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Biomedical subjects

N Marks

Publications and source records attributed to N Marks.

At least 109 records · Page 6Linked to original sources
PubMed1965

Separation of acid and neutral proteinases of brain.

1. Cerebral proteinases were separated on Sephadex G-100 columns into acid and neutral fractions free from cross-contamination. Acid proteinases were more stable and were purified by additional steps with salt and pH5.0 precipitations, column chromatography on DEAE- or CM-cellulose and free-flow electrophoresis. 2. The separation made it possible to study the properties of the partially purified enzyme fractions. Some of these properties, such as K(m) with selected protein substrates, pH optima and temperature-dependence in the presence and absence of substrates, are described. 3. No requirement for metal ions or added cofactors was demonstrated. Neutral-proteinase activity was more sensitive to inhibition by heavy-metal ions; its activity could be increased by thioglycollate and glutathione, and inhibited by thiol reagents. Neutral and acid proteinases were inhibited by the chymotrypsin inhibitor chloromethyl l-2-phenyl-1-toluene-p-sulphonamidoethyl ketone. 4. In the presence of the appropriate synthetic substrates no cathepsin A activity was found, and only trace quantities of cathepsin B or C activities, which were more than 50-fold less than cathepsin D-like activity.

Journal Article↗
PubMedDate not supplied

Metabolism of a heptapeptide opioid by rat brain and cardiac tissue.

Met-enkephalin-Arg6-Phe7 (E7) is converted to Met-enkephalin by rat striatal membranes; in contrast, Leu-enkephalin (E5) is inactivated by cleavage at the Tyr-Gly (aminopeptidase) and Gly-Phe sites (metalloendopeptidase). Conversion of E7 is inhibited by MK-421, and inactivation of E5 is inhibited by bestatin and Thiorphan. Purified brain angiotensin converting enzyme (EC 3.4.15.1) also converts E7 but a purified metalloendopeptidase acts on both E5 and E7 at the Gly-Phe site. Cleavage of E7-amide by metalloendopeptidase leads to release of Phe-Met-Arg-Phe-amide, a cardioactive neuropeptide. Rat heart, a potential target organ, does not convert E7-amide to release the cardioactive peptide but cleaves the Met5-Arg6 bond to release Met-enkephalin by an enzyme sensitive to MK-421.

Animals↗
PubMedDate not supplied

Enkephalin analogs as substrates for the assay of brain cysteine proteinase (Cathepsin L) and its endogenous inhibitors.

A series of enkephalin-like peptides (X-Tyr-Gly-Gly-R-Pro) were synthesized for assay of cathepsin L and papain. Enzymes acted only at the Gly-Gly bond to release N-terminal dipeptides. When X = dansyl and R = Phe(NO2) the substrate was suited for continuous fluorimetric assay of rat brain cathepsin L (Km 45 microM, kcat/Km 1333 mM-1 sec-1). The substituted pentapeptides provided information on the influence of P2, P2' residues on rates of Gly-Gly cleavage. The synthetic substrate provided rapid and sensitive assays for the brain cathepsin L and its interaction with 13-14 kDa (cerebrocystatin) and 70 kDa (T-kininogen) rat brain inhibitors. The suppression of cathepsin L- or papain-mediated hydrolysis of substrates by inhibitors may be the result of competition between their binding domains at the enzyme catalytic center.

Animals↗
PubMedDate not supplied

Synthetic domains of cystatins linked to enkephalins are novel inhibitors of brain cathepsins L/B.

Cystatin domains or homologous sequences were synthesized and tested as inhibitors of papain, and rat brain cathepsins B and L. These domains included: I, an enzyme substrate binding site containing a -GG- cleavage site (YGGFL); II, known cystatin consensus sequences (-QVVAG- or -QLVSG-); and III, the proposed ancillary site for binding of chicken cystatin to papain (-IPWLN-). A Domain II analog QVVAG(K-NH2) inhibited cathepsin L and papain with Ki 1-4 X 10(-4) M but was inactive towards cathepsin B. A peptide containing Domains I and II, YGGFL-QVVAG(K-NH2), inhibited papain and cathepsin B with Ki 10(-4)-10(-5) M, and cathepsin L with Ki 10(-6) M. The presence of Domain III in the analog YGGFL-QVVAG-IPWLN(K-NH2) resulted in a 10-fold increase in potency towards papain. These data demonstrated that putative cystatin domains are: 1) probably proximal in the intact cystatins; 2) can be linked directly to each other to yield smaller peptides active as inhibitors; 3) showed some specificity towards the three cysteine proteinases.

Amino Acid Sequence↗
PubMedDate not supplied

Biodegradation of alpha-MSH and derived peptides by rat brain extracts, and by rat and human serum.

The peptides alpha-MSH and MSH/ACTH 4-10 were degraded by rat brain extracts and serum to yield free amino acids among the end-products. Breakdown of these two peptides was double that of a related synthetic hexapeptide Met (0)-Glu-His-Phe-D-Lys-Phe. No significant breakdown of the hexapeptide occurred after incubation with human serum; it also had almost negligible pigmentary effects in vivo and in vitro when compared to alpha-MSH. The patterns of amino acid release indicate possible endopeptidase cleavage at Phe-Arg in alpha-MSH followed by secondary exopeptidase action to release free amino acids. For the hexapeptide, the primary cleavage point occurred at the -His3-Phe4 bond. The stability of this analog in human sera, coupled with its lower rate of degradation in the CNS, may contribute to its more potent behavioral actions in vivo.

Adrenocorticotropic Hormone↗
PubMedDate not supplied

Metabolism of potent enkephalin analogs (FK 33-824, D-Ala2, pentafluorophenylalanine-4-enkephalinamide and a dimer of D-Ala2-enkephalin) and D-amino acid substituted derivatives of human beta-endorphin.

New analogs of the opiate peptides containing novel substitutions were compared in terms of their metabolic stability in the presence of an ultrafiltrate of mouse brain. The enkephalin analog FK 33-824 was more stable at short incubation periods (30 min) than Met-enkephalin but less stable than D-Ala2-enkephalinamide at longer periods (180 min) as shown by the complete release of N-terminal Tyr. In contrast, D-Ala2-enkephalinamide substituted in position 4 with pentafluorophenylalanine was completely stable at the time periods tested. A dimer of D-Ala2-enkephalin was relatively stable at short periods but subject to a 30% hydrolysis (vs. 100% for FK 33-824) in terms of Tyr release at longer periods of incubation. A doubly substituted human beta-endorphin (D-Leu17, D-Lys29-beta-endorphin) showed greater stability than the native peptide based on release of internal residues after incubation with the ultrafiltrate of brain. The presence of D-Leu17 blocked release of intermediate sized endorphins but did not affect liberation of Tyr. The additional presence of D-Thr in position 6 of a triply substituted beta-endorphin (D-Thr6, D-Leu17, D-Lys29-beta-endorphin) did not affect liberation of Tyr, indicating that formation of gamma-endorphin (cleavage of Leu17-Phe) and of enkephalin (cleavage of Met5-Thr) need not occur before the action of brain peptidases. The results demonstrate the feasibility of altering the resistance of analogs of enkephalin and endorphin to degradation by brain enzymes.

Amino Acids↗
PubMedDate not supplied

Protein phosphorylation in human synaptosomal membranes: evidence for the presence of substrates for cyclic nucleotide guanosine 3'-5'-monophosphate dependent protein kinases.

Synaptosomal membranes prepared from different anatomical regions of postmortem human brain readily incorporate phosphate when incubated with labelled ATP in vitro. Separation of proteins on SDS slab gels indicated up to 30 protein bands stained by Coomassie blue of which ten incorporated label, as detected by radioautography (mol. wt. range 16-110 K with a major double band at 49-50 K). Incorporation into the 110 K region appeared to increase in older persons, and that into the 49-50 K region decreased. Human but not rat membranes subjected to similar conditions of treatment failed to respond to the addition of cAMP at 10(-5) - 10(-7) M but were highly sensitive to the addition of cGMP at 10(-6) M, which led to intensification of existing bands and the appearance of a major band at 60 K. The 110 K band present in high concentration in white matter striatum, caudate nucleus, and putamen was insensitive to cGMP addition. The presence of cGMP-dependent protein kinase substrates has not previously been reported in synapses, and it may be of significance in view of the known sensitivity of this system to biological regulatory agents.

Animals↗
PubMedDate not supplied

Brain cathepsin B but not metalloendopeptidases degrade rAPP751 with production of amyloidogenic fragments. Comparison with synthetic peptides emulating beta- and gamma-secretase sites.

Lysosomal cathepsin B but not L degraded rAPP751 to yield C-terminal 19-25 kDa fragments containing beta A4, reinforcing the view that acidic proteases participate in endosomal-lysosomal processing to yield amyloidogenic fragments in situ. This mechanism is consistent with fragmentation of endogenous APPs within clathrin-coated vesicles (CVs) by vesicular hydrolases, with the appearance of C-terminal amyloidogenic fragments following incubation at pH 6.5. A neutral endopeptidase resembling NEP 24.11 (PS-NEP) purified from detergent extracts of human brain degraded rAPP751; however, breakdown was not blocked robustly by metal chelators or phosphoramidon, suggesting the presence of an alternative processing enzyme. Effects of other inhibitors showed that breakdown was mediated by serine-protease-like component(s). A phosphoramidon-insensitive metalloendopeptidase (PI-NEP) partially purified from rat brain P2 using detergents, and resembling NEP 24.15, showed no activity towards rAPP751. Peptides containing putative beta- or gamma-secretase sites were synthesized for purposes of examining their metabolism by the brain enzymes. Those containing beta-secretase sites were hydrolysed at one or more sites by the four enzymes, but only PI- and PS-NEP acted at the Met-Asp site of Ac-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg.NH2. In the case of substrates containing the gamma-site, these two categories of enzymes were the only ones degrading N-Ac-Ile-Ala.NH2. These data imply that the brain metalloendopeptidases, while inactive towards intact precursors, may be involved in turnover of intermediates containing beta- or gamma-sites.

Amino Acid Sequence↗
PubMedDate not supplied

Chimeric enkephalin-cystatins: opioid binding and structure-activity relationships of inhibitory domains.

A chimeric inhibitor of cysteine proteinases, YGGFLQVVAGK.amide, was synthesized for use in examining SAR of its cystatin and opioid domains. Analogs were prepared by solid-phase syntheses and evaluated for inhibition of rat brain cathepsin L (E.C. 3.4.22.15) and papain (E.C.3.4.22.2), or binding to rat brain opioid receptors using tritiated DSLET (delta), DAGO (mu) and U69593 (kappa) in competitive binding assays. Cystatin consensus analogs QVVAGK- or N-Ac.QVVK.amide were weakly active as inhibitors, but were enhanced 10-fold to 20-fold with N-terminal L-, FL-, GFL-, GGFL-, or YGGFL, yielding Ki 3 microM and 26 microM for cathepsin L and papain, respectively. YGGFL itself was inactive, but expressed inhibition with N-terminal Q-, QV-, QVV-, etc. Highest activity was found with YGGFLQVK.amide (Ki, 2 microM). N-Dns analogs differentially increased inhibition for papain, whereas an N-Ac-cyclic peptide, N-Ac-CKYGGFLQVVKC.amide, was 2-fold to 10-fold less potent than the two-domain inhibitor. Chimeric peptides containing YGGFL were equipotent as delta-ligands (Ki, 1.7-3.7 nM), but were 2-fold to 10-fold more potent as mu ligands (3.7-11 nM) versus YGGFL itself (37 nM). Two analogs with -QVVAK- and -QVK.amide expressed kappa-binding properties. These data demonstrate the feasibility of using chimeric peptides as probes for exploring enzyme catalysis, and the potential for targeting inhibitors to endosomal or lysosomal compartments via receptor-mediated uptake in cells.

Amino Acid Sequence↗