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G Borin

Publications and source records attributed to G Borin.

At least 37 records · Page 2Linked to original sources
PubMed1989

Conformation and ion binding properties of peptides related to calcium binding domain III of bovine brain calmodulin.

The conformational and ion binding properties of the sequences 93-104, 96-104, and 93-98 of domain III of bovine brain calmodulin (CaM) have been studied by CD and Tb3+-mediated fluorescence. In aqueous solution the interaction of all fragments with Ca2+ and Mg2+ ions is very weak and without any effect on the peptide conformation, which remains always random. In trifluoroethanol the interaction is very strong and the different fragments exhibit very distinct binding properties. In particular, the dodecapeptide fragment 93-104, and its N-terminal hexapeptide 98-104, bind calcium and magnesium with a very high binding constant (Kb greater than 10(5) M-1), undergoing a substantial conformational change. The structural rearrangement is particularly evident in the hexapeptide fragment, which tend to form a beta-bend. The C-terminal nonapeptide fragment 96-104 interacts with calcium and magnesium more weakly, and the binding process causes a decrease of ordered structure. These results suggest that, even in the entire dodecapeptide sequence corresponding to the loop of domain III of CaM, the calcium binding site is shifted toward the N-terminal hexapeptide segment. This interpretation is consistent with the results of crystallographic studies of CaM, which show that the calcium ions are located toward the amino terminal portion of the loop.

Amino Acid Sequence↗
PubMed1988

Substrate-specificity determinants for a membrane-bound casein kinase of lactating mammary gland. A study with synthetic peptides.

A tissue-specific casein kinase, purified from the Golgi-enriched-membrane fraction of guinea-pig lactating mammary gland (GEF-CK), readily phosphorylates the synthetic peptide Ser-Glu5, a good substrate of casein kinase-2, and several derivatives varying for the number and position of acidic residues on the C-terminal side of serine, except those lacking an acidic side chain at position +2. The least acidic peptide, still significantly affected by GEF-CK, is Ser-Ala-Glu-Ala3 which is not a substrate for CK-2. Conversely, the peptides Ser-Ala2-Glu-Ala2, Ser-Ala2-Glu3, Ser-Ala2-Glu5 and Ser-Glu-Ala-Glu3, all of which are more or less readily phosphorylated by CK-2, are not appreciably affected by GEF-CK. On the other hand the presence of additional glutamyl residues, besides the one in the second position, improves the affinity of the peptide substrate for GEF-CK, as indicated by the Km values of Ser-Glu5, Ser-Glu2-Ala3 and Ser-Ala-Glu-Ala3 which are 80, 950 and 3950 microM respectively. It is concluded that although both CK-2 and GEF-CK require, for optimal activity, rather extended acidic clusters on the C-terminal side of the target serine, the most critical residue in the case of GEF-CK is not the one at position +3, which is required for CK-2 catalyzed phosphorylation [Marin, O. et al. (1986) Eur. J. Biochem. 160, 239-244], but the one lying at position +2. Additional differences, concerning the site specificities of these enzymes, have been outlined using the threonyl derivative of Ser-Glu5 and the peptide Arg-Ser-Glu3-Val-Glu. The former is still phosphorylated by CK-2 but not to any appreciable extent by GEF-CK, which apparently is strictly specific for seryl residues. On the contrary, the presence of an N-terminal basic residue, which greatly reduces phosphorylation by CK-2, is tolerated rather well by GEF-CK. On the other hand a C-terminal basic residue, interrupting the acidic cluster, compromises phosphorylation by GEF-CK, as indicated by the extremely high Km value of Ser-Glu3-Lys-Glu vs Ser-Glu3-Val-Glu (13,000 and 170 microM, respectively).

Amino Acid Sequence↗
PubMed1988

Synthetic peptide substrates for casein kinase 2. Assessment of minimum structural requirements for phosphorylation.

Unlike the peptides SAEAAA and SEEAAA which are not substrates for casein kinase 2 (CK-2) their analogs SAAEAE and SAAEAA are still significantly phosphorylated. Their Km values, however, (13.3 and 18.9 mM, respectively) are almost two orders of magnitude higher than that of SEEEEE and their Vmax values are 3- and 14-fold lower than that of SAAEEE. The peptide ESEEEEE, but not ASEEEEE, is a slightly better substrate than SEEEEE, while both RSEEEEE and SEEEKE are very poor substrates compared to ASEEEEE and SEEEAE, respectively. SAAEAE is much more responsive to polylysine stimulation and polyphosphate inhibition than is SEEEEE. Taken together these data show that a single acidic residue at the third position from the C-terminal side of the phosphorylatable amino acid represents not only a necessary, but also a sufficient condition for site recognition by CK-2. Optimal phosphorylation efficiency, however, requires an extended C-terminal cluster of several acidic residues, and can be compromised by the presence of only a basic residue either inside the acidic cluster or adjacent to the N-terminal side of the phosphoacceptor amino acid. The structure of the phosphoacceptor site can greatly influence the efficacy of substrate-directed effectors of CK-2.

Amino Acid Sequence↗
PubMed1987

Synthesis of human [15-norleucine]little-gastrin-II and des-1-tryptophan-[12-norleucine]minigastrin-II.

By the use of a newly developed procedure for the synthesis of tyrosine-O-sulfate peptides based on the direct incorporation of the suitably N alpha-protected tyrosine-O-sulfate residue along the synthetic route, the synthesis of two human gastrin-II analogues was successfully accomplished. Thereby acid labile side chain protection was applied in combination with the N alpha-benzyloxycarbonyl group in the intermediate chain elongation steps. Despite the pronounced acid-lability of the sulfate ester moiety, its hydrolysis during the final acidolytic deprotection step was significantly reduced under optimized conditions. Subsequent chromatographic purification led to the two gastrin analogues in satisfactory yields as highly pure compounds as judged by various indicative analytical assays.

Amino Acid Sequence↗
PubMed1986

Site specificity of casein kinase-2 (TS) from rat liver cytosol. A study with model peptide substrates.

The factors determining the site recognition and phosphorylation by rat liver casein kinase-2 (CK-2) have been explored with a set of 14 related hexapeptides each including a single phosphorylatable amino acid and five acidic plus neutral residues. Such peptides are different from each other in the following features: the nature of the phosphorylatable amino acid, if any; its position relative to the critically required acidic residues; the extension and the structure of the acidic cluster. All of them were tested as substrate and/or competitive inhibitors of CK-2, and their kinetic and inhibition constants were determined. The results suggest the following conclusions. Under strictly comparable conditions Ser is by far preferred over Thr. Tyr not being affected at all. In order to carry out its role of structural determinant the critical acidic cluster must be located on the C-terminal side of the target residue, though not necessarily adjacent to it. The affinity for the protein-binding site, as deduced from Km and/or Ki values, is largely dependent on the number of acidic residues but it is also significantly enhanced if a hydroxylic residue is located on their N-terminal side. An acidic residue at position +3 relative to serine plays an especially important role for triggering phosphorylation, the peptide Ser-Glu-Glu-Ala-Glu-Glu having similar Km but negligible Vmax compared to Ser-Glu-Ala-Glu-Glu-Glu and Ser-Glu-Glu-Glu-Ala-Glu. These data provide a rationale for the substrate specificity of CK-2 and will give a helpful insight into the structure of the protein-binding site of this enzyme.

Amino Acids↗
PubMed1986

Synthesis of human des-tryptophan-1,norleucine-12-minigastrin-II and its biological activities.

A human minigastrin-II analog was prepared by conventional methods in solution using N-benzyloxycarbonyltyrosine O-sulfate as starting material in the synthetic route. Upon removal of the acid-labile protecting groups and purification by preparative reversed-phase chromatography the sulfated gastrin peptide was obtained in satisfactory overall yields as homogeneous material. It was found to be about twice as active as the non-sulfated form in stimulating gastric acid secretion and to exhibit a 10-fold higher affinity to gastrin receptors of purified parietal cells.

Animals↗
PubMed1985

Antinociceptive effect of some carboxypeptidase A inhibitors in comparison with D-phenylalanine.

It had previously been shown that D-phenylalanine and hydrocinnamic acid, two in vitro inhibitors of carboxypeptidase A, possess an analgesic action when injected i.p. in mice. We have studied the in vivo effects of indole-3-acetic acid, another carboxypeptidase A inhibitor, and of the following analogs of D-phenylalanine substituted in position 4: D-tyrosine, p-fluoro-D-phenylalanine and trifluoroacetyl-p-fluoro-D-phenylalanine. Whereas indole-3-acetic acid caused a higher and shorter analgesia in comparison with D-phenylalanine, p-fluoro-D-phenylalanine and its N-trifluoroacetyl derivative yielded both a greater and a much longer lasting analgesic effect. Since the latter compound showed only slight inhibitory activity on carboxypeptidase A in vitro, we suggest that inhibition of this enzyme and analgesia might not be directly correlated.

Analgesics↗
PubMed1985

Distinct structural requirements of Ca2+/phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase as evidenced by synthetic peptide substrates.

Protein kinase C, purified to near homogeneity from the brain, has been tested toward a variety of synthetic peptide substrates including different phosphorylatable residues. While it proved totally inactive toward the tyrosyl peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly, as well as toward several more or less acidic seryl peptides, it phosphorylates with a Ca2+/phospholipid-dependent mechanism, at seryl and/or threonyl residues, many basic peptides, some of which are also good substrates for cAMP-dependent protein kinase (A-kinase). Among the peptides tested, however, the best substrate for protein kinase C, with kinetic constants comparable to those of histones, is the nonapeptide Gly-Ser-Arg6-Tyr, which is not a substrate for A-kinase. Moreover, although the peptide Pro-Arg5-Ser-Ser-Arg-Pro-Val-Arg is a good substrate for both kinases, its derivative with ornitines replacing arginines is phosphorylated only by protein kinase C. Some typical substrates of A-kinase on the other hand, like the peptides Phe-Arg2-Leu-Ser-Ile-Ser-Thr-Glu-Ser and Arg2-Ala-Ser-Val-Ala, are phosphorylated by protein kinase C rather slowly and with unfavourable kinetic constants. It is concluded that, while both protein kinase C and A-kinase need basic groups close to the phosphorylatable residues, their primary structure determinants are quite distinct.

Animals↗
PubMed1985

Are calcitonins analgesic and/or hyperalgesic?

The acetylcholine writhing test and the hot plate test were used in mice to evaluate peripheral and central analgesia after porcine, salmon and human calcitonin administrations. ICV and IV injection of calcitonins caused a peripheral analgesia that persisted for at least 2 hr. SC injection of calcitonins did not produce peripheral analgesia. However, when administered by IP route, an increasing peripheral analgesia was observed. Although it had a slow onset, it was as powerful as after ICV or IV administration. Employing the hot plate test to determine the entity of a central analgesic response, the IP administration of calcitonin surprisingly revealed a hyperalgesic effect. It started soon after the injection, reaching its maximum after about 2 hr. At this point the hyperalgesic effects were fully antagonized by EDTA.2 Na ICV, or by Ca++ICV. Moreover only the latter produced a strong and long acting analgesia. Applying a central or peripheral test, calcium seems to play different roles on the algesia variations induced by calcitonins.

Analgesics↗
PubMed1985

Synthetic and binding studies on the calcium binding site I of bovine brain calmodulin. II. Synthesis and CD studies on the cyclic 20-31 sequence.

The synthesis of the cyclic 20-31 sequence of bovine brain calmodulin corresponding to the loop of the hypothetical calcium binding site I of the protein has been accomplished by classical solution methods. The interaction of the synthetic cyclic fragment with calcium ions has been investigated by CD spectroscopy in water and in 98% trifluoroethanol solution. Calcium ions have no effect on the dichroic absorption of aqueous solution of the cyclic dodecapeptide in the wavelength range 200-250 nm. In 98% trifluoroethanol the CD spectrum of the cyclic compound in the absence of calcium ions is almost identical to that of the linear dodecapeptide in the presence of saturation concentrations of calcium. This result supports our previous hypothesis of a folding of the linear sequence upon interaction with the metal ion. The cyclic peptide also interacts with calcium ions in 98% trifluoroethanol forming a 1:1 complex.

Amino Acid Sequence↗
PubMed1984

Synthetic peptides including acidic clusters as substrates and inhibitors of rat liver casein kinase TS (type-2).

The hexapeptides AcSer-Glu-Glu-Glu-Val-Glu and Ser-Glu-Glu-Glu-Glu-Glu, reminiscent of the sites phosphorylated by type-2 casein kinase TS in troponin T and glycogen synthase, respectively, have been synthesized and tested as phosphorylatable substrates for casein kinase TS as well as for other protein kinases. Both peptides are readily phosphorylated by casein kinase TS but not, to any detectable extent, by either cAMP-dependent protein kinase or phosphorylase kinase. Phosphorylation by type-1 casein kinase S was almost negligible. On the other hand the hexapeptide Ser-Glu-Glu-Glu-Ala-Ala is phosphorylated much more slowly and the hexapeptide Ser-Glu-Glu-Ala-Ala-Ala is almost unaffected by casein kinase TS. While the Vmax values of casein kinase TS with the acidic hexapeptides are comparable to those obtained with the corresponding protein substrates, the apparent Km values for the peptides are about two orders of magnitude higher than those for the protein substrates. The heptapeptide Arg-Ser-Glu-Glu-Glu-Val-Glu is a very poor substrate of casein kinase TS in comparison with the corresponding hexapeptide lacking the N-terminal Arg; it is, however, a competitive inhibitor toward the protein substrates, exhibiting a Ki similar to those of Ser-Glu-Glu-Glu-Glu-Glu and (Glu)5 which, in turn, are one order of magnitude higher than that of (Glu)10. It is concluded that the minimum structural requirement of type-2 casein kinases consists of a phosphorylatable residue followed by an acidic cluster, whose length is critical for the binding to the enzyme. Additional residues on the N-terminal side are not required, but their nature can influence the transphosphorylation reaction considerably.

Amino Acid Sequence↗
PubMed1984

Opposite and mutually incompatible structural requirements of type-2 casein kinase and cAMP-dependent protein kinase as visualized with synthetic peptide substrates.

The synthetic hexapeptide Ser-Glu-Glu-Glu-Val-Glu and its N-acetylated derivative are readily and specifically phosphorylated by rat liver casein kinase TS (type-2), while the derived heptapeptide with an additional N-terminal Arg is a very poor substrate. Conversely, the substitution of Glu for Val5 in the synthetic peptide Arg-Arg-Ser-Thr-Val-Ala, which is a good substrate for cAMP-dependent protein kinase by virtue of the N-terminal arginyl residues, prevents its phosphorylation by this enzyme. These data indicate that the site specificities of these two classes of protein kinases, requiring acidic and basic residues on the C- and N-terminal sides of the target residue(s), respectively, are mutually incompatible.

Animals↗
PubMed1984

[Synthesis of peptides with gastrinlike activity. Studies on the structure-activity relationship of the natural hormone human little gastrin I].

To identify the role of the block of glutamic acid residues characteristic for the gastrin molecule, a series of shortened peptides related to the human little-gastrin-I sequence were synthesized. The biological activities of these gastrin peptides strongly suggest in the pentaglutamic acid sequence a specific information for a pronounced amplification of the hormonal activity.

Amino Acid Sequence↗
PubMed1983

Polyglutamyl peptides: a new class of inhibitors of type-2 casein kinases.

Casein kinase-TS (Ck-TS), a type-2 casein kinase purified from rat liver cytosol which phosphorylates seryl and threonyl residues N-terminal to acidic clusters, is specifically inhibited by polyglutamyl peptides which are ineffective both on type-1 casein kinase and on cAMP-dependent protein kinase. The inhibition is competitive toward the protein substrate and non-competitive toward ATP. Among the polyglutamates tested (Glu)70 is the most effective (Ki 0.11 microM). (Glu)10 and (Glu)5 are also inhibitors, though less powerful than (Glu)70, while (Glu)3, (Glu)2 and free glutamic acid up to 5 mM are ineffective. These results disclose the possibility that naturally occurring polypeptides containing long stretches of acidic residues may act as physiological inhibitors of type-2 casein kinases.

Adenosine Triphosphate↗
PubMed1983

Analgesic properties of N-terminal substituted phenylalanyl-alanine.

Phenylalanylalanine, an in vitro inhibitor of enkephalinase, and some of its N alpha-derivatives are shown to possess an analgesic action when injected i.p. and i.c.v. into mice in the presence or absence of Leu5-enkephalin. In the second case a synergistic response is observed. The intensity of the analgesic response depends markedly on the nature of the N-terminal substituent which affects the hydrophobic character of the resulting dipeptide, its subsequent transport and probably its rate of biotransformation by cleaving enzymes.

Acylation↗