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I Lindberg

Publications and source records attributed to I Lindberg.

At least 73 records · Page 4Linked to original sources
PubMed1989

A putative processing enzyme for proenkephalin in bovine adrenal chromaffin granule membranes. Purification and properties.

A putative processing enzyme for proenkephalin, with activity directed toward basic residues, was purified over 2000-fold from washed bovine adrenal medullary chromaffin granule membranes. The molecular mass of this membrane-bound adrenal trypsin-like enzyme (mATLE) is 31 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme is extremely basic, binding to carboxymethyl-Sephadex at pH 8.5. The pH optimum of mATLE using t-butoxycarbonyl-Glu-Lys-Lys-aminomethylcoumarin as a substrate is 8.5-8.7, and its Km value for this substrate is 2.2 mM. mATLE activity was inhibited by soybean trypsin inhibitor, lima bean trypsin inhibitor, and aprotinin but not by metal chelators or thiol-directed reagents. Sequencing of cleavage products released from Peptide B revealed that the enzyme preferentially cleaves between and following the paired basic residues at positions 23 and 24 of Peptide B (thus generating [Met-enkephalin]-Arg-Phe and Arg-[Met-enkephalin]-Arg-Phe). Dynorphin A was cleaved following a single lysine at position 11 but not at the paired arginine site. Our results suggest that mATLE is a trypsin-like serine protease with the specificity appropriate to that of a proenkephalin processing enzyme.

Adrenal Medulla↗
PubMed1989

Purification and assay of opioid activity of low molecular weight enkephalin-immunoreactive peptides generated by peptic digestion of rat plasma proteins.

A low molecular weight Methionine enkephalin-immunoreactive peptide (MEIP) and two Leucine-enkephalin immunoreactive peptides (LEIP) generated by peptic digestion of rat plasma were purified through gel filtration followed by five sequential reverse phase HPLC gradients in different solvent systems. Binding experiments of these peptides to opioid receptors of rat brains were performed. The two LEIPs were able to inhibit binding of [3H]naloxone to opioid receptors in rat brain membranes. No inhibition was found with the MEIP (which represented the only MEIP present in the low molecular weight fraction of pepsin-digested rat plasma). Sequencing revealed that the MEIP is a six residue peptide with the following sequence: Gly-Glu-Tyr-Gly-Phe-Gln. This sequence corresponds to that of residues 422-427 of rat serum albumin.

Amino Acids↗
PubMed1988

Evidence for the phosphorylation of a proenkephalin-derived peptide, peptide B.

The potential phosphorylation of a proenkephalin-derived peptide, Peptide B, was investigated in primary cultures of bovine adrenal chromaffin cells and fresh adrenal medullary tissue. Cultures were labeled with [32P]phosphate for 24 h and extracts subjected to immunoprecipitation using affinity-purified anti-serum directed against the carboxyl terminus of Peptide B. A 4.6-kDa-labeled peptide was observed in autoradiograms of immunoprecipitates separated by sodium dodecyl sulfate-polyacrylamide electrophoresis; this peptide was not observed when excess antigen was present during the immunoprecipitation. Radioimmunoassay of extracts prepared from adrenal medullary tissue and separated by isoelectric focusing revealed the presence of four isoelectric forms of Peptide B-immunoreactive peptides; these peptides also exhibited Met-enkephalin-Arg-Phe immunoreactivity. The isoelectric points of these peptides (4.5, 4.3, 4.1, and 3.9) were consistent with the predicted pI values for phosphorylated derivatives of Peptide B. Treatment of samples with alkaline phosphatase prior to isoelectric focusing resulted in the conversion of the more acidic forms to the least acidic form. The presence of phosphate in the more acidic peaks was additionally verified by isoelectric focusing of 32P-labeled immunoprecipitates; the pI values of the radioactive peptides corresponded precisely to the peaks of immunoreactivity. In adrenal medullary tissue, the relative contributions of the various phosphorylated species to the total Peptide B immunoreactivity were as follows: unphosphorylated form, 13%; singly phosphorylated, 31%; doubly phosphorylated, 37%; and triply phosphorylated, 17%. Thus more than 85% of the Peptide B molecules present in the bovine adrenal medulla are phosphorylated.

Adrenal Medulla↗
PubMed1988

Characterization of enkephalin-immunoreactive peptides generated by peptic digestion of rat plasma proteins.

Met-Enkephalin-immunoreactive peptides (MEIP) and Leu-enkephalin-immunoreactive peptides (LEIP), generated from plasma proteins after digestion with pepsin, were characterized with gel filtration, reverse phase HPLC, and isoelectric focusing. Pooled rat plasma was found to generate 170 pmol LEIP and 76 pmol MEIP/ml plasma. Two peaks of enkephalin (enk)-immunoreactive peptides (EIP) were observed after gel filtration of plasma digests; the early eluting peak (which contained 90% of the total LEIP and 75% of the total MEIP) eluted close to, but not in, the void volume of the column, whereas the elution volume of a later eluting peak (which contained 25% of the total MEIP and about 5% of the total LEIP) was close to that of authentic enk. Five peaks of EIP with pI values of 3.7, 5.8, 7.3, 8.0, and 9.4, were observed after isoelectric focusing of plasma digests; with the exception of the pI 3.7 peak, all of these immunoreactive species could be detected with greater efficiency using the Leu-enk RIA. Peptic digestion of rat serum albumin generated a MEIP with the same pI and HPLC retention time as the plasma pI 3.7 MEIP. No other EIP could be generated from rat serum albumin. No immunoreactive peptides were found which coeluted with synthetic Met-enk or its sulfoxide on HPLC; however, a portion of low mol wt LEIP was eluted with the retention time of authentic Leu-enk. With certain exceptions, these results support and extend the original findings of Singer and Carraway concerning the ability of pepsin to generate extremely high concentrations of EIPs from plasma protein(s).

Animals↗
PubMed1987

Enkephalins in the rat pituitary gland: immunohistochemical and biochemical observations.

The immunohistochemical distribution of opioid peptides derived from proenkephalin A in the rat pituitary was studied by indirect immunofluorescence; immunoreactive peptides were also characterized by column chromatography followed by specific RIAs. Nerve terminals in the neural lobe were immunoreactive (ir) for Tyr-Gly-Gly-Phe-Met-Arg-Phe (YGGFMRF), Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu (YGGFMRGL), and met-enkephalin [Tyr-Gly-Gly-Phe-Met (YGGFM)]. All cells in the intermediate lobe were ir for YGGFMRF, while only occasional cells exhibited YGGFMRGL-like immunoreactivity, and YGGFM-ir cells were not detected in this lobe. In the anterior lobe, some large ovoid cells, identified as gonadotrophs, were immunoreactive for enkephalins. The number of YGGFMRF-ir cells was larger than the number of YGGFMRGL- and YGGFM-ir cells, and these opioid peptides were present in cells that did not contain beta-endorphin immunoreactivity. Twenty times more YGGFMRF than YGGFMRGL-immunoreactivity was present in the anterior lobe, whereas the neurointermediate lobe obtained 4 times more ir YGGFMRF than YGGFMRGL. Pituitary lobe extracts contained substantial amounts of high mol wt forms of ir YGGFMRF and YGGFMRGL, but not of YGGFM or Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu). Low mol wt ir peptides present in both lobes consisted largely of the authentic peptides when analyzed by HPLC; however, an unidentified YGGFMRF-ir peptide was also detected. The results indicate that the proenkephalin A molecule may be processed differentially in the various compartments of the pituitary gland and that opioid peptides derived from this precursor may have functional roles in all three lobes. The relatively large amount of YGGFMRF immunoreactivity, which was detected both biochemically and immunohistochemically, indicates that YGGFMRF-ir peptides may be important proenkephalin A-derived products in the pituitary gland.

Animals↗
PubMed1987

Proenkephalin gene expression in the PC12 pheochromocytoma cell line: stimulation by sodium butyrate.

The differentiation promoter sodium butyrate increases the content of Met5-enkephalin-Arg6-Gly7-Leu8 (Met5-enk-RGL)-immunoreactive peptides in PC12 pheochromocytoma cells, which, unlike mature adrenomedullary chromaffin cells, contain exceedingly low levels of opioid peptides. These butyrate-induced enkephalin-immunoreactive peptides, which are specific products of the proenkephalin gene, consist principally of two high mol wt forms of amino-terminally extended Met5-enk-RGL. These high mol wt peptides, with apparent mol wt of 20,000 and 10,000, are approximately the same size as the two major immunoreactive peptides found in adult New England Deaconess Hospital rat adrenal. The low mol wt Met5-enk-RGL-immunoreactive peptide found in butyrate-treated cells is similar in size to authentic Met5-enk-RGL, which is not found in the adrenal medulla of the adult rat. When PC12 cells are grown as a tumor in vivo, the amount of Met5-enk-RGL-immunoreactive peptide increased only slightly above the level found in control cells grown in vitro and consisted exclusively of the highest mol wt immunoreactive species. In PC12 cells, the butyrate-stimulated elevation in the content of Met5-enk-RGL-immunoreactive peptides may involve changes in transcription, since the peptide increase is preceded by a 2- to 3-fold increase in the level of proenkephalin mRNA. These results suggest that the PC12 cell line may be useful for investigating those factors that control the initial expression and processing of proenkephalin-derived peptides during embryogenesis.

Adrenal Gland Neoplasms↗
PubMed1986

Reserpine-induced alterations in the processing of proenkephalin in cultured chromaffin cells. Increased amidation.

We have used antisera directed towards eight different portions of the proenkephalin molecule to examine the processing rates and patterns of proenkephalin-derived peptides in chromaffin cell cultures in the presence and absence of reserpine. Reserpine treatment produced profound effects on the molecular weight profile of nearly all enkephalin-containing peptides. Increased production of low molecular weight immunoreactive [Met5]enkephalin, [Leu5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, and [Met5]enkephalin-Arg6-Phe7 was observed in reserpine-treated cultures; immunoreactivity corresponding to several intermediate sized enkephalin-containing peptides such as Peptide B and the high molecular weight [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactive peptide was decreased. The production of two amidated opioid peptides, amidorphin and metorphamide, was greatly accelerated in the presence of reserpine. The increased levels of low molecular weight enkephalins could not be accounted for by assuming decreased basal release. These results indicate that reserpine treatment is able to increase the extent of post-translational processing of proenkephalin within chromaffin cells.

Adrenal Medulla↗
PubMed1986

Distribution of immunoreactive peptide B in the rat brain.

Peptide B represents one of the most highly conserved sequences in proenkephalin. To investigate the potential presence of this peptide in the mammalian nervous system, an antiserum raised to this peptide was used to measure the distribution and molecular weight forms of immunoreactive Peptide B in the rat brain. Peptide B-immunoreactivity (ir) was found to be most concentrated in the hypothalamus and the striatum, with lower concentrations in the midbrain and medulla-pons. Characterization of Peptide B-ir by gel filtration demonstrated that the major immunoreactive peak in the hypothalamus corresponded to a peptide with the approximate molecular weight of Peptide B. The major immunoreactive peptide exhibited a retention time on HPLC indicative of a peptide slightly more hydrophilic than bovine Peptide B. The results suggest that proenkephalin in rat brain can be processed to peptides related to bovine Peptide B.

Amino Acid Sequence↗
PubMed1986

Reptilian enkephalins: implications for the evolution of proenkephalin.

To shed light on the evolution of the opioid peptide precursor proenkephalin, we have investigated the profile of immunoreactive enkephalins in reptilian brain using six antisera directed toward different portions of the proenkephalin molecule. Three different orders of reptiles--turtles, alligators, and lizards--were studied; these orders represent lineages diverging more than 250 million years ago. Reptilian brain was found to contain large quantities of Met5-enkephalin (Met5-enk), Leu5-enkephalin (Leu5-enk), and Met5-enk-Arg6-Phe7 (MERF); gel filtration and reverse-phase chromatography of acid extracts prepared from turtle brain indicated that nearly all of the immunoreactivity corresponding to these peptides could be ascribed to the authentic penta- to heptapeptides. Immunoreactive metorphamide, but not amidorphin, was present in all three species. Unlike mammalian brain, reptilian brain does not contain immunoreactive Met5-enk-Arg6-Gly7-Leu8 (MERGL). These results indicate that reptilian proenkephalin, while similar to mammalian enkephalin in containing Met5-enk, Leu5-enk, MERF, and metorphamide, nevertheless differs from mammalian proenkephalin. Reptilian proenkephalin either may contain the MERGL sequence in a form not recognized by the MERGL antibody, or may lack the MERGL sequence entirely.

Alligators and Crocodiles↗
PubMed1986

Distribution and characterization of the opioid octapeptide met5-enkephalin-arg6-gly7-leu8 in the gastrointestinal tract of the rat.

The distribution and characterization of the opioid octapeptide met5-enkephalin-arg6-gly7-leu8 (met5-enk-arg6-gly7-leu8) within the gastrointestinal tract of the rat has been determined by immunohistochemistry and radioimmunoassay by use of a newly developed antibody to met5-enk-arg6-gly7-leu8. With both techniques, met5-enk-arg6-gly7-leu8-immunoreactivity (met5-enk-arg6-gly7-leu8-IR) was detected in all regions of the gastrointestinal (GI) tract except the esophagus. The highest concentration of immunoreactive met5-enk-arg6-gly7-leu8 was observed in the colon, while intermediate concentrations were found in the stomach, duodenum, jejunum, and ileum. Immunostained somata were observed chiefly in the myenteric plexus; immunostained processes were present primarily in the myenteric plexus and the circular muscle layer. This distribution pattern is similar to that previously observed with antiserum to met5-enkephalin-arg6-phe7 (met5-enk-arg6-phe7). Chromatographic analysis of met5-enk-arg6-gly7-leu8-immunoreactive peptides extracted from the GI tract revealed the presence of an immunoreactive peptide of high molecular weight which accounted for approximately three-quarters of met5-enk-arg6-gly7-leu8-IR in both stomach and colon. These findings suggest a role for peptides related to the octapeptide met5-enk-arg6-gly7-leu8 in the regulation of GI function.

Animals↗
PubMed1985

Release of high molecular weight forms of met5-enkephalin-arg6-gly7-leu8 from rat brain.

The release of various molecular weight forms of met5-enk-arg6-gly7-leu8 (MERGL) from slices prepared from rat medulla-pons and hypothalamus was studied. The release of MERGL-IR from both medulla-pons and hypothalamic slices was elevated four- to ten-fold in response to potassium depolarization. Gel of 8 to 10 kilodalton MERGL-IR peptide(s), as well as MERGL itself, were released in response to stimulation with 50 mM K+.

Animals↗
PubMed1984

Distribution of Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactive peptides in rat brain: presence of multiple molecular forms.

An antiserum to the opioid octapeptide met5-enk-arg6- gly7 -leu8 was used to measure the distribution and molecular weight heterogeneity of met5-enk-arg6- gly7 -leu8-immunoreactive (IR) peptides in rat brain. High concentrations of met5-enk-arg6- gly7 -leu8-IR peptides were found in the striatum and hypothalamus; low concentrations were observed in the cortex and cerebellum. Intermediate levels of immunoreactivity were found in other brain regions. Thus the distribution of met5-enk-arg6- gly7 -leu8-IR peptides closely parallels the distribution of met5-enkephalin. The immunoreactivity present in brain regions was characterized by gel filtration and reverse-phase high pressure liquid chromatography. In the striatum, more than 85% of the met5-enk-arg6- gly7 -leu8-IR peptides eluted from a Biogel P-30 column at the position of the authentic octapeptide; the identity of this material was further confirmed by HPLC. Gel filtration of extracts prepared from the medulla-pons resulted in two peaks of immunoreactive material of equal size; one eluted at approximately 8000 daltons and one eluted in the position of the octapeptide. Enzymatic digestion (trypsin + carboxypeptidase B) of the 8000 dalton-IR peptide resulted in the generation of met5-enkephalin-IR. Extracts prepared from other brain regions contained varying amounts of this high molecular weight form of met5-enk-arg6- gly7 -leu8.

Animals↗
PubMed1984

Further characterization of an enkephalin-generating enzyme from adrenal medullary chromaffin granules.

An adrenomedullary protease capable of generating Met5-enkephalin from endogenous precursor(s) has been purified 1,000-fold using affinity chromatography in combination with gel filtration. This trypsin-like enzyme has an apparent molecular weight of 20,000 daltons by gel filtration. The reactivity of the enzyme toward several fluorogenic peptides, Peptides E and F, and the heptapeptides, Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Arg7, was examined. The two heptapeptides and the fluorogenic compounds were poor substrates for the adrenal enzyme; in contrast, Peptides E and F were cleaved. The low molecular weight products of Peptide F digestion were identified by HPLC as Arg1-Met6-enkephalin, Met5-enkephalin, and Met5-enkephalin-Lys6, while digestion of Peptide E resulted in the production of Leu5-enkephalin and Met5-enkephalin-Arg6-Arg7. [3H]-beta m-Lipotropin was not hydrolyzed by the adrenal enzyme. These results indicate that this adreno-medullary protease is capable of cleaving adrenal opioid peptides at the paired basic sites and thus represents a possible candidate for a proenkephalin-converting enzyme.

Adrenal Medulla↗
PubMed1983

A high molecular weight form of met5-enkephalin-arg6-gly7-leu8 in rat brain and bovine adrenal chromaffin granules.

An antiserum against met5-enkephalin-arg6-gly7-leu8 (MAGL) has been used to identify various molecular weight forms of this immunoreactive material in brain and adrenal tissue. The predominant immunoreactive species in acid extracts of chromaffin granules eluted on Biogel P-30 as an approximately equal to 8,000 dalton peptide. Acid extracts prepared from rat brain medulla-pons also contained 8,000 dalton immunoreactive material which exhibited an identical retention time on HPLC to the corresponding immunoreactive material prepared from chromaffin granules.

Adrenal Medulla↗
PubMed1981

Development of enkephalinergic neurons in the gut of the chick.

The content and distribution of Met-enkephalin immunoreactivity in the developing chick gut was studied by radioimmunoassay and immunocytochemistry. Met-enkephalin was detected by radioimmunoassay in the duodenum of the 5-day chick embryo. The concentration in this region increased 4-fold by 13 days of incubation and declined thereafter to the levels found in the 4-week chicken. The concentration of enkephalin in the midgut increased about 2-fold between 9 and 13 days of incubation and remained constant until hatching. In the 7-day duodenum, metenkephalin immunoreactivity was found in a network of darkly stained nodes (accumulations of ganglion cells) faintly stained internodal nerve bundles; this network of immunoreactivity was localized to the myenteric plexus. By 9 days of incubation, the network was more extensive and the intensity of staining was increased. At 13 days of incubation, varicosities were found in the region of the ganglion cells and in internodal nerve bundles. At this time, immunoreactivity was clearly visualized in the submucosal plexus. In the newly hatched chicken, met-enkephalin was found in nerves within the circular smooth muscle, as well as the myenteric and submucosal plexuses. The early appearance of met-enkephalin in the developing chick enteric nervous system suggests this peptide may act as a primary neurotransmitter in the organization and control of gut motility.

Animals↗
PubMed1981

Characterization of enkephalin release from rat striatum.

Using antisera specific for methionine- and leucine-enkephalin, we studied the characteristics of the release of these peptides from rat striatal slices. Only 2-3% of the total tissue stores of enkephalin could be released by potassium-depolarization; similar percentages were released from globus pallidus, thalamus, and nucleus accumbens. Enkephalin release from hippocampus could not be detected. The striatal release of both enkephalins was affected similarly by changes in potassium and calcium levels in the superfusion medium. Lithium has no effect on either basal or potassium-stimulated release; tyr-arg did not affect basal release of either peptide. Striatal enkephalin levels were stable during the short-term incubation periods used in these experiments.

Animals↗