Proline Residue In Position Research Articles

Apolipoprotein A-I variants. Naturally occurring substitutions of proline residues affect plasma concentration of apolipoprotein A-I.

Six unrelated families with genetically determined structural variants of apo A-I were found in the course of an electrophoretic screening program for apo A-I variants in dried blood samples of newborns. The following structural variations were identified by the combined use of HPLC, time-of-flight secondary ion mass spectrometry (TOF-SIMS), and automated gas phase sequencing: Pro3----Arg (1x), Pro4----Arg (1x), and Pro165----Arg (4x). All variant carriers were heterozygous for their mutant of apo A-I. Subjects heterozygous for apo A-I(Pro165----Arg) (n = 12) were found to exhibit lower mean values for apo A-I (109 +/- 16 mg/dl) and HDL cholesterol (37 +/- 9 mg/dl) than unaffected family members (n = 9): 176 +/- 41 and 64 +/- 18 mg/dl, respectively (P less than 0.001). In 9 of 12 apo A-I(Pro165----Arg) variant carriers the concentrations of apo A-I were below the fifth percentile of sex-matched controls. By two-dimensional immunoelectrophoresis as well as by densitometry the relative concentration of the variant apo A-I in heterozygous carriers of apo A-I(Pro165----Arg) was determined to account for only 30% of the total plasma apo A-I mass instead of the expected 50%. Thus, the observed apo A-I deficiency may be largely a consequence of the decreased concentration of the variant apo A-I. In the case of the apo A-I(Pro3----Arg) mutant, densitometry of HDL apolipoproteins demonstrated a distinctly increased concentration of the variant proapo A-I relative to normal proapo A-I. This phenomenon was not observed in the apo A-I(Pro4----Arg) mutant or in other mutants. This suggests that the interspecies conserved proline residue in position 3 of mature apo A-I is functionally important for the regular enzymatic conversion of proapo A-I to mature apo A-I.

Efficient production of native, biologically active human cystatin C by Escherichia coli

A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the λ P R promoter, an optimized Shine-Dalgarno sequence and the λ cI 857 repressor. When induced at 42°C, such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized. All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated. The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.

Circular dichroism studies of some arginine-vasopressin analogues.

CD spectra of arginine-vasopressin (AVP) and of its analogues substituted in position 1 and/or 7 were measured in aqueous solution at different pH values. The shapes of the CD spectra of AVP analogues substituted in position 1 are strongly influenced by the type of group attached to the beta-carbon of residue 1. The substitution of the proline residues in position 7 by N-methylalanine also leads to a change in conformation of the peptide. The differences in the CD spectra are interpreted in terms of conformational changes, which are due to the interaction of the tyrosine side chain with neighbouring residues (for 1-substituted analogues of AVP), or to that between the hexapeptide ring and acyclic tripeptide chain (for 7-substituted analogues).

Regulation of fibroblast cyclooxygenase synthesis by interleukin-1.

We have prepared polyclonal antiserum against sheep seminal vesicle prostaglandin H synthase (also termed cyclooxygenase) which cross-reacted with human cyclooxygenase, thereby enabling us to directly determine the synthetic rate of cyclooxygenase protein and its modulation by the monokine interleukin-1 (IL-1). Cultured human dermal fibroblast cells were labeled with [35S]methionine, and the membrane-bound cyclooxygenase was solubilized and immunoprecipitated 35S-labeled fibroblast cyclooxygenase migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular size of approximately 73,000 daltons, similar to that of native sheep cyclooxygenase and of cyclooxygenase covalently labeled by [3H]aspirin, i.e. [3H]acetylcyclooxygenase. Additional validation of the immunoprecipitated 35S-labeled cyclooxygenase band indicated that it was specifically displaced by unlabeled sheep cyclooxygenase. N-terminal amino acid radiosequence analysis of [3H]proline-labeled cyclooxygenase revealed [3H]proline residues in positions 3, 6, and 8, consistent with the previously reported N-terminal sequence of sheep cyclooxygenase. Endoglycosidase H treatment of 35S-labeled fibroblast cyclooxygenase caused a decline in apparent molecular size (due to removal of mannose residues) which was similar to that seen with the native sheep cyclooxygenase. [35S]Methionine pulse-chase experiments indicated a half-life of 1 h for fibroblast cyclooxygenase. The monokine interleukin-1 stimulated fibroblast cyclooxygenase synthesis in a time- and dose-dependent fashion; as little as 0.03 unit/ml of IL-1 produced significant stimulation of 35S-labeled cyclooxygenase synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with 0.3 unit/ml of IL-1 for 12-16 h. IL-1 treatment of cells yielded parallel dose-response curves for stimulation of prostaglandin E2 formation, increased cellular cyclooxygenase activity, and increased synthetic rate of newly formed cyclooxygenase, suggesting that the IL-1 effect is mediated mainly, if not solely, via induction of cyclooxygenase synthesis.

Angiotensin analogues that selectively augment the force of contraction of the isolated heart.

Angiotensin II (Ang II) produces a positive inotropic effect on the heart; however, its usefulness as an inotropic agent is limited because of its inherent vasoconstrictor action. We therefore designed Ang II analogues that are potent, positive inotropic agents with minimal myotropic properties. Replacement of the proline residue in position 7 with alanine reduced the pressor and vascular contractile response to less than 1% of Ang II. In spite of negligible vascular actions, however, [7-alanine]Ang II produced 50% of the inotropic activity of Ang II in the cat papillary muscle. The results of pharmacological evaluation of various position 7-substituted analogues were as follows: 1) Replacement of proline in position 7 of angiotensin I (Ang I) and Ang II with primary amino acids produced cardiac-specific, positive inotropic properties. 2) The selectivity of positive cardiac inotropic activity of position 7-substituted analogues of Ang II was dependent upon the nature of the amino acid in position 1. Replacement of aspartic acid in position 1 with sarcosine increased vasoconstrictor activity, thereby diminishing cardiac selectivity. However, this change did not affect cardiac selectivity in Ang I analogues. 3) Introduction of any type of steric hindrance in position 7 (e.g., replacement of alanine with N-methyl- or alpha-methylalanine) led to a considerable loss in inotropic activity. In conclusion, contrary to rigid, structural requirements (solution conformation) for the pressor action of Ang II, a less organized structure or a random conformation at the carboxyl terminus appears to favor cardiac-selective contractile response (or positive inotropic response).

Synthesis and some pharmacological properties of oxytocin and vasopressin analogues with sarcosine or N-methyl-L-alanine in position 7.

Eight analogues of oxytocin and arginine-vasopressin were synthesized, in which the proline residue in position 7 was replaced by either sarcosine or N-methylalanine; some of the pharmacological properties of these analogues were evaluated. In peptides containing a beta-mercaptopropionic acid residue in position 1, the additivity of the effects of deletion of the amino group in position 1 and of the above-noted replacements in position 7 on biological properties of these analogues was ascertained. All of the analogues were found to be potent in either antidiuretic or uterine activity and also selective in action. From the point of view of pharmacological properties, substitution of sarcosine in position 7 of oxytocin gave analogues with higher oxytocic and milk-ejecting activities than did the substitution of N-methylalanine. The opposite structure-activity relationship was observed with arginine-vasopressin, where the N-methylalanine-containing analogues were more potent than the sarcosine-containing analogues with respect to pressor activity and also, if not deaminated, with respect to antidiuretic activity.

Studies of the enzymatic degradation of β-casomorphins

β-Casomorphins (β-CMs), although known to be highly resistant to proteolytic enzymes, are demonstrated to be rapidly degraded in bovine or rat plasma. Degradation of these peptides consisting of the amino acid sequence TYR-PRO-PHE-GLY-PRO-ILE and C-terminally shortened fragments thereof, may be due to an enzyme identical with or similar to the dipeptidyl-peptidase IV (DP IV) which is known to cleave dipeptide fragments from the N-terminus of peptides after proline residues. This assumption is compatible with the finding that β-casomorphin (β-CM) analogues in which the proline residue in position two has been replaced by D-alanine, seem to be completely resistant to enzymatic attack in the plasma.

Immunochemistry of cytochrome c. Identification of antigenic determinants through the study of hybrid molecules.

Antibodies elicited in rabbits against horse cytochrome c cross-react in varying degree with the cytochromes c of other species. Radioimmunoassay experiments are described in which 23% of the immunoglobulin shown to bind to horse cytochrome c fails to react with beef cytochrome c, and 14% of that bound by beef cytochrome c does not react with rabbit cytochrome c. Studies of hybrid cytochromes, prepared by a reconstitution procedure involving the cleavage and re-formation of peptide bond 65-66, show that these differences result from single amino acid replacements, Beef cytochrome c differs in binding capacity from horse cytochrome c as a result of the substitution of a glycine for a lysine residue in sequence position 60, and the difference between rabbit cytochrome c and beef cytochrome c reflects the substitution of a valine for a proline residue in position 44. Reconstituted horse cytochrome c and reconstituted beef cytochrome c have binding capacities indistinguishable from those of the parent proteins. The presence of a homoserine rather than a methionine residue in position 65 results, in the case of the reconstituted horse molecule, in a slightly lower affinity for the antibody population directed against the lysine-60 region. A corresponding difference is not observed in the case of beef cytochrome c and its reconstitution product, which do not bind the population in question.

NMR evidence for a type I β‐turn in (Pro2)‐tetrapeptides and interdependence of cis:Trans isomerism, ring flexibility, and backbone conformation

AbstractTetrapeptides with proline in position 2, asparagine or leucine in position 3, and glycine in positions 1 and 4, with end groups free or blocked on the N‐terminal side, were studied in their various ionic states in 2H2O and in Me2SO‐d6 by 1H‐ and 13C‐nmr. In order to clarify or refine some details, successive substitutions of the residues in these peptides with amino acids enriched to 85% in 13C, or to 85% 13C plus 97% 2H were carried out. The 1H and 13C chemical shifts as well as the 1H‐1H, 13C‐13C, and 13C‐1H coupling constants and the signal intensities show strong similarity of behavior between the tetrapeptides of asparagine and leucine. The main conformational characteristics are (1) the almost total stabilization of the trans conformer in the type I β‐turn structure when the peptide is in the zwitterion state dissolved in Me2SO. This is deduced from the 3J and the 3J coupling constants, which both furnish a dihedral angle of ϕ3 = −90°, and from the positive value of the temperature coefficient of the glycine‐4 amide protons, which suggests a type 4 → 1 hydrogen bond; (2) the evolution of cis and trans isomer fractions which change with the ionic state of the peptides in Me2SO, whereas they remain constant in aqueous solution; and (3) the conformation of the pyrrolidine ring as it follows the variations in cis:trans isomer populations together with the side‐chain rotamer fractions of the residue in position 3. In the β‐turn conformation the isomer cis is less abundant and the pyrrolidine ring is more flexible; this explains the perfect accommodation of the proline residue in position 2 of a bend. The interdependence of these phenomena where interactive forces play a predominant role underlines the importance of cooperative effects in the molecule. The results also suggest that the cis isomer of proline can adapt itself just as well as the trans isomer to position 2 of a type I β‐turn.

Antagonists of angiotensin II containing N-methyl-L-alanine and DL-nipecotic acid in position 7.

[1-sarcosine, 7-N-methyl-L-alanine, 8-isoleucine]-Angiotensin II and [1-sarcosine, 7-DL-nipecotic acid, 8-isoleucine]-angiotensin II were synthesized by the solid-phase method and purified by cation-exchange chromatography and high-pressure liquid chromatography. In the isolated rat uterus these analogs and less than 0.1% of the myotropic activity of angiotensin II and inhibited angiotensin II with pA2 values of 8.2 and 7.8, respectively. In the rat pressor assay (vagotomized ganglion blocked rat) these analogs had 0.9 and 2.8%, respectively, of the pressor activity of angiotensin II. The results show that the proline residue in position 7 of [Sar1,Ile8]-angiotensin II may be replaced by other secondary amino acids without disrupting interactions at angiotensin II receptors.

Inhibition effect of rosette formation between human lymphocytes and sheep erythrocytes by specific heptapeptide isolated from uremic fluid and its analogs

One of middle molecular substances (H-His-Pro-Ala-Glu-Asn-Gly-Lys-OH) and its two analogs, in which the proline residue in position 2 was replaced by glycine and the alanine residue in position 3 was replaced by valine exert a inhibition effect on in vitro E-rosette formation. Its synthetic two analogs showed diminished biological activity compared to native heptapeptide.

Immunological Aspects of Secretin. Substance P, and Vip

Secretin, substance P, and vasoactive intestinal peptide (VIP) were studied from the immunological point of view using synthetic hormones and their related peptides which were prepared by the conventional method for peptide synthesis. Immunological properties of these hormones were characterized by radioimmunoassays specific to the respective hormones. Antisecretin antisera (NCC-R-1 and R-801) were generated in rabbits with synthetic porcine secretin absorbed on polyvinylpyrrolidone. Antiserum to substance P (R-400) was produced in a rabbit with synthetic substance P-human alpha-globulin conjugate. Generation of anti-VIP antiserum (R-502) was carried out by immunizing rabbits with synthetic VIP absorbed on polyvinylpyrrolidone. Synthetic polypeptides related to the three hormones that were examined in this study include secretin(4-27), secretin(5-27), secretin(7-27), secretin(11-27), secretin(14-27), secretin(18-27), secretin(1-22)amide, secretin(7-22)amide, Nalpha-tyrosyl-secretin, [1-Tyr]secretin, [4-Ala]secretin, [4-D-Ala]secretin, [4-Ala,5-Val]secretin, [6-Tyr]secretin, substance P(2-11), substance P (3-11), substance P(4-11), substance P(5-11), substance P(6-11), Nalpha-tyrosyl-substance P, [1-Tyr]substance P, [8-Tyr]substance P, [11-Leu]substance P, des-11-Met-substance P, VIP(7-28), VIP(11-28), VIP(18-28), VIP(1-18)amide, and VIP(1-22)AMIDE. The results revealed two antigenic regions at the amino- and carboxylterminal portions of the secretin and VIP molecules. As to substance P, the major antigenic region was located within the 3 to 11 sequence. The proline residue in position 4 and methionine in position 11 seemed to be of special importance. The immunoassays demonstrated the existence of immunoreactivities of these hormones in hot water extracts from various porcine tissues. In the pituitary, VIP and substance P immunoreactivities were detected, whereas secretin was not. Secretin, VIP, and substance P were found in the pancreas, but at low concentrations. Distributions of these hormones in various sites of the gastrointestinal tract were also demonstrated.

Oxytocin analogues with combined high smooth muscle and negligible antidiuretic activities. Investigation of position 7 in neurohypophyseal hormones

The proline residue in position 7 of oxytocin occupies one of the four corner positions in the two beta turns proposed for the preferred conformation of the pituitary hormone. It has been suggested that synthetic modifications of the residues in these corner positions will yield analogues in which one or more of the biological activities of the parent hormone is highly accentuated in terms of potency relative to other activities. In a continued effort to test this hypothesis the following analogues of oxytocin were prepared: [7-glycine]oxytocin, [1-beta-mercaptopropionic acid,7-glycine]oxytocin, [7-alanine]oxytocin, and [1-beta-mercaptopropionic acid,7-alanine]oxytocin. These peptides were found to possess the following specific activities, respectively: rat uterotonic, 65 +/- 2, 355 +/- 3, 22 +/- 1, 123 +/- 4; avian vasodepressor, 5.3 +/- 0.8, 17 +/- 0.4, 4.8 +/- 0.1, 9.8 +/- 0.5; rat antidiuretic, less than0.01, 0.062, 0.081 +/- 0.01, 0.17 +/- 0.01; rat pressor, 0.3, 0.5, 0.4, 0.5 unit/mg. Thus the analogues retain high uterotonic activity but exhibit strongly diminished renal and vascular activities relative to oxytocin. Especially noteworthy is [1-beta-mercaptopropionic acid,7-glycine]oxytocin with its high uterotonic activity but very low antidiuretic and pressor activities. The activity profile of this analogue combined with the fact that it is only slowly enzymatically degraded warrants further investigations of this peptide for clinical applications.

Specificities of protocollagen hydroxylases from different sources

1. 1. Protocollagen hydroxylase from calf embryo skin is able to hydroxylate only proline residues in position I of synthetic ( l-Pro-Gly- l-Pro) n substrates. 2. 2. The corresponding enzyme from earthworm body wall is less specific. It converts proline residues in both position 1 and 3 of the polymeric tripeptide substrates to hydroxyproline. In both positions a high amount of 3- l-Hyp is synthesized besides the 4-isomer. 3. 3. When protocollagen is used as a substrate, 3-hydroxyproline is formed by both hydroxylases. The amounts are close to the values of 3-Hyp found in the collagens of the respective tissues from which these enzymes were extracted. 4. 4. Hydroxylase preparations from both sources lose half of their activity when once frozen and thawed. The original level of activity may be restored by addition of α-ketoglutarate.

The structure of sheep hemoglobins: II. The amino acid composition of the tryptic peptides of the non-α chains of hemoglobins A, B, C, and F

This paper contains detailed data of the amino acid compositions of the peptides from tryptic digests of the non-α chains of the three adult sheep hemoglobins A, B, and C and of sheep fetal hemoglobin. Sequential arrangements of amino acids of some of these peptides are also presented. The peptides were isolated from 2- and 24-hour tryptic digests by Dowex-50 chromatography and purified on Dowex-1 resin. The β chains of the adult hemoglobins A and B and the γ chains of fetal hemoglobin contain likely 145 residues with methionine in amino terminal position. The minimal number of differences between the two adult chains is 6; these differences include a methionine residue in position 76 and a proline residue in position 58. The β chain of hemoglobin C, which replaces the β chain of hemoglobin A in sheep homozygous for hemoglobin A during severe anemia, may contain 141 residues with proline in amino terminal position. Excluding the first 8 residues from the comparison, at least 12 differences are likely to exist between the β chains of the hemoglobins A and C. The γ chain differs from the β chain of hemoglobin B minimally in 27 residues. A comparison of the putative structures of these chains with the corresponding bovine polypeptide chains revealed a greater resemblance than noted with any other non-α chain from species studied. Since the structural differences between the non-α chains of the hemoglobins A, B, and C are located in various positions in the total chain structures, it seems indicated (a) that the allelic genes governing the structural synthesis of the β chains of the hemoglobins A and B are greatly different in structure, and (b) that the synthesis of the β chain of hemoglobin C is controlled by a structurally different and “silent” gene, which is activated during severe anemia.