₂-terminal hexapeptide from the -chain. The glycation of tetrameric human Hb by glyceraldehyde was found to be dependent on the ligation state of the protein since deoxy-HbA reacted about 50% more with glyceraldehyde than did liganded HbA. The enhanced glycation of deoxy HbA was mainly attributable to the more extensive reaction at the NH₂-terminal of the -chain. The presence of a histidine adjacent to the NH₂-terminal at this site may facilitate the Amadori rearrangement. The glycation of horse Hb in which the second residue is glutamine was not increased under anaerobic conditions.     

A new method of coupling proteins to insoluble polymers

The A chain isolated from biologically active human thrombin is thirteen residues shorter from the amino terminus than the A chain isolated from bovine thrombin. Automated Edman degradation of the reduced, alkylated A chain permitted the direct identification of 34 out of the 36 residues and established all tryptic overlaps. The NH₂-terminal residue is threonine. This residue is homologous to threonine-14 of the A chain of bovine thrombin. Human thrombin A chain has one half cystine linking it to the larger B chain. Methionine, histidine, valine and tryptophan are not present. There are nine acid residues (Glu, Asp) but none of their respective amides.     

Dimerization of Translationally Controlled Tumor Protein Is Essential For Its Cytokine-Like Activity

Background

Translationally Controlled Tumor Protein (TCTP) found in nasal lavage fluids of allergic patients was named IgE-dependent histamine-releasing factor (HRF). Human recombinant HRF (HrHRF) has been recently reported to be much less effective than HRF produced from activated mononuclear cells (HRFmn).

Methods and Findings

We found that only NH₂-terminal truncated, but not C-terminal truncated, TCTP shows cytokine releasing activity compared to full-length TCTP. Interestingly, only NH₂-terminal truncated TCTP, unlike full-length TCTP, forms dimers through intermolecular disulfide bonds. We tested the activity of dimerized full-length TCTP generated by fusing it to rabbit Fc region. The untruncated-full length protein (Fc-HrTCTP) was more active than HrTCTP in BEAS-2B cells, suggesting that dimerization of TCTP, rather than truncation, is essential for the activation of TCTP in allergic responses. We used confocal microscopy to evaluate the affinity of TCTPs to its putative receptor. We detected stronger fluorescence in the plasma membrane of BEAS-2B cells incubated with Del-N11TCTP than those incubated with rat recombinant TCTP (RrTCTP). Allergenic activity of Del-N11TCTP prompted us to see whether the NH₂-terminal truncated TCTP can induce allergic airway inflammation in vivo. While RrTCTP had no influence on airway inflammation, Del-N11TCTP increased goblet cell hyperplasia in both lung and rhinal cavity. The dimerized protein was found in sera from allergic patients, and bronchoalveolar lavage fluids from airway inflamed mice.

Conclusions

Dimerization of TCTP seems to be essential for its cytokine-like activity. Our study has potential to enhance the understanding of pathogenesis of allergic disease and provide a target for allergic drug development.     

Isolation,structure and biologic activity of chicken intestinal neurotensin

Using a radioimmunoassay towards bovine neurotensin (NT), chicken NT has been purified to homogeneity from extracts of intestine and its amino acid sequence determined to be: <Glu-Leu-His-Val-Asn-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu-OH. The molecule is identical to the bovine peptide except for the 3 amino acid substitutions located in its NH₂-terminal half and italicized above (His/Tyr; Val/Glu; Ala/Pro). The structure for chicken NT is consistent with earlier immunochemical studies which indicated a COOH-terminal homology with bovine NT [1]. The peptide isolated was shown to be near equipotent with bovine NT in its ability to induce hypotension, hyperglycemia, and cyanosis in the anesthesized rat, underscoring the importance of the COOH-terminal residues in NT for biological activity.     

Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells

Summary The aspartic protease gene of a zygomycete fungus Mucor pusillus was expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter. A putative preproenzyme with an NH₂-terminal extension of 66 amino acids directed by the gene was processed in yeast cells and the mature enzyme, whose NH₂-terminus was identical to that of the Mucor enzyme, was efficiently secreted into the medium at a concentration exceeding 150 mg/l. The enzyme secreted from the recombinant yeast was more glycosylated than the native Mucor enzyme but its enzymatic properties were almost identical with those of the native enzyme, which has been used as a milk coagulant in cheese manufacture.     

Aspartate transaminase from E. coli: amino acid sequences of the NH2-terminal 33 residues and chymotryptic pyridoxyl tetrapeptide.

The amino acid sequences of pyridoxal-binding tetrapeptide and the NH₂-terminal portion of aspartate transaminase from $\text{E.}$$\text{coli}$ B were analyzed and compared with those of the corresponding parts of the cytosolic and mitochondrial isozymes from pig heart. After borohydride reduction and chymotryptic digestion of the $\text{E.}$$\text{coli}$ enzyme, a pyridoxal-containing peptide was isolated, showing the sequence, Ser-Lys(Pxy)-Asn-Phe, identical with that of the cytosolic isozyme. The NH₂-terminal sequence was determined up to 33 residues with a liquid phase sequence analyzer. Nearly the same degree of homology was observed among the NH₂-terminal sequences of the three aspartate transaminases.     

Enzymatic Probe for Accessibility of NH<Subscript>2</Subscript>-Terminal Residues in Immunoglobulins

ARGINYL-tRNA-protein transferase is a soluble enzyme from mammalian tissues which catalyses the transfer of arginine from arginyl-tRNA into peptide linkage specifically with NH₂-terminal aspartic or glutamic acid residues of protein acceptors^1,2. Molar equivalents of arginine are transferred to appropriate NH₂-terminal residues². The reaction differs in many respects from the transfer of amino-acids associated with protein synthesis de novo and is thought to be a mechanism for regulating the activity of acceptor proteins³. Many immunoglobulins possess aspartic or glutamic acid in the NH₂-terminal position and it was interesting to examine whether arginylation of such residues might result in an alteration of activity of these proteins, for there is considerable evidence that the antibody-combining site is contained in the NH₂-terminal region of both light^4–7 and heavy^8,9 chains.     

Specific cleavage of the native type III collagen molecule with trypsin. Similarity of the cleavage products to collagenase-produced fragments and primary structure at the cleavage site.

Solutions of native Type III collagen (chain composition, [α1(III)]₃) exhibit a rapid and dramatic decrease in relative viscosity when incubated with trypsin. Cleavage products of the reaction were precipitated with ammonium sulfate and isolated in denatured form by molecular sieve chromatography. They were found to be comprised of: α1(III)-T1 (molecular weight, 71,000) derived from the NH₂-terminal portion of the Type III molecule; and α1(III)-T2 (molecular weight, 24,000) from the COOH-terminal portion of the molecule. Determination of the amino acid sequence at the NH₂-terminal portion of α1(III)-T2 as well as at the COOH-terminus of α(III)-T1 demonstrated that the products arose from specific cleavage of the type III molecule at an arginine-glycine bond corresponding to residues 780–781 in the repetitive triplet sequence of the α1(III) chain. The results suggest that the trypsin-susceptible bond in the native Type III collagen molecule resides in a region characterized by a relative lack of the normal collagen helicity.     

Conformational studies on substance P. C-terminal pentapeptide pGlu-Phe-Phe-Gly-Leu-Met NH2

The conformational behavior of the active C-terminal pentapeptide of substance P(SP), pGlu-Phe-Phe-Gly-Leu-Met NH₂ [pGlu-SP(7–11)] was investigated using empirical energy calculations. A sequential approach was used to display the specific contribution of each residue to induce stable conformations of the whole pentapeptide. The most stable conformations include the α_R helix and some partially helical structures; some conformations with glycyl residue in a C₇^eq and C₇^ax configurations (γ and $\text{γ}$ turns) are also favoured. Helical conformations provide a good accessibility of side-chains which play an important role in interacting with the receptor. Fully extended structures and β turns are not specially stable. Such helical stable structures would favour a “lock and key” model of binding.