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Biomedical subjects

I Lindberg

Publications and source records attributed to I Lindberg.

At least 19 recordsLinked to original sources
PubMed1992

Fluorometric assay of a calcium-dependent, paired-basic processing endopeptidase present in insulinoma granules.

A novel fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC (RSKR-AMC) was used to characterize Ca(++)-activated proteolytic activity present in purified insulinoma secretory granules. Secretory granules efficiently cleaved this substrate in a time- and protein-dependent manner; the hydrolysis rate was between 2 and 4 pmol/min/ug of protein, with an apparent Km of 55 microM. Greater than 90% of the activity against this substrate was dependent on the presence of Ca++, with half-maximal stimulation obtained at 100 microM Ca++. The pH optimum of enzymatic activity was 5.5-6, and the profile of inhibition by various proteinase inhibitors was similar to that previously described for the type I and II proinsulin processing enzymes. These biochemical characteristics and co-elution of the RSKR-AMC processing activity with the type II endopeptidase activity on anion-exchange chromatography suggest that the new assay selectively detects the Lys-Arg-directed, or type II, proinsulin processing endopeptidase. This fluorogenic assay is more quantitative, sensitive and rapid than methods previously used, and therefore presents a significant improvement for the study of similar Ca(++)-activated processing endopeptidases.

Amino Acids, Diamino↗
PubMed1992

Posttranslational processing of proenkephalin in SK-N-MC cells: evidence for phosphorylation.

SK-N-MC cells have recently been shown to be a rich source of proenkephalin and/or the proenkephalin-derived peptide, peptide B. We have investigated the synthesis and the posttranslational processing of proenkephalin in these cells. SK-N-MC cells retain very little of the proenkephalin synthesized; greater than 99% of the immunoreactive enkephalin synthesized within a 48-h period is secreted into the medium rather than contained intracellularly. When medium samples were subjected to gel filtration and assayed for the various enkephalins present within proenkephalin, only two major molecular-weight classes of peptides, with molecular weights and immunoreactive profiles consistent with those of proenkephalin and the 3.6-kDa carboxyl-terminal fragment peptide B, were observed. The proenkephalin-like peptide present in medium samples was shown by western blot procedures to consist of a 32-kDa protein with a slight amount of a higher-molecular-weight immunoreactive component above it. Only proenkephalin-sized peptides were present within cell extracts. Radiolabeled proenkephalin added to cell cultures was also cleaved to products similarly sized to those found in medium extracts; radiolabeled proenkephalin incubated in the absence of cells was not cleaved. Cleavage of exogenous proenkephalin thus probably at least partially occurs following secretion. Cell radiolabeling experiments with [32P]orthophosphate demonstrated that SK-N-MC proenkephalin is phosphorylated. Microheterogeneity of proenkephalin was also observed using isoelectric focusing coupled with western blotting. Our results suggest that the SK-N-MC cell line represents a useful model to study the earliest steps of the posttranslational processing of human proenkephalin in a neuronal cell type.

Cell Line↗
PubMed1992

Characterization of proenkephalin-cleaving proteinases in bovine adrenal chromaffin granules using [35S]proenkephalin copolymerized into sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [35S]methionine-labeled recombinant rat proenkephalin as a selective substrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteinase radiozymography. This technique was used for the screening of subcellular fractions, general characterization of pH optima, and the mechanistic characterization of proteinases with both reversible and irreversible inhibitors. Two enzymes with approximate molecular masses of 76 and 30 kDa were shown to be localized to the highest-density fractions of chromaffin granules by sucrose density gradient fractionation. Both were enriched in a 1 M NaCl wash of purified chromaffin granule membranes, were active at high pH, and were characterized as serine proteinases based on inhibition by soybean trypsin inhibitor. The 30-kDa enzyme was also inhibited by diisopropyl fluorophosphate, D-Phe-Pro-Arg-CH2Cl, and D-Val-Phe-Lys-CH2Cl and appeared to be the previously described adrenal trypsin-like enzyme. A third enzyme, of 66 kDa, was also associated with the 1 M NaCl wash of purified chromaffin granule membranes but was not localized exclusively to chromaffin granules in sucrose gradients. This proteinase was found to be Ca2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, or pepstatin.

Adrenal Glands↗
PubMed1992

Posttranslational processing of proenkephalin in AtT-20 cells: evidence for cleavage at a Lys-Lys site.

Proteolytic processing of proenkephalin was examined in several subclones of AtT-20 cells stably transfected with rat proenkephalin cDNA (AT/PE cells). Proenkephalin is synthesized in both N-glycosylated and unglycosylated forms, as demonstrated by treatment with tunicamycin. RIAs and Western blot studies showed that AT/PE clones process proenkephalin at some, but not all, Lys-Arg sequences in a limited processing profile reminiscent of bovine adrenal chromaffin cells. Pulse-chase studies using Met5-enkephalin-Arg-Gly-Leu antiserum demonstrated that 50% of the precursor is processed within 1 h, and processing is complete after 2.5 h with the production of the 5.3-kilodalton (kDa) peptide. Further cleavage to the octapeptide Met5-enkephalin-Arg-Gly-Leu is minimal. Radiosequencing results verified the efficient cleavage of a Lys-Lys site within proenkephalin that resulted in the production of the 5.3-kDa peptide. Proenkephalin cleavage products stored within cells, which included the 5.3-kDa peptide, could be released upon stimulation of cells with BaCl2 (2-fold above basal levels), 8-bromo-cAMP or CRF (7- and 8-fold above basal levels, respectively), and a mixture of BaCl2 and 8-bromo-cAMP (20-fold above basal levels). An important difference between the processing of proenkephalin and the ACTH/endorphin precursor (POMC) in AtT-20 cells is efficient cleavage of a Lys-Lys site in proenkephalin and not in POMC. The ability of AT/PE to process proenkephalin in a natural manner makes it a suitable model system to investigate elements involved in the processing of proenkephalin at Lys-Lys sites.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells.

A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Posttranslational modifications of rat proenkephalin overexpressed in Chinese hamster ovary cells.

Rat proenkephalin was overexpressed in Chinese hamster ovary cells using the dihydrofolate reductase-coupled genetic amplification method. About 2 mg purified protein could be obtained from 250 ml conditioned medium; multiple successive harvests could be obtained from the same roller bottle. Degradation of proenkephalin released into the conditioned medium was reduced significantly in the presence of 2% fetal bovine serum. Forty-eight percent of recombinant proenkephalin was glycosylated; glycosylation could be entirely prevented by the addition of tunicamycin. Two-dimensional isoelectric focusing experiments showed that recombinant proenkephalin exhibited considerable charge heterogeneity, with two major unglycosylated isoelectric forms and six or seven glycosylated isoelectric forms. The estimated isoelectric points of the major unglycosylated proenkephalins were 6.0 and 6.1, while glycosylated proenkephalins ranged in pI from 5.7-6.1. Some of this isoelectric heterogeneity is due to phosphorylation; [32P] orthophosphate was readily incorporated into serine residues within newly synthesized proenkephalin.

Animals↗
PubMed1991

Partial purification and characterization of a putative prohormone-processing enzyme complex from bovine pituitary.

A putative prohormone-processing enzyme complex with specificity toward basic residues was partially purified from whole bovine pituitary glands. The complex is basic, binding to S-Sepharose at pH 8.2. The pH optimum of the enzyme is around 8.0. The enzyme is capable of cleaving proenkephalin and is present in at least three forms with relative molecular masses of about 36,000, 58,000, and 90,000 Da. The proteinase complex is inhibited by soybean trypsin inhibitor, limabean trypsin inhibitor, and aprotinin, but not by inhibitors of thiol proteinases or metal chelators. Our results indicate that this proteinase is a trypsin-like serine esterase with properties appropriate to that of a prohormone-processing enzyme.

Animals↗
PubMed1990

Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes.

A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.

Adrenal Medulla↗
PubMed1990

Cleavage of proenkephalin by a chromaffin granule processing enzyme.

Human proenkephalin generated by means of a recombinant vaccinia virus expression vector was used as the substrate for a putative processing enzyme obtained from bovine adrenal chromaffin granules. The adrenal enzyme successfully cleaved proenkephalin to generate low mol wt enkephalins as well as other enkephalin-containing intermediates. Radioactively labeled proenkephalin prepared with this system was also cleaved; however, under identical conditions bovine proinsulin was not cleaved. These results provide support for the notion that the adrenal trypsin-like enzyme is involved in the processing of proenkephalin in vivo and demonstrate the usefulness of protein substrates prepared by expression vector systems in testing the reactivity and specificity of proposed prohormone processing enzymes in vitro.

Amino Acid Sequence↗
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↗