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J A Biggins

Publications and source records attributed to J A Biggins.

At least 19 recordsLinked to original sources
PubMed1992

Human brain peptidase activity with the specificity to generate the N-terminus of the Alzheimer beta-amyloid protein from its precursor.

The synthetic peptide acetyl-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-NH2, which spans the cleavage site (Met-Asp) required to generate the N-terminus of the Alzheimer beta-amyloid protein from its precursor, was used to search for human brain peptidases which may be involved in this potentially amyloidogenic process. In both soluble and particulate fractions from human brain the primary cleavage point of the peptide was the Met-Asp bond. Purification and characterization of the activity from the soluble fraction showed that it was metalloendopeptidase 24.15 (EC3.4.24.15). This enzyme is therefore a candidate for the generation of the N-terminus of beta-amyloid protein from its precursor.

Alzheimer Disease↗
PubMed1991

Human brain leucyl aminopeptidase: isolation, characterization and specificity against some neuropeptides.

In order to obtain a greater understanding of the role of aminopeptidases in the degradation of peptides and proteins in the nervous system, we have isolated and characterized leucyl aminopeptidase (EC 3.4.11.1) from human cerebral cortex and studied its action on some physiologically important neuropeptides. The enzyme has a low specificity constant for the hydrolysis of Leu-7-amido-4-methylcoumarin (69s-1M-1) but the peptides Tyr-Gly-Gly and Tyr-Gly-Gly-Phe-Leu (Leu5-enkephalin) were much better substrates (specificity constants 8300 and 18050s -1M-1 respectively). Optimum activity for the degradation of Leu-enkephalin was obtained at pH10.5 in the presence of 5mM-Mn++. A sharp drop in specificity constant occurred with increasing chain length in the series Leu-enkephalin, dynorphin 1-8, 1-10 and 1-13, suggesting that the enzyme functions only as an oligopeptidase. Other neuropeptides were poor substrates (cholecystokinin octapeptide, angiotensin-I) or not hydrolysed at all (somatostatin, Arg8-vasopressin).

Amino Acid Sequence↗
PubMed1991

Multicatalytic, high-Mr endopeptidase from postmortem human brain.

The main high molecular weight (650K) multicatalytic endopeptidase has been purified from postmortem human cerebral cortex. As in other tissues and species, this enzyme is composed of several subunits of 24-31K and has three distinct catalytic activities, as shown by the hydrolysis of the fluorogenic tripeptide substrates glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin, benzyloxycarboxyl-Gly-Gly-Arg-7-amido-4-methylcoumarin, and benzyloxycarboxyl-Leu-Leu-Glu-2-naphthylamide with hydrophobic (Phe), basic (Arg), and acidic (Glu) residues in the P1 position, respectively. These activities are distinguishable by their differential sensitivity to peptidase inhibitors. The enzyme hydrolysed neuropeptides at pH 7.4 at multiple sites with widely differing rates, ranging from 113 nmol/min/mg for substance-P, down to 2 nmol/min/mg for bradykinin. The enzyme also had proteinase activity as shown by the hydrolysis of casein. For the hydrolysis of the Tyr5-Gly6 bond in luteinizing hormone-releasing hormone, the Km was 0.95 mM and the specificity constant (kcat/Km) was 4.7 X 10(3) M-1 s-1. The bond specificity of the enzyme at neutral pH was determined by identifying the degradation products of 15 naturally occurring peptide sequences. The bonds most susceptible to hydrolysis had a hydrophobic residue at P1 and either a small (e.g., -Gly or -NH2) or hydrophobic residue at P'1. Hydrolysis of -Glu-X bonds (most notably in neuropeptide Y) and the Arg6-Arg7 bond in dynorphin peptides was also seen. Thus the three activities identified with fluorogenic substrates appear to be expressed against oligopeptides.

Brain↗
PubMed1988

Purification and characterization of two soluble Cl(-)-activated arginyl aminopeptidases from human brain and their endopeptidase action on neuropeptides.

Two closely related Cl(-)-activated arginyl aminopeptidases (I and II) were purified from a soluble extract of postmortem human cerebral cortex by anion-exchange chromatography and preparative gel electrophoresis. The electrophoretic mobility of II was approximately 80% that of I; the molecular mass of both enzymes was approximately 70 kilodaltons (kDa) (gel filtration). The aminopeptidase action of I and II on aminoacyl-7-amido-4-methylcoumarin (AMC) substrates was restricted to the Arg and Lys derivatives. Both enzymes had significant endopeptidase activity, hydrolysing several biologically active peptides including neurotensin, bradykinin, angiotensin-I, substance P, luliberin, and somatostatin at internal bonds. Other peptides [Leu-enkephalin, proctolin, thyroliberin, adrenocorticotropin18-39 (ACTH18-39), ACTH11-24, and dynorphin (1-13)] were not appreciably hydrolysed. The amino- and endopeptidase activities had pH optima at 6.5 and 7, respectively, and were both inhibited by metal ion chelators and sulphydryl group blocking agents. The aminopeptidase activity was stimulated 20-fold by Cl- ions, whereas the endopeptidase activity was unaffected by the latter. Km values for neurotensin degradation were 20 microM (I) and 37 microM (II) and for Arg-AMC hydrolysis they were 167 microM (I) and 125 microM (II). The endopeptidase activity was not inhibited by the aminopeptidase inhibitors arphamenine or bestatin (IC50 = 9 nM and 0.1 microM, respectively, with Arg-AMC substrate).

Aged↗
PubMed1987

Ectopic corticotrophin-releasing-factor and growth hormone releasing factor secretion: diagnosis using human pituitary cell culture.

Cell culture of human pituitary tissue has been used to diagnose a patient with Cushing's syndrome due to ectopic secretion of corticotrophin-releasing factor (CRF; case 1) and a case of acromegaly associated with ectopic secretion of a growth-hormone releasing factor (GRF; case 2). In both patients a pituitary tumour was not detected. Case 1 had a small cell carcinoma and symptoms of the ectopic ACTH syndrome, but in culture the carcinoma failed to secrete detectable ACTH. However, the culture medium used to maintain this carcinoma in vitro was found to contain a substance which stimulated ACTH secretion by human pituitary corticotrophs in cell culture. Radioimmunoassays and HPLC indicated that this substance had similar elution characteristics to human CRF and cross-reacted with antiserum to ovine CRF. Case 2 was found to have a lung tumour, the removal of which led to regression of her acromegalic symptoms. In culture, this tumour did not secrete GH, but did secrete a GRF. We conclude that the Cushing's syndrome and acromegaly, in cases 1 and 2, respectively, were due to ectopic secretion of CRF and GRF leading to hyperstimulation of the pituitary gland.

Acromegaly↗
PubMed1986

Stability of [D-Trp11]-neurotensin to rat brain peptidases.

The stability of neurotensin (NT) and a potent, long lasting analogue, [D-Trp11]-NT, to rat brain peptidases was compared by incubating the peptides with subcellular fractions (synaptosomes, synaptic membranes) and a purified endopeptidase from rat brain. Degradation of the peptides with time was followed by high performance liquid chromatography (HPLC). The rates of degradation (pmol/min/mg prot.) in synaptosomes were 890 (NT) and 59 [D-Trp11]-NT), and in synaptic membranes were 1180 (NT) and 12 ([D-Trp11]-NT). The main products of the degradation of [D-Trp11]-NT by synaptic peptidases (isolated by HPLC and characterized by amino acid analysis) were the 1-3, 1-4 and 6-13 fragments implying cleavage of [D-Trp11]-NT at the Tyr3-Glu4, Glu4-Asn5 and Asn5-Lys6 bonds. The rates of degradation of NT and [D-Trp11]-NT by the purified endopeptidase from rat brain were 27.2 and 0.76 pmol/min/microliter of enzyme solution respectively. This endopeptidase, which hydrolyses NT at Arg8-Arg9, may be responsible along with other endopeptidases for NT degradation at nerve terminals.

Animals↗
PubMed1986

Molecular forms of somatostatin, substance P and neurotensin in fresh human cerebral cortex.

The molecular forms of somatostatin, substance P and neurotensin in fresh normal human cerebral cortex have been investigated using high performance liquid chromatography and radioimmunoassay. For each peptide most of the immunoreactivity measured corresponds to a single molecular form co-eluting with the authentic peptide. Small differences in the minor peaks of somatostatin and substance P immunoreactivity are seen when compared to the results of similar studies on post mortem human brain. A substantial difference in the molecular forms of neurotensin was seen which suggests that degradation of this peptide may occur post mortem.

Adult↗
PubMed1986

Neuropeptides in gliomas: identification of somatostatin 14 in a medulloblastoma.

Forty nine gliomas were analysed for the following neuropeptides: somatostatin (SS), substance P (SP), neurotensin (NT) and vasoactive intestinal polypeptide (VIP) and the pituitary peptide, adrenocorticotrophin (ACTH). A significant amount of authentic SS was found in a medulloblastoma, and low concentrations of SP and NT immunoreactivity in an ependymoma and cerebellar astrocytoma respectively. The majority of the other gliomas did not contain detectable levels of these five neuropeptides. Low levels of neuropeptides were found in some specimens probably due to contamination with cerebral cortex.

Astrocytoma↗
PubMed1985

Specificity of neuropeptide degradation by two calcium-activated neutral proteases from human skeletal muscle.

Two calcium-activated neutral proteases (CAPI & II) were purified from human skeletal muscle by anion exchange, gel filtration and affinity (antipain-Sepharose and Blue Ultrogel A4R) chromatography. The enzymes were homogenous as judged by polyacrylamide gel electrophoresis, and have similar properties with the exception of the Ca2+ concentration required for optimum activity (CAP I = 0.1 mM; CAP II = 1 mM). Both enzymes hydrolysed a wide variety of neuropeptides. In six cases, the products were separated and identified by hplc and amino acid analysis. Neurotensin was hydrolysed at Tyr3-Glu4; dynorphin1-13 at Arg8-Arg9; LH-RH at Gly6-Leu7; CCK-8 at Phe8-NH2, substance-P at Met10-NH2; somatostatin at Thr10-Phe11. Although differences in the rates of neuropeptide degradation were noted for the two CAP's the specificity was the same for these six peptides. It is suggested that conformational requirements may be more important than side chains adjacent to the cleavage site in directing the specificity of CAP.

Amino Acids↗
PubMed1985

Somatostatin immunoreactivity in cortical and some subcortical regions in Alzheimer's disease.

Reverse phase HPLC analysis of somatostatin immunoreactivity in the cerebral cortex in elderly normal individuals revealed that the majority of the immunoreactivity co-eluted with synthetic somatostatin-14. While an immunoreactive peak corresponding to somatostatin-28 was not detected there was a peak of immunoreactivity which eluted after somatostatin-14. In cases of senile dementia of Alzheimer type (SDAT), where abundant neurofibrillary tangles and senile plaques (density greater than 30 per 1.3-mm2 field) were present in the cerebral cortex, somatostatin immunoreactivity was found to be significantly decreased in either the frontal or temporal cortex. Chromatographic analysis, however, revealed that both the major immunoreactive peaks detected in the normal group were reduced in SDAT in the temporal and frontal cortex. Using a punch microdissection technique somatostatin immunoreactivity has been assessed in the nucleus of Meynert and amygdala of SDAT and elderly normal cases. While there was no change in somatostatin immunoreactivity in the nucleus of Meynert in the SDAT group, tissue punches taken from the amygdala revealed a selective decrease in somatostatin immunoreactivity in the basal nucleus, in the SDAT cases.

Aged↗
PubMed1984

N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) affects locomotor activity without producing a nigrostriatal lesion in the rat.

Drug addicts abusing heroin substitutes contaminated with N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and perhaps those who work with this substance, may develop symptoms similar to those seen in Parkinson's disease [7, 12, 13]. We describe the results of a study in which rats were given daily injections of MPTP for two weeks. A progressive suppression of activity was seen, but the subjects rapidly recovered when treatment ceased. The animals were then injected with D-amphetamine or apomorphine; the former drug enhanced activity, to levels seen in control (non-MPTP treated) subjects. Apomorphine had no effect, either on control or MPTP-treated subjects. The effects of acute (0, 2.5, 5.0 and 10.0 mg per rat) administration of MPTP were also studied. The two lower doses significantly decreased activity, but the highest dose did not. Histological examination showed that 2 weeks' treatment with MPTP did not produce neuronal degeneration in the pars compacta of the substantia nigra (SN). In these animals, there were no changes in levels of dopamine, 5-hydroxytryptamine, or their metabolites in either the SN or the caudate nucleus. MPTP had no effect on the levels of neurotensin, somatostatin and substance P in several brain areas. It is concluded that MPTP has reliable effects on locomotor activity in rats without producing measurable histological or neurochemical changes in the nigrostriatal dopaminergic system.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1984

Chromatographic characterization of neuropeptides in post mortem human brain.

Reverse phase high performance liquid chromatography was used to establish the immunoreactive species of five neuropeptides (thyrotropin-releasing hormone, luteinising hormone-releasing hormone, neurotensin, substance-P and somatostatin) in three areas of post mortem human brain--the hypothalamus, amygdala and cortex. In the majority of cases the major immunoreactive peak corresponded to the authentic peptide, although other peaks of immunoreactivity were observed in several instances. It was established that somatostatin-14 was present as the major immunoreactive form and that somatostatin-28 did not occur in any of the three brain areas, although other somatostatin-immunoreactive peaks of unknown structure were detected. In addition to authentic neurotensin in the cortex, a substantial peak of immunoreactivity corresponding to the elution time of neurotensin (1-11) was observed. LH-RH was not detected in the amygdala, but was present in the cortex as a minor component of overall immunoreactivity. The major peak of substance-P immunoreactivity in all three brain areas corresponded to authentic substance-P; in addition immunoreactive material eluting in the region of [Met-O] substance-P, substance-P (5-11) and substance-P (6-11) were detected. TRH occurred as the major peak in all three areas, although minor peaks of immunoreactivity were seen in the amygdala.

Adult↗
PubMed1983

Post mortem levels of thyrotropin-releasing hormone and neurotensin in the amygdala in Alzheimer's disease, schizophrenia and depression.

The levels of neurotensin and thyrotropin-releasing hormone (TRH) in normal post mortem human amygdala have been compared with those in cases of schizophrenia, Alzheimer's disease and depression. Amongst various factors which can influence post mortem human brain biochemistry (including age, sex, post mortem delay, time of death, disease status and severity), sex difference appeared to be responsible for the most extensive variation. The levels of both peptides were nearly doubled in males compared with females and this increase was significant in the case of neurotensin. There was also a positive correlation between neurotensin and TRH levels. Although levels of neurotensin and TRH tended to be lower in the disease groups these trends did not reach significance.

Aged↗
PubMed1982

Characterization of corticotropin-like immunoreactive peptides in rat brain using high performance liquid chromatography.

The molecular nature of corticotropin (ACTH)-related peptides in rat brain has been studied using high performance liquid chromatography (HPLC) and radioimmunoassay. The major ACTH-immunoreactive species in rat hypothalamic extracts coelutes with corticotropin-like intermediate lobe peptide (CLIP); ACTH18-39) on two HPLC solvent gradients, and has 3-4 times more C-terminal than N-terminal immunoreactivity. N-terminal ACTH-immunoreactivity is composed of a number of peaks on HPLC with less than 10% eluting at the position of ACTH. Hypothalamic C-terminal ACTH immunoreactivity is also heterogeneous and resembles in some respect that seen in the rat neuro-intermediate lobe. Around 90% of the alpha-MSH immunoreactivity in the hypothalamus elutes as a single peak in the position of des[N-acetyl] alpha -MSH (ACTH1-13-NH2).

Adrenocorticotropic Hormone↗