Beta-casomorphins and their significance in behavioural processes.
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Amino Acid Sequence↗
Biomedical subjects
F Nyberg
Publications and source records attributed to F Nyberg.
Opioid peptides in psychiatric disorders.
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Endorphins↗
Characterization of prolactin receptors in human choroid plexus.
The specific binding of 125I-human prolactin (hPRL) was studied in different areas of the human brain. Particularly high binding affinity of the hormone was found in the choroid plexus and this tissue was therefore selected for further studies. The hippocampus, the hypothalamus and the pituitary were among other regions containing prolactin-binding sites. In the choroid plexus the amount of PRL receptors was significantly higher in females than in males and was also found in both sexes to decrease with age. The binding affinity of 125I-hPRL to choroid plexus was 3.0 x 10(9) M-1 and the binding capacity was 10.3 pmol per mg protein. Following solubilization with Triton X-100 the PRL receptor fraction retained its hormone-binding properties and upon molecular sieve chromatography it behaved as a protein with a molecular mass of approximately 250,000. Cross-linking of 125I-hPRL to receptors from choroid plexus and subsequent sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis indicated a major hormone-binding unit of M(r) 44,000. This value is about 7,000 smaller than that reported earlier by us for the growth hormone receptors from the same tissue, following cross-linking to 125I-human growth hormone (hGH). By affinity column chromatography a complete separation of the hPRL and hGH binding units was achieved. It was thus shown that in choroid plexus the binding sites for GH and PRL occur as discrete entities.
Aging↗
Role of serotonin in heat adaptation: an experimental study in the conscious young rat.
The possibility that serotonin may play a role in heat adaptation was examined in a rat model of heat stress (HS). Subjection of animals to acute 4 h HS (but not to 1 h and 2 h duration) in a biological oxygen demand (BOD) incubator (relative humidity 50-55%, wind velocity 20-25 cm/s) resulted in profound hyperthermia (41.68 +/- 0.25 degrees C) associated with behavioural salivation and prostration. This experimental condition simulates the out door environmental conditions of Varanasi, India during the month of June. Measurement of serotonin (5-hydroxytryptamine, 5-HT) content in the plasma and brain showed a profound increase of the amine by 462% and 187% respectively from the control group. Morphological examination in these animals revealed marked cell changes in the brain at both light and electron microscopic level. Thus chromatolysis, shrunken and dark neurons along with a general sponginess of the gray and white matter was quite frequent in all the brain regions. At ultrastructural level, perivascular edema, damage to pre- and postsynaptic terminals, swollen neurons and glial cells, splitting of myelin and vacuolation was very common throughout the whole brain. The microvessels were mainly collapsed and a diffuse infiltration of lanthanum ion within the endothelium was common. Occasionally, lanthanum could also be seen within the basement membrane. However, the tight junctions were mainly intact. On the other hand, when animals exposed to chronic heat stress of 1 h duration at the same intensity for 7 days and then subjected to a 4 h HS on the 8th day, resulted in only minor symptoms. Thus these 4 h heat stressed animals did not exhibit hyperthermia or increased serotonin levels in plasma and brain as compared to the animals exposed to chronic HS of 1 h duration until alone 7th day. These heat adapted animals did not show any marked cell changes in the brain. However, the values of circulating 5-HT level and rectal temperature of these chronically heat stressed animals (for 1 h daily) were mild but significantly elevated on the 7th day as compared to the intact control animals. These results show that: 1. chronic HS of short duration induces heat tolerance; 2. the circulating 5-HT level some how appears to play an important role in the pathophysiology of HS; hand in the physiological mechanism(s) of secondary heat adaptation.
Adaptation, Physiological↗
A comparison of human lung, brain, CSF and plasma angiotensin-converting enzyme with regard to neuropeptide metabolism.
Human ACE obtained from different tissues and body fluids was assayed with regard to degradative action on tachykinins and various opioid peptides. Substance P (1-9) was easily cleaved, whereas substance P and neurokinin A seemed stable against ACE activity. However, endopeptidase-24.11 easily degraded both of these amidated peptides. When the same peptides were assayed as potential inhibitors of the hydrolysis of hippuryl-His-Leu (specific substrate for ACE activity), substance P and its (1-9) fragment were equally potent, whereas neurokinin A was inactive. The beta-casomorphins, beta-casein derived opioid peptides, with a proline residue at their C-terminus also showed inhibitory action on ACE activity, without being cleaved by the enzyme. These results indicate a modulatory action of these peptides. No differences between ACE originating from different tissues or body fluids could be demonstrated in this regard.
Amino Acid Sequence↗
Approach to studying proteinase specificity by continuous-flow fast atom bombardment mass spectrometry and high-performance liquid chromatography combined with photodiode-array ultraviolet detection.
Fast atom bombardment mass spectrometry (FAB-MS) and high-performance liquid chromatography using a photodiode-array ultraviolet detector were applied to study a dynorphin-converting endopeptidase from the human pituitary gland. The specificity of the enzyme was tested towards various opioid peptides derived from the prodynorphin precursor, i.e. dynorphin A, dynorphin B and alpha-neoendorphin. Peptide fragments were analysed directly by continuous-flow FAB-MS and those containing aromatic amino acids were detected independently by the photodiode-array ultraviolet detector. The results obtained suggest a similar processing of these structure-related substrates and it appears that the enzyme recognizes the dibasic stretch in their sequence. It is also clear from this study that the combination of the above techniques provides a powerful tool for studies of enzymatic conversion among the prodynorphin-derived peptides and it should be applicable to studies of similar mechanisms in other peptide systems.
Amino Acid Sequence↗
Hemorphins derived from hemoglobin have an inhibitory action on angiotensin converting enzyme activity.
The hemorphins are opioid active peptides, which are enzymatically released from the beta-chain of hemoglobin. In this paper we report an inhibitory effect of these peptides on angiotensin converting enzyme (ACE) activity, known to be involved in blood pressure regulation. The hemorphins were found to be quite stable in tissue extracts containing ACE, and their importance as naturally occurring ACE inhibitors is discussed.
Amino Acid Sequence↗
Fingerprinting of molecular components in individual human cerebrospinal fluid samples with a new micropurification system.
This paper reports a rapid and sensitive microtechnique for fingerprinting as little as 20 microliters of cerebrospinal fluid (CSF) from individual humans. Different molecular components were also isolated from the fluid. The SMART system, a new system optimized for high-recovery micropurification, was used for this purpose. The CSF sample, obtained by lumbar puncture, was applied directly into the system, and the patterns recorded for different individuals under various physiological conditions were compared. The results indicate that the procedure provides a powerful tool for the identification or recovery of CSF components and may also be of importance for diagnostic use.
Biogenic Monoamines↗
Isolation and characterization of a hemoglobin-derived opioid peptide from the human pituitary gland.
An opioid nonapeptide was isolated from fresh frozen human pituitaries. Its primary structure (Leu-Val-Tyr-Pro-Trp-Thr-Gln-Arg) was identical to fragment 32-40 of the beta-, delta-, gamma- and epsilon-chains of human hemoglobin. A larger peptide of about 4.5 kDa, which generated a fragment containing the nonapeptide on trypsin digestion, showed an amino acid composition similar to fragment 1-41 of the beta-chain of human hemoglobin. The nonapeptide interacted with mu-opioid receptors in rat brain homogenates using [3H]-(D-Ala2, MePhe3, Gly-ol5)-enkephalin and with sigma-receptors using (+)-[3H]-3-(3-hydroxyphenyl)-N-1-(propyl)piperidine, respectively. The affinities for mu-opioid receptors were in the same range as those observed for the structurally related beta-casomorphins. However, the isolated peptide showed markedly higher affinity at sigma-binding sites when compared to the beta-casomorphins or other opioid peptides. The opioid potency of this peptide as determined in the guinea-pig ileum myenteric plexus muscle preparation, was significant but less than that observed for the beta-casomorphins.
Amino Acid Sequence↗
Purification and characterization of endoproteases from human choroid plexus cleaving prodynorphin-derived opioid peptides.
An endoprotease converting the dynorphins and alpha-neoendorphin has been purified to apparent homogeneity from soluble extracts of human choroid plexus. The purified enzyme was stained as a single band after sodium dodecyl sulphate polyacrylamide gel electrophoresis with an apparent molecular weight of around 54,000 Da. The enzyme potently cleaves dynorphin A, dynorphin B and alpha-neoendorphin at consecutive pairs of basic amino acid residues generating Leu-Enk-Arg6, but it is less active on other neuropeptides containing dibasic stretches. It is optimally active at neutral pH, sensitive to EDTA and slightly affected by the serine protease inhibitors DFP and PMSF. A similar membrane-bound enzyme present in the same tissue was solubilized with 0.5% Triton X-100 and isolated with the same purification procedure. This latter enzyme showed almost identical properties with the soluble peptidase, except for a slightly higher molecular weight.
Amino Acid Sequence↗
Characterization of putative growth hormone receptors in human choroid plexus.
The ability to bind iodine-labelled human growth hormone ([125I]hGH) was measured in different parts of the human brain. The choroid plexus contained the highest amount of binding sites (receptors) and was therefore selected for further studies. The binding between [125I]hGH and the receptor was saturable, of high affinity (Ka = 0.63 nM-1) and pH- as well as time-dependent. After solubilization with Triton X-100 the receptors retained their hormone-binding properties and eluted in the high molecular weight range (greater than 500,000) upon molecular sieve chromatography. Analysis by an affinity cross-linking technique indicated a hormone-binding unit of molecular weight 51,000. The molecular characteristics of the identified binding sites are discussed in comparison to those of growth hormone receptors of human and animal origin.
Adult↗
Analysis of tyrosine- and methionine-containing neuropeptides by fast atom bombardment mass spectrometry.
A simple and unambiguous method for the detection of the amino acids tyrosine and methionine in peptide structures has been developed. The procedure, which was applied in studies of opioid peptides, is based on continuous-flow fast atom bombardment mass spectrometry (CF-FAB-MS) following chemical modification of the residue to be analyzed. Thus, for the detection of tyrosine, modification reactions such as acetylation or non-radioactive iodination were performed prior to analysis by CF-FAB-MS. O-Acetylation of the tyrosine residue with N-acetylimidazole was accompanied by a shift of 42 Da in the molecular mass of the peptide under investigation. This modification was reversed by treatment with hydroxylamine hydrochloride. Incorporation of iodine resulted in a molecular weight shift of 126 Da per iodine atom. Methionine residues were detected in methionine-enkephalin-containing peptides following S-oxidation with hydrogen peroxide. The procedures described may have a wide application in peptide chemistry, particularly for the identification of peptide fragments containing the above residues, e.g. in studies of processing or degradation of the enkephalins or other neuropeptides (e.g. endorphins and tachykinins).
Amino Acid Sequence↗
Application of photodiode array detection and fast atom bombardment mass spectrometry for the identification of the arginine residue in neuropeptides.
Chemical derivatization by phenylglyoxal (PGX) was applied to the identification of arginine in the neuropeptides dynorphin A (1-6) and substance P. The obtained products were separated on a short reversed phase C18 column and analysed on-line with the photodiode array UV technique. The selective attachment of a chromogenic molecule into the arginine residue resulted in significant change in the absorbance spectra around 250 nm, depending on the number of PGX molecules attracted. Further analysis employed fast atom bombardment mass spectrometry (FAB MS) and C-terminal sequencing for detailed verification of the derivatives formed during modification with PGX. The results clearly demonstrated that the photodiode array technique, when combined with chemical modification of certain amino acids, provides new possibilities for the analysis of peptide structures.
Amino Acid Sequence↗
Analysis of human pituitary growth hormone and its charge variants by fast-atom bombardment mass spectrometry.
There is evidence that even highly purified preparations of human growth hormone are not homogenous, but contain charge as well as size variants. The charge heterogeneity was suggested to be due to deamidation of the native hormone. To verify this we have applied peptide mapping followed by fast-atom bombardment mass spectrometry (FAB-MS), in order to identify fragments containing the altered amino acids. Growth hormone was purified from human pituitaries and the differently charged forms were separated by column electrophoresis in agarose suspension. The isolated components were treated with trypsin and analysed directly by FAB-MS without prior separation by reversed-phase high-performance liquid chromatography (RP-HPLC). Using this technique, approximately 80% of the hormone structure was recovered and two deamidation sites were found in the fragment T15 (FDTNSHNDDALLK). The results clearly elucidated the potential use of FAB-MS for the fast screening of other variants of the growth hormone which are known to exist.
Amino Acid Sequence↗
Inhibition of proteases with enkephalin-analogue inhibitors.
N-peptidyl-O-acyl hydroxylamines have proven to be effective and selective mechanism-based inhibitors of serine and cysteine proteases as demonstrated using enzymes with specificities for hydrophobic amino acids at the cleavage site. Here, we report for the first time the inhibition of proteases able to accommodate cationic amino acid side chains in their binding pockets using compounds of this inhibitor class. Trypsin and papain are inactivated by enkephalin-analogue diacyl hydroxylamines in a time-dependent and irreversible manner exhibiting second-order rate constants in the range of 100-1000 M-1.s-1. In contrast, human cerebrospinal fluid dynorphin-converting enzyme (hCSFDCE) is inhibited only moderately by these inhibitors. Mechanistic implications have been derived.
Amino Acid Sequence↗
Studies of specificity and inhibition of human cerebrospinal fluid dynorphin converting enzyme.
Dynorphin-converting activity was recently discovered in human cerebrospinal fluid. This enzyme (hCSF-DCE) cleaves dynorphin A, dynorphin B and alpha-neoendorphin to release Leu-enkephalin-Arg6. To characterize the enzyme further we used several protease inhibitors, including N-peptidyl-O-acyl hydroxylamines which are known to act as potent irreversible inhibitors of serine and cysteine proteinases. No irreversible inactivation occurred but strong, reversible effects on the dynorphin-converting activity by some of the inhibitors tested could be observed. Although, hCSF-DCE binds its substrates (dynorphin A and B) in the microM-mM concentration range, it exhibits high specificity in recognizing and cleaving the linkage between the two basic amino acids in the substrate sequence.
Amino Acid Sequence↗
Isoforms of a dynorphin B converting enzyme isolated by column electrophoresis in agarose suspension.
A dynorphin B converting enzyme previously purified from bovine spinal cord was subjected to column electrophoresis in agarose suspension. By this technique combined with HPLC gel filtration it was possible to resolve and recover several isoforms of the proteinase. All these isoenzymes were associated with a similar molecular size but apparently they differed with regard to their net charges. No significant difference between their inhibitory profiles or their Km values for the release of Leu-enkephalin-Arg6 from dynorphin B was observed.
Animals↗
Molecular heterogeneity of angiotensin converting enzyme in human cerebrospinal fluid.
Three different molecular forms of angiotensin converting enzyme (ACE) (approximately Mr 150,000, 80,000 and 40,000, respectively), have been recovered from human cerebrospinal fluid. All three enzymes were inhibited by captopril and enalapril and their activity was potentiated by chloride ions. They were capable of degrading Leu-enkephalin-Arg6 and substance -P, but gave no conversion of neurokinin A. In all these aspects, the CSF enzymes were identical with the human pulmonary enzyme. The Mr 40,000 form of ACE is the smallest active form of the enzyme hitherto reported and is likely to represent a fragment of the C-terminal part of native ACE, where its active center is located.
Amino Acid Sequence↗