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Biomedical subjects

J Silberring

Publications and source records attributed to J Silberring.

At least 73 records · Page 4Linked to original sources
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1990

Fast atom bombardment mass spectrometric analysis of arginine-containing neuropeptides.

A new procedure using fast atom bombardment mass spectrometry for the determination of arginine residues in neuropeptides is described. The technique is based on the modification of the arginine side-group with 1,2-cyclohexanedione. This novel procedure may have a potential use in the study of neuropeptides and their processing and degrading enzymes.

Amino Acid Sequence↗
PubMed1990

Conversion of the dynorphins to Leu-enkephalin in human spinal cord.

This study describes three different endopeptidases in human spinal cord with high affinity for the dynorphins. The enzymes, with strict specificity towards paired basic residues, release Leu-enkephalin and its C-terminal extensions - Arg6 and -Arg6 -Arg7 from the substrate peptides. They differ with regard to molecular size, charge properties, and inhibitory profile. Two of the enzymes show optimal activity at neutral pH, whereas the third enzyme has its activity optimum at pH 5.6. They are all suggested to be of importance for the formation of Leu-enkephalin from prodynorphin.

Amino Acid Sequence↗
PubMed1989

A novel bovine spinal cord endoprotease with high specificity for dynorphin B.

An endopeptidase that converts the opioid peptide dynorphin B (Tyr-Gly-Gly-Phe-Leu-Arg-aRg-Gln-Phe-Lys-Val-Val-Thr) to its bioactive fragment Leu-enkephalin-Arg6 was isolated from bovine spinal cord. The enzyme was purified about 230-fold from a concentrated spinal cord extract. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it stained as a protein of Mr 55,000. The purified enzyme is optimally active at around pH7 and has essential thiol groups. It appears to be highly specific for dynorphin B (Km = 11 microM) but not for alpha-neoendorphin or dynorphin A, two other opioids included in the prodynorphin precursor. From its specificity, molecular size, and inhibitory spectrum, this enzyme is different from other known dynorphin-converting or -degrading enzymes and appears to be a unique and novel endoprotease.

Amino Acid Sequence↗
PubMed1989

Application of high performance liquid chromatography combined with diode-array detection for analysis of proteins and peptides in human cerebrospinal fluid.

Different strategies for HPLC separation, including molecular sieving, ion-exchange, and hydrophobic interaction as well as reversed phase chromatography, were used to study molecular components in human cerebrospinal fluid (CSF). The separations were followed by photodiode-array UV detection, which is a recently developed technique allowing a direct and rapid discrimination between peptides and proteins differing in their content of aromatic amino acids. By the various HPLC techniques in conjunction with diode-array detection it was possible to identify and characterize several protein and peptide components present in CSF. The procedure also allowed quantitative analysis of CSF proteins using minute amounts of the fluid.

Cerebrospinal Fluid Proteins↗
PubMed1988

Noncatalytic protein component of elastase from horse leucocytes. A protein with regulatory function.

1. Noncatalytic protein component (NPC), a strongly acidic protein (pH = 4.5) was separated from native horse leucocyte elastase 1. 2. This protein reduces elastinolytic properties of elastases: 1 and 2A probably by decreasing their isoelectric points. 3. A possible regulatory role of this protein may be inferred from a higher affinity of elastase 1 to NPC rather than to elastin.

Animals↗
PubMed1988

Kinetics of [3H]-prazosin binding to the rat cortex during aging.

Kinetics (Bmax, KD, k on, k off) of [3H]-prazosin binding to the rat cortex was measured at different ages of animals (2-4, 13-15, 20-24 months old). Additionally, an agonist (phenylephrine) and an antagonist (phentolamine) inhibition of [3H]-prazosin binding was performed at two different rat ages (2-4 and 20-24 months old). The number of cortical alpha 1-adrenoceptors was significantly reduced in 20-24-month-old rats, when compared with 2-4-month ones. Association (k on) and dissociation (k off) rate constants were also reduced in the oldest group, but the dissociation constant (KD) was similar in all the age groups tested. Affinity of phenylephrine, but not of phentolamine, was reduced in the oldest group (20-24 months old). The obtained data suggest that changes in the brain alpha 1-adrenoceptors, correlated with the animal age, are connected to the number of binding sites, rate constants and the affinity of an agonist.

Aging↗
PubMed1987

The in vitro release of immunoreactive dynorphin and alpha-neoendorphin from the perfused rat duodenum.

The release of immunoreactive (ir) dynorphin (DYN) and alpha-neoendorphin (ir-ANEO) from the isolated perfused rat duodenum was demonstrated using specific radioimmunoassays (RIAs). Depolarization of the tissue by increasing the potassium (K+) concentration up to 108 mM enhanced the release of ir-DYN and ir-ANEO in Ca2+-dependent manner. Administration of the serotonin-releasing agent fenfluramine (10(-6) M) and the serotonin receptor agonist m-chlorophenylpiperazine (m-CPP, 10(-6) M) stimulated the release of ir-DYN and ir-ANEO from the duodenum. A subsequent study revealed that serotonin (5-HT, 10(-6)-10(-4) M) induced a dose-dependent increase in the release of ir-DYN and ir-ANEO from the duodenum. The effect of 5-HT on the release of ir-DYN and ir-ANEO from the duodenum was antagonized by 5-HT antagonist cyproheptadine (10(-6) M). The presence of dynorphin and the related peptides in the gastrointestinal tract (GIT) and their release from the duodenum in vitro indicate that these peptides may act as transmitters involved in some GIT functions. Furthermore, our results suggest that at least part of 5-HT effects on the GIT may be mediated by the release of dynorphin and the related peptides.

Animals↗
PubMed1987

The influence of chronic stress on multiple opioid peptide systems in the rat: pronounced effects upon dynorphin in spinal cord.

Recurrent exposure to intermittent electrical foot-shock (30 min, twice daily) for 7 days caused an increase in immunoreactive (ir) dynorphin and ir-alpha-neo-endorphin in lumbar and cervical (but not thoracic) spinal cord as measured 16 h following the final session. At this time the level of ir-Met-enkephalin-Arg6-Gly7-Leu8 (MEAGL) was also increased at the lumbar level. An acute foot-shock depleted spinal cord dynorphin in chronically stressed but not in naive rats. No alterations in levels of ir-dynorphin or ir-MEAGL were seen in discrete brain tissues. In contrast to the brain, where no effects were seen, the levels of beta-endorphin increased in both lobes of the pituitary. This change, however, was not accompanied by an alteration in levels of beta-endorphin in plasma. These data show that chronic foot-shock stress selectively influences particular pools of opioid peptides, predominantly those derived from proenkephalin B in the spinal cord and from proopiomelanocortin in the anterior pituitary. It is suggested that alterations observed in the spinal cord reflect enhanced activity of the proenkephalin B system in response to chronic nociceptive stimulation.

Animals↗
PubMed1986

A modified radioimmunoassay for arylsulfatase A in human serum and urine.

A radioimmunoassay was developed for the determination of arylsulfatase A (EC 3.1.6.1) in human serum and urine. An isoenzyme of arylsulfatase A purified from human urine was used as a standard antigen. The enzyme was radioiodinated with 125I using the Chloramine T method and was stable for about 4 wk. Antibody-bound enzyme was separated from free enzyme by means of a double antibody technique in the presence of polyethylene glycol (PEG). The working range of the method was 0.15-5.0 ng of arylsulfatase A per assay. The within-assay CV was about 8% for both biological fluids and the between-assay CV for serum was 14.1%. Analytical recoveries were 93.2 +/- 9.1% and 97.8 +/- 5.5% for serum and urine, respectively, and the sensitivity was 0.040 ng of arylsulfatase per assay. Serum samples of 50 healthy blood donors were assayed to establish the normal serum level of immunoreactive enzyme, which was found to be 8.3 ng/ml +/- 1.8 ng/ml of serum. Storage of frozen serum was shown to have no significant effect on results obtained using this RIA.

Adult↗
PubMed1986

Release of opioid peptides from the spinal cord of rats subjected to chronic pain.

Chronic localized pain increased the level of the opioid peptides, dynorphin (DYN), alpha-neoendorphin (ANEO), Met-enkephalin (MET) and Mets-enkephalin-Arg6-Gly7-Leu8 (MEAGL), in the lumbar enlargement of the rat spinal cord. It was accompanied with a reduction of the spontaneous and K+-stimulated release of ANEO and MEAGL from spinal cord slices in vitro and a decreased release of ANEO from the spinal cord in vivo. The results indicate that the reduction in the activity of endogenous opioid peptide systems might occur in the spinal cord of rats subjected to chronic pain.

Animals↗
PubMed1986

Elastinolytic activity of horse leukocyte proteinases. Comparison with elastases from human leukocytes and porcine pancreas.

Three proteinases from the azurophilic granules of horse leucocytes are typical elastases degrading elastin at neutral pH. Both proteinases: 1 and 2A exhibit similar elastinolytic activity, comparable with human leucocyte elastase (HLE). In relation to human enzyme, elastase 2B shows several-fold higher activity, which is comparable to the porcine pancreatic elastase activity (PPE). Similarly to HLE elastinolytic activity of the horse proteinases increases at higher ionic strength: twofold in case of 1 or 2A and fivefold for 2B. Significant activity observed during degradation of homologous lung elastin, implies the possible role of these enzymes during pathological injury of connective tissue in the lower respiratory tract and suggests similar pathogenesis of horse and human pulmonary emphysema.

Animals↗
PubMed1984

Alpha 2-macroglobulin from horse plasma. Purification, properties and interaction with certain serine proteinases.

alpha 2-macroglobulin was isolated by polyethylene glycol precipitation, gel filtration on Sephacryl S-300 and DE-52 cellulose chromatography, with 20% yield. The preparation obtained was homogenous as tested by biochemical and immunological criteria. Its molecular mass was estimated at 800,000, comprising of four identical subunits. The isoelectric point of our preparation was 4.8 and two molecules of serine proteinases per 1 molecule of inhibitor were bound.

Animals↗