Biomedical subjects
Publications and source records attributed to F Nyberg.
Calcitonin gene-related peptide (CGRP) was found to potently inhibit a substance P endopeptidase isolated from human CSF. CGRP potentiated substance P irritant actions; a possible mechanism is interaction for a common metabolic step. Somatostatin is another peptide capable of competing with substance P endopeptidase.
An enzyme radioimmunoassay procedure has been used to identify (Leu)- and (Met)enkephalin containing polypeptides in human cerebrospinal fluid (CSF). A large volume of lumbar CSF collected from 15-20 neurologic subjects was fractionated by ion exchange chromatography on Aminex Q-15 and subsequently on Sephadex G-50. The lyophilized fractions were treated with trypsin and the generated enkephalyl hexapeptides were isolated by SP-Sephadex ion-exchange chromatography and quantified by radioimmunoassay specific for (Met)enkephalin-Arg6/Lys6 and (Leu)enkephalin-Arg6, respectively. Confirmation of the structures of the recovered fragments was obtained by HPLC. In total, the procedure yielded about 100 fmole (Met)-enkephalyl and 40 fmole (Leu)enkephalyl hexapeptides per ml CSF. Our results also suggest that the main part of the (Leu)enkephalin related activity emerged from prodynorphin.
Tryptic digestion followed by radioimmunoassay for (Leu)enkephalin-Arg6 has been used in this study as a general method to detect the presence of all possible products containing the enkephalin sequence from the opioid peptide prohormone, proenkephalin B. Tissue extracts of human hypothalamus and pituitary were examined. Gel filtration was used to separate the different precursor products according to molecular weight. The elution profile was also monitored with highly sensitive radioimmunoassays for dynorphin A and dynorphin B, respectively. Immunoreactive dynorphin A appeared in three peaks with the approximate molecular weight of 1000, 2000 and 5000. Immunoreactive dynorphin B partly occurred in other peaks, 1500, 5000 and 10 000 dalton. Profiles obtained by measuring immunoreactive (Leu)enkephalin-Arg6 in all fractions from gel filtration after trypsin digestion showed a more complex pattern compared to the profiles of immunoreactive dynorphin A and dynorphin B. The major peaks coincided with dynorphin A and dynorphin B but high levels of immunoreactive (Leu)enkephalin-Arg6 were also generated from higher molecular weight regions (MW greater than 5000).
Substance P prohormones were identified in the caudate nucleus, hypothalamus, and substantia nigra of human brain. A polypeptide fraction of acidic brain extracts was fractionated on Sephadex G-50. The lyophilized fractions were sequentially treated with trypsin and a substance P-degrading enzyme with strong preference toward the Phe7-Phe8 and Phe8-Gly9 bonds. The released substance P(1-7) fragment was isolated by ion-exchange chromatography and quantitated by a specific radioimmunoassay. Confirmation of the structure of the isolated radioimmunoassay-active fragment was achieved by electrophoresis and HPLC. By using this enzymatic/radioimmunoassay procedure, two polypeptide fractions of apparent Mr 5000 and 15,000, respectively, were identified. The latter component was the major one of the two but was estimated to account for only about 5% of total substance P radioimmunoassay activity. Because it is of the size predicted from the nucleotide sequences of cDNA for substance P prohormones in bovine brain, the Mr 15,000 component may represent the full-length prohormone.
Differently charged isomers of human pituitary prolactin were subjected to stability studies under various conditions. The study was guided by analytical electrophoresis and radioimmunoassay. In alkaline medium (pH 10) it was found that each isohormone was converted into faster-migrating components, probably due to deamidation. A quantitative study of this alteration was made by measuring the rate constant for this conversion assuming first-order kinetics. The result indicated that the rate constant decreased with increased acidity of the examined prolactin components. Furthermore, this alteration was also found to be paralleled by a decreased immunoactivity of the components. At neutral pH the conversion reaction was studied both in 0.9% NaCl and in human serum. For the least acidic isohormone the rate constant in serum was calculated to be about four times higher than that in the saline solution which in turn was comparatively low. It was concluded that the alteration observed at pH 10 might be attributed to non-enzymic deamidation. The conversion of prolactin that occurred in human serum indicated that this reaction could be caused by enzymes. A study of the immunoactivity of human prolactin as a function of time at various storage conditions is also included in this work. The stability of the hormone was found to be strictly concentration dependent and also affected by buffer ionic strength. In the presence of ethylene glycol (50%, v/v) at -20 degrees C and at a protein concentration in the range of 0.1-2 mg/ml the hormone was found to be both immunologically and electrophoretically stable for several years.
A substance P-hydrolyzing endopeptidase has been purified from a large quantity of human cerebrospinal fluid by ion exchange chromatography (DEAE-Sepharose CL-6B) and molecular sieving (Sephadex G-100 and Sephacryl S-200). The purification was monitored by measuring the conversion of synthetic substance P using a radioimmunoassay specific for its (1-7) fragment. The enzyme has an apparent molecular weight of 43,000. It cleaves predominantly at the Phe7-Phe8 and Phe8-Gly9 bonds but gives no or negligible conversion of the other tachykinins, neuromedin K and L (substance K).
The authors measured opioid receptor-active components in the CSF of 11 women with postpartum psychosis, 11 healthy lactating women, and 16 healthy women who were not lactating. Activity that eluted with 0.2 M acetic acid 0.7-0.9 times the total volume of the column (fraction II activity) was significantly higher in the CSF of both healthy and psychotic women in the puerperium than in that of the lactating women. Very high levels of fraction II activity were seen in four psychotic patients. Material from these patients was further characterized by electrophoresis and high-performance liquid chromatography: The material migrated as bovine beta-casomorphin. Receptor-active material with the same characteristics was also found in the plasma of these four patients. The authors conclude that certain cases of postpartum psychosis are associated with the occurrence in plasma and CSF of unique opioid peptides probably related to bovine beta-casomorphin.
Using a radioimmunoassay procedure substance P-like activity was measured in samples of human CSF obtained from 12 patients with major depressive disorder, 12 with schizophrenia, and 15 control cases diagnosed as psychiatrically normal. Levels were significantly higher in CSF of patients with depressive disorder as compared with schizophrenic patients or controls. Chemical characterization revealed that the measured activity was less basic than substance P itself and might be due to C-terminal fragments. A component reacting with an antiserum highly specific for the substance P[1-7]fragment was found in CSF of patients with depressive disorder. The results indicate that substance P-related peptides may be biological markers in psychoses, particularly in major depressive disorder.
Rat growth hormone (rGH) (100 micrograms/kg) produced 2-4 h after its i.v. injection a rapid reduction of catecholamine stores and turnover in the subependymal layer and in the medial and lateral palisade zone of the median eminence. It is suggested that rGH may inhibit its own secretion partly via reduction of DA synthesis and release in the median eminence leading to increased somatostatin release and partly via reduced noradrenaline synthesis and turnover in the median eminence leading to reduced secretion of a growth hormone releasing factor.
Opioid peptides have been fractionated by column electrophoresis in agarose suspension. The procedure provides a high resolution efficiency and compares favorably with high-performance liquid chromatography. Peptides were recovered from the column in 70% or higher yields when run in the femtomole to the nanomole range. By choosing different pHs it is possible to get complete resolution of a number of different endorphins. The electrophoreses were performed in volatile buffers, which are easily evaporated, and the residues are suitable for direct screening by radioreceptor- or radioimmunoassays. In combination with these assays the separation procedure makes it possible to detect and identify very low concentrations of endorphins in samples of biological origin.
Using the spinal superfusion procedure, in anesthetized rats and cats, the presence of active factors which displace dihydromorphine in brain opiate binding studies, has been observed. Separation of this activity on a Sephadex G-10 column reveals the presence of two fractions which occur before (Fraction I) and after (Fraction II) the salt peak which account for over 70% of the observed dihydromorphine-displacing activity. The ratio of activity in Fraction II/Fraction I is 33 and 21, in the resting spinal perfusates of the rat and cat, respectively. High intensity, bilateral stimulation of the sciatic nerve in cats, results in a 30- and 5.4-fold increase in the levels of Fraction I and Fraction II, respectively, over pre-stimulation levels. In rat, bilateral stimulation of the hind paws, resulted in a frequency-dependent increase in the levels of Fraction I (1.9- and 3.2-fold at 5 and 50 Hz, respectively). Dynorphin 1-13 fragment elutes at least partly in Fraction I. With regard to Fraction II, the peak co-chromatographs with hexapeptide derivatives of enkephalin. Met- and Leu-enkephalin (Fraction III), elute off the column at a point where opiate receptor displacing activity is relatively small. Electrophoretic separation of Fraction I radioreceptor activity of alkaline and acid pH on agarose columns revealed two principle peaks which co-migrated with alpha-neoendorphin and dynorphin 1-13. Fraction II activity appeared primarily in a single peak which was isographic with enkephalin hexapeptides. Using radioimmunoassays, detectable levels of dynorphin and Met-enkephalin were observed and sciatic nerve stimulation resulted in significant increases. Neither column-coupled radioreceptor assays nor radioimmunoassays revealed the presence of beta-endorphin. The present experiments demonstrate the releasability by high intensity somatic stimulation of a variety of opioid peptides present in spinal terminals. Significantly, however, the majority of this activity appears to be found in fractions different from those of the pentapeptide enkephalins.
Opioid peptides have been purified from large pooled samples of human cerebrospinal fluid. The purification steps involved chromatography on Sephadex G-10 and electrophoresis in agarose suspension. The purified material was further characterized by HPLC and radioimmunoassay. All procedures were guided by a specific radioreceptor assay. The Sephadex G10 fractionation yielded receptor activity in two discrete fractions, Fraction I (FI) and Fraction II (FII). A second Sephadex run of FII gave a partial resolution of two components, one of which was larger (FIIA). Electrophoresis resolved these fractions into several components, most of which showed a more basic behaviour than the enkephalins. Thus, FI separated into at least 4 components and FIIB into two components while FIIA remained a single peak. These components appeared to migrate as distinct peaks and some of them also chromatographed on a HPLC-column as single components. Considering their behaviour in electrophoresis and on HPLC, two components are suggested to represent known endorphin structures. The predominant FII component (FIIA) was thus indistinguishable from Met-enkephalin-Lys6 in all chromatographic systems and one of the most basic FI components showed close similarity with dynorphin. Each of these components occurs at a higher concentration than Met- or Leu-enkephalin, dynorphin or beta-endorphin.
Spinal superfusion was performed in anesthetized cats before and during sciatic nerve stimulation. The superfusates were fractionated on Sephadex G-10 columns and thereafter on electrophoresis and HPLC. The endorphin activity was monitored by radioreceptor and radioimmunoassays. In additional experiments, chromatographic fractions were subjected to enzymatic digestion prior to radioimmunoassay. Nerve stimulation caused a release of at least three different endorphins which separated on electrophoresis, one of which comigrated with [Met]enkephalin-Lys6. The identity of this peptide was further supported by HPLC analysis and radioimmunoassay. Furthermore, enzymatic degradation experiments provided evidence for the presence of enkephalin sequences in all three components released by stimulation. There were also increased dynorphin concentrations during stimulation. These findings suggest that at least two different endorphin systems (enkephalin and dynorphin) are functionally present in spinal cord and may be activated by somatic stimulation.
A method has been described for the isolation of three differently charged isohormones of rat prolactin from a discard fraction obtained after extraction of gonadotropins, thyrotropin and growth hormone from homogenized frozen pituitaries. The procedure involved extraction at pH 9.8, ammonium sulphate fractionation, molecular sieve chromatography on Sephadex G-100, and column electrophoresis in agarose suspension. The purification was monitored by radioimmunoassays and the recovered components were all found to possess a specific immunoactivity exceeding that of the standard preparation (RP-1) supplied by the NIAMDD, Bethesda, U.S.A. Increased acidity among these isohormones was found to be paralleled by significantly decreased immunopotency. Each component showed biological activity in radioreceptor assay. A high degree of purity of the isolated components was shown by analytical electrophoresis in polyacrylamide gel. Sodium dodecyl sulphate electrophoresis in the same medium showed no size heterogeneity and yielded a value of approximately 25 000 for the molecular weight of the isohormones. In addition a large form of prolactin, suggested to represent a dimer, was isolated by a further extraction step (pH 10.5) followed by molecular sieve chromatography on Sephadex G-100 and electrophoresis in agarose suspension. The large form was associated with both biopotency and immunopotency. The electrophoresis resolved the prolactin activity into three or four immunoactive components. This pleomorphism of the large prolactin was confirmed by analytical polyacrylamide gel electrophoresis. Amino acid analyses revealed a close similarity between the three monomers and the major dimeric form of the hormone.
Rat prolactin produces in a dose of 100 micrograms/kg a rapid and marked increase of dopamine turnover in the medial palisade zone of the median eminence and of noradrenaline turnover in the posterior periventricular hypothalamic region as well as a possible minor increase of noradrenaline turnover in the magnocellular part of the paraventricular hypothalamic nucleus of the hypophysectomized male rat. The rat prolactin serum levels measured 2 h following injection were within the upper physiological range of lactating rats. The findings suggest that dopamine in the medial palisade zone may be released as a prolactin inhibitory factor and respond rapidly to increases in serum prolactin levels. Furthermore, rat prolactin can acutely modify noradrenergic mechanisms in discrete areas of the hypothalamus indicating that prolactin can participate in the regulation of types of hypothalamic functions other than regulation of its own secretion.
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Three forms of human pituitary prolactin, separable at alkaline pH in a highly purified preparation, were isolated by means of column electrophoresis in agarose suspension. The most acidic component showed a significantly lower radioimmunological activity but a higher bioactivity than the other two components, which were approximately equipotent in both assays. Consequently, in both assays the most acidic component differed markedly from the other components. Amino acid analysis indicated close similarity between the three components and no size heterogeneity was observed by sodium dodecyl sulphate electrophoresis in polyacrylamide gel. A high degree of purity of the isolated components was demonstrated by analytical electrophoresis in polyacrylamide gel at alkaline pH. Runs in the same medium also revealed that the difference in electrophoretic migration velocity remained after reduction and alkylation of the isohormones. A comparison of the migrations of the S-carboxymethylated and the S-carbamidomethylated derivatives with those of the unmodified components indicated that the diversity in electrophoretical behaviour of two adjacent isohormones was consistent with a difference in one single net charge. By analytical isoelectric focusing the component of intermediate migration velocity was resolved into two distinct bands proposed to contain isohormones differing only in the exchange of one residue of aspartic acid for one of glutamic acid. The isoelectric points of the prolactin isomers were all in the range of 5.7--5.9.
A process developed earlier for the extraction of human follitropin, lutropin, thyrotropin and growth hormone from homogenized frozen pituitaries provided a residue utilized for the isolation of prolactin. The isolation procedure involved extraction at pH 9.8, molecular sieve chromatography on Sepharose CL-6B, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B, molecular sieve chromatography on Sephadex G-100 Superfine, and ion-exchange chromatography on DEAE-Sepharose CL-6B using a convex gradient. The progressive purification was guided by radioimmunoassays. The final product was obtained in yields of 31 microgram/gland, and was equipotent with a pituitary preparation (VLS-3) supplied by the National Pituitary Agency (NIH, Bethesda, U.S.A.). Contamination hormones negligible (less than 0.05%). No heterogeneity of the isolated prolactin was observed by sedimentation-equilibrium analysis in the ultracentrifuge, by SDS electrophoresis in polyacrylamide gel or by molecular sieve chromatography in 6 M guanidine hydrochloride. These different techniques gave values in the range of 21 000-23 000 for the molecular weight of prolactin. In free zone electrophoresis, and also in polyacrylamide gel electrophoresis the prolactin preparation was, however, heterogeneous and resolved at alkaline pH into three distinct components. The former technique permitted isolation and assay of the components, indicating that they were all fully active.