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1.
Biochim Biophys Acta ; 454(3): 514-23, 1976 Dec 13.
Article in English | MEDLINE | ID: mdl-999915

ABSTRACT

Near-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1epsilon, 7epsilon-diguanidino-[Tyr8]-,1epsilon,7epsilon-diguanidono-[Asn14]-, [Phe(F)8, Orn10]- and 1epsilon, 7epsilon-diguanidino-S-peptide, with S-protein. Environmental alterations of Phe-8 in the S-peptide and Tyr-25 in the S-protein, derived from the association process, lead to strong optical signals whose location and magnitude were clearly defined by means of a comparative analysis of the above spectra. Additionally, the spectroscopic effects resulting from insertion of a tyrosyl residue into an hydrophobic environment in the presence or absence of hydrogen-bonding partners were identified and compared with similar findings obtained from the model compound p-cresol.


Subject(s)
Peptides , Ribonucleases , Circular Dichroism , Guanidines , Ornithine , Phenylalanine/analogs & derivatives , Protein Binding , Protein Conformation , Spectrophotometry, Ultraviolet
2.
Biochim Biophys Acta ; 454(3): 524-38, 1976 Dec 13.
Article in English | MEDLINE | ID: mdl-999916

ABSTRACT

Far-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1epsilon, 7epsilon-diguanidino-[Tyr8]-, 1epsilon, 7epsilon-diguanidino-[Asn14]-, [Phe(F)8, Orn10]-, [Cha8, Orn10]- and 1epsilon, 7epsilon-diguanidono-S-peptide, with S-protein. The aromatic chromophores contributions to the absorption spectra in the 220-250 nm wavelengths interval strongly exceed the hyperchromism due to the random coil to right handled alpha-helix transition of the S-peptide, accompanying the association process. Contributions resulting from peptide transitions constitute a large portion of the total dichroism, nevertheless substitution of the non aromatic cycloexylalanine residue in position 8 of the S-peptide with phenylalanine, p-fluorophenylalanine and tyrosine leads to CD negative maxima located at 215, 215 and 212,5 nm, respectively. The strongest ellipticity increment (28%), relative to that of the Cha-derivative, was observed at 212,5 nm for the [Tyr8]-S-peptide analog, while in the narrow 222 nm range minimal differences were found upon insertion into the position 8 of the S-peptide of the above aromatic residues. Information derived from above data and from a comparison of RNAase A, S and S-protein CD spectra enabled us to assign the positive CD band at 240 nm in RNAase A spectrum to transitions of phenylalanines and inaccessible tyrosines.


Subject(s)
Peptides , Ribonucleases , Circular Dichroism , Guanidines , Protein Binding , Protein Conformation , Spectrophotometry, Ultraviolet
3.
Biochim Biophys Acta ; 576(2): 429-39, 1979 Feb 26.
Article in English | MEDLINE | ID: mdl-427200

ABSTRACT

The three main components YI, YII, and Z of clupeine, a protamine from herring, have been purified and characterized. The conformational preferences of clupeines have been examined as a funciton of pH, temperature, added salts, and presence of structure-disrupting agents and helix-supporting solvents using circular dichroism. It was found that these small basic proteins assume predominantly an unordered conformation in aqueous solution. Addition of counter ions, in particular perchlorate, and 2-chloroethanol induces in various amounts the onset of the right-handed alpha-helical conformation. Urea favors the statistical coil state. It was also demonstrated that in the 0.1--4.0 . 10(-1) M range, in contrast to clupeines YI and Z, the circular dichroic properties of the YII component do not seem to be sensitive to the addition of mono- and diphosphate.


Subject(s)
Clupeine , Protamines , Amino Acids/analysis , Circular Dichroism , Protein Conformation , Spectrophotometry, Ultraviolet
4.
Biochim Biophys Acta ; 971(3): 332-8, 1988 Oct 07.
Article in English | MEDLINE | ID: mdl-3167103

ABSTRACT

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.


Subject(s)
Oligopeptides/chemical synthesis , Protein Kinases/metabolism , Amino Acid Sequence , Animals , Casein Kinases , Indicators and Reagents , Kinetics , Liver/enzymology , Phosphorylation , Rats , Substrate Specificity
5.
Biochim Biophys Acta ; 1012(2): 191-5, 1989 Jul 11.
Article in English | MEDLINE | ID: mdl-2500978

ABSTRACT

The octapeptide E-E-K-E-Y-H-A-E, corresponding to the amino acid sequence 841-845 of EGF receptor, whose tyrosine-845 is homologous to the main phosphorylation site of pp60v-src, has been synthesized together with seven shorter peptides encompassing variable segments around the tyrosine residue. The peptides have been employed as model substrates for inspecting the local structural determinants of three tyrosine protein kinases (TPKs), namely; TPK-IIB and TPK-III, isolated from lymphoid cells (Eur. J. Biochem. 172, 451-457 (1988] and the TPK encoded by the oncogene of Abelson murine leukemia virus. The phosphorylation order with the different peptide substrates is variable depending on the TPK used: in particular, the lysine residue at position -2 relative to tyrosine proved especially harmful with TPK-IIB, the peptides K-E-Y-H and K-E-Y-H-A-E being very poor substrates compared with their shorter derivatives devoid of the N-terminal lysine (E-Y-H and E-Y-H-A-E, respectively). Conversely, such a basic residue is well tolerated by the other two TPKs. The negative effect of the N-terminal lysine on TPK-IIB-catalyzed phosphorylation is accounted for by an increase of Km and can be overcome by the presence of additional glutamic acid(s) on that side. On the other hand, the C-terminal acidic doublet Ala-Glu specifically impairs the phosphorylation efficiency of abl-TPK, by lowering the Vmax value, the heptapeptide E-K-E-Y-H-A-E being much less readily phosphorylated than E-K-E-Y-H. Collectively, these results would indicate that the site specificity of tyrosine protein kinases results from the balance of positive and negative determinants whose influence on the catalytic activity of the individual enzymes can differ greatly.


Subject(s)
ErbB Receptors/metabolism , Peptide Fragments/metabolism , Protein-Tyrosine Kinases/metabolism , Retroviridae Proteins/metabolism , Amino Acid Sequence , Molecular Sequence Data , Oncogene Protein pp60(v-src) , Peptide Fragments/chemical synthesis , Phosphorylation , Substrate Specificity
6.
Biochim Biophys Acta ; 1091(1): 123-6, 1991 Jan 10.
Article in English | MEDLINE | ID: mdl-1995063

ABSTRACT

The previously isolated spleen tyrosine protein kinase, conventionally termed TPK-IIA, displaying activation by either positively or negatively charged polyelectrolytes has been further characterized. TPK-IIA is immunologically related with the tyrosine protein kinase encoded by the lyn gene, a member of src subfamily and is dramatically activated by very high NaCl concentration. The stimulatory effects of NaCl and polylysine, which are not additive, are accounted for by increased Vmax values, the Km being virtually unchanged, suggesting that both effectors probably interact with the same site(s). Stimulation of TPK-IIA by heparin appears to be partially additive to that promoted by NaCl and possibly occurring through a different mechanism. The NaCl activatory effect correlates with the electrolytic nature of synthetic peptides used as substrates, being much more consistent with neutral peptides as compared with acidic ones. Of the other three spleen tyrosine protein kinases, TPK-I shows similar biochemical and immunological features, suggestive of close relatedness with TPK-IIA, while TPK-IIB and TPK-III are neither related with the lyn protein nor with the products of three other oncogenes of the src subfamily, namely lck, hck and fyn.


Subject(s)
Protein-Tyrosine Kinases , Protein-Tyrosine Kinases/isolation & purification , Spleen/enzymology , src-Family Kinases , Animals , Cross Reactions , Heparin/pharmacology , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck) , Polylysine/pharmacology , Protein-Tyrosine Kinases/immunology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins c-fyn , Proto-Oncogene Proteins c-hck , Rats , Sodium Chloride/pharmacology , Substrate Specificity
7.
FEBS Lett ; 184(1): 72-7, 1985 May 06.
Article in English | MEDLINE | ID: mdl-3157599

ABSTRACT

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.


Subject(s)
Protein Kinases/analysis , Animals , Cattle , Kinetics , Peptides , Phosphorylation , Protein Kinase C , Structure-Activity Relationship
8.
FEBS Lett ; 162(2): 235-8, 1983 Oct 17.
Article in English | MEDLINE | ID: mdl-6195015

ABSTRACT

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.


Subject(s)
Peptides/pharmacology , Polyglutamic Acid/pharmacology , Protein Kinase Inhibitors , Adenosine Triphosphate/pharmacology , Animals , Binding Sites , Casein Kinases , Cytosol/enzymology , Liver/enzymology , Phosphorylation , Polyglutamic Acid/analogs & derivatives , Protein Kinases/isolation & purification , Rats , Structure-Activity Relationship
9.
FEBS Lett ; 171(2): 211-4, 1984 Jun 11.
Article in English | MEDLINE | ID: mdl-6586496

ABSTRACT

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.


Subject(s)
Oligopeptides/metabolism , Protein Kinases/metabolism , Animals , Arginine/metabolism , Casein Kinases , Glutamine/metabolism , Kinetics , Liver/enzymology , Protein Conformation , Rats , Substrate Specificity
10.
FEBS Lett ; 206(2): 203-7, 1986 Oct 06.
Article in English | MEDLINE | ID: mdl-3758347

ABSTRACT

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.


Subject(s)
Gastrins/chemical synthesis , Animals , Chemical Phenomena , Chemistry , Chromatography, High Pressure Liquid , Gastric Acid/metabolism , Gastrins/pharmacology , Humans , Male , Parietal Cells, Gastric/metabolism , Rabbits , Rats , Receptors, Cholecystokinin/metabolism , Sulfates
11.
Peptides ; 6 Suppl 3: 277-82, 1985.
Article in English | MEDLINE | ID: mdl-3938532

ABSTRACT

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.


Subject(s)
Analgesics , Calcitonin/pharmacology , Pain/physiopathology , Animals , Calcium/pharmacology , Edetic Acid/pharmacology , Female , Humans , Mice , Salmon , Structure-Activity Relationship , Swine
12.
Brain Res ; 862(1-2): 83-9, 2000 Apr 17.
Article in English | MEDLINE | ID: mdl-10799672

ABSTRACT

Glutathione (GSH) is a key component of the cellular defence cascade against injury caused by reactive oxygen species. Kainic acid (KA) is a potent central nervous system excitotoxin. KA-elicited neuronal death may result from the generation of ROS. The present study was undertaken to characterize the role of GSH in KA-induced neurotoxicity. Cultures of cerebellar granule neurons were prepared from 8-day-old rats, and used at 8, 14 and 20 days in vitro (DIV). Granule neurons displayed a developmental increase in their sensitivity to KA injury, as quantified by an ELISA-based assay with the tetrazolium salt MTT. At DIV 14 and 20, a 30-min challenge with KA (500 microM) reduced cell viability by 45% after 24 h, significantly greater (P<0.01) than the 22% cell loss with DIV 8 cultures. Moreover acute (30 min) KA exposure concentration-dependently reduced intracellular GSH and enhanced reactive oxygen species generation (evaluated by 2', 7'-dichlorofluorescein diacetate). In comparison to control, KA (500 microM) lowered GSH levels in DIV 8 granule neurons by 16% (P=0. 0388), and by 36% (P=0.0001) in both DIV 14 and DIV 20 neurons, after 30 min. Preincubation of granule neurons with the membrane permeant GSH delivery agent, GSH ethyl ester (5 mM), for 30 min significantly increased intracellular GSH content. Importantly, GSH ethyl ester reduced the toxic effects of KA, becoming significant at 1 mM (P=0.007 vs. KA-treated group), and was maximal at >/=2.5 mM (P<0.0001). GSH ethyl ester displayed a similar dose-dependence in its ability to counteract KA-induced depletion of cellular GSH. The data strengthen the notion that cellular GSH levels have a fundamental role in KA-induced neurotoxicity.


Subject(s)
Cerebellum/cytology , Excitatory Amino Acid Agonists/toxicity , Glutathione/analysis , Kainic Acid/toxicity , Nerve Degeneration/chemically induced , Neurons/chemistry , Animals , Cell Survival/drug effects , Cerebellum/chemistry , Cerebellum/metabolism , Fluoresceins , Glutamic Acid/toxicity , Glutathione/analogs & derivatives , Glutathione/pharmacology , N-Methylaspartate/toxicity , Nerve Degeneration/drug therapy , Nerve Degeneration/metabolism , Neurons/cytology , Neurons/drug effects , Neuroprotective Agents/pharmacology , Neurotoxins/toxicity , Oxidative Stress/drug effects , Oxidative Stress/physiology , Radiation-Protective Agents/pharmacology , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism
13.
Eur J Pharmacol ; 116(3): 287-92, 1985 Oct 22.
Article in English | MEDLINE | ID: mdl-4076340

ABSTRACT

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.


Subject(s)
Analgesics , Carboxypeptidases/antagonists & inhibitors , Phenylalanine/pharmacology , Animals , Carboxypeptidases A , Female , Hypnotics and Sedatives , Indoleacetic Acids/pharmacology , Mice , Phenylalanine/analogs & derivatives , Phenylpropionates/pharmacology , Time Factors , Tyrosine/pharmacology
14.
Life Sci ; 33(16): 1575-80, 1983 Oct 17.
Article in English | MEDLINE | ID: mdl-6355734

ABSTRACT

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.


Subject(s)
Analgesics , Dipeptides , Acylation , Animals , Dipeptides/pharmacology , Drug Synergism , Enkephalin, Leucine/pharmacology , Female , Kinetics , Mice , Neprilysin , Protease Inhibitors , Structure-Activity Relationship
15.
Adv Exp Med Biol ; 467: 207-15, 1999.
Article in English | MEDLINE | ID: mdl-10721058

ABSTRACT

The physiological roles of the pineal hormone melatonin are still not completely clarified. Recently it has been shown that melatonin is a potent, endogenous scavenger of reactive oxygen species suggesting that it might interfere with neurodegenerative processing involving free-radical formation and excitatory aminoacid release. These neuroprotective effects of melatonin may result, at least in part, from a sparing of glutathione reductase, which is decreased following administration of the neurotoxic agent kainate (KA) in rats. Moreover, KA causes a rapid decrease in glutathione (GSH) content of cultured cerebellar granule neurons but not in astrocytes. These cell types both express functional KA receptors, but only the former is sensitive to reactive oxygen species-dependent KA injury. Melatonin counteracts the changes in GSH, induced by KA, in cultured cerebellar granule neurons.


Subject(s)
Brain/physiology , Melatonin/pharmacology , Melatonin/physiology , Neuroprotective Agents/pharmacology , Animals , Astrocytes/drug effects , Astrocytes/physiology , Brain/drug effects , Free Radical Scavengers/pharmacology , Glutathione/metabolism , Glutathione Reductase/metabolism , Humans , Kainic Acid/toxicity , Neurons/drug effects , Neurons/physiology , Rats
16.
J Exp Anal Behav ; 62(2): 169-84, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7964364

ABSTRACT

Rats in a laboratory foraging paradigm searched for sequential opportunities to drink in two water patches that differed in the bar-press price of each "sip" (20 licks) of water within a bout of drinking (Experiment 1) or the price and size (10, 20, or 40 licks) of each sip (Experiment 2). Total daily water intake was not affected by these variables. The rats responded faster at the patch where water was more costly. However, they accepted fewer opportunities to drink, and thus had fewer drinking bouts, and drinking bouts were smaller at the more costly patch than at the other patch. This resulted in the rats consuming a smaller proportion of their daily water from the more costly patch. The size of the differences in bout frequency and size between the patches appears to be based on the relative cost of water at the patches. The profitability of each patch was calculated in terms of the return (in milliliters) on either effort (bar presses) or time spent there. Although both measures were correlated with the relative total intake, bout size, and acceptance of opportunities at each patch, the time-based profitability was the better predictor of these intake measures. The rats did not minimize bar-press output; however, their choice between the patches and their bout sizes within patches varied in a way that reduced costs compared to what would have been expended drinking randomly. These data accord well with similar findings for choices among patches of food, suggesting that foraging for water and food occurs on the basis of comparable benefit-cost functions: In each case, the amount consumed is related to the time spent consuming.


Subject(s)
Conditioning, Operant , Drinking Behavior , Motivation , Animals , Choice Behavior , Male , Mental Recall , Rats , Rats, Sprague-Dawley , Reaction Time
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