Isolation and quantitation of several new peptides from the canine neurotensin/neuromedin N precursor

Using antisera towards the bioactive peptides, neurotensin and neuromedin N, as well as towards the N-terminal and C-terminal regions of their shared 170-residue precursor, peptides representing various portions of the precursor were isolated from extracts of canine ileum. In total, seven peptides were isolated, two of which had not been previously identified. One was the C-terminal tail of the precursor (Gly-Ser-Tyr-Tyr-Tyr) and the other was the tail peptide extended N-terminally to include neurotensin (Glp-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-Lys-Arg-Gly-Ser-Tyr-Tyr-Tyr). By comparing the measured concentrations for each of the identified peptides, it was established that processing at the three Lys-Arg cleavage sites within the precursor did not occur to the same extent. Cleavage at the N-terminus of neuromedin N was 10% complete, that between neurotensin and the tail was 90% complete, and that between neuromedin N and neurotensin was 95% complete. Three immunoreactive proteins were also identified by immunochemical and chromatographic analyses: N-terminally extended neuromedin N (125 residues), N-terminally extended neurotensin (140 residues), and the entire 147-residue precursor. It was concluded that neurotensin, tail and large molecular neuromedin N were the primary products of processing for this precursor in canine ileum, while minor products included neuromedin N, neurotensin tail, and large molecular neurotensin.     

VIP receptors on canine submucosal synaptosomes

The present study characterized [¹²⁵I]VIP binding to synaptosomes from the submucosa of canine small intestine. Studies of saturation, competition binding, and kinetic studies revealed high- and low-affinity binding sites. Studies with GTP-γ-S and cholera toxin suggested that the receptor was coupled to a G-protein, possibly G_s. Competition with VIP analogs suggested that the N-terminal end of the molecule played the major role in determining affinity and that this receptor was for VIP, not PACAP. Cross-linking VIP to the receptor revealed a single peptide (M_r 60,000). We suggest that VIP may act to modulate mediator release from enteric nerve endings.     

The effect of N-terminal substitutions on the biological activity of MSH fragments.

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of -melanotropin, [Glp⁵]-MSH(5–10), [Glp⁵, -Phe⁷]-MSH(5–10), [Sar⁵, -Phe⁷]-MSH(5–10), [Nle⁴, -Phe⁷]-MSH(4–10), [N-carbamoyl]-MSH(5–10), and formyl and acetyl derivatives of -MSH(5–10), [Gly⁵]-MSH(5–10) and [Gly⁵, -Phe⁷]-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.     

Some studies on the metabolism of labelled molybdenum compounds in cattle

An initial experiment showed that [99Mo]di- and trithiomolybdates could be detected in bovine plasma after the introduction of [99Mo]molybdate into the rumen. It was felt that this justified the use of [99Mo]trithiomolybdate for the subsequent studies made of plasma thiomolybdate metabolism in vivo in cattle. Rapid intravenous injection of [99Mo]trithiomolybdate into cattle showed that doses of 50 mg Mo were subject to extensive hydrolysis over the first few minutes postinjection, but at lower dose rates this was reduced so that tracer doses (less than 1.5 mg Mo) were relatively stable. The plasma metabolism was unaffected by copper status within the limits of the experiments (that is, liver copper levels down to 9 mg/kg d.m.) The disappearance of [99Mo] and [35S]trithiomolybdate (1 mg Mo) from plasma was delayed for up to 10 hr by the immediate preinjection of copper, although no chemical modification of the thiomolybdate appeared to occur.     

The N-terminal domain of desmin is not involved in intermediate filament formation: evidence from thrombic digestion studies

Thrombic digestion of chicken gizzard desmin resulted in the cleavage of six arginyl bonds in the desmin molecule (at arginine residues 27, 33, 48, 59, 67 and 96), all of which are located in the N-terminal globular domain of the molecule. The large thrombic fragment (residues 97–463) of desmin isolated from this digest, which contains the central rod and C-terminal non-helical regions of desmin, was found to form filamentous structures indistinguishable from those of intact desmin. These results indicate that, in contrast to previous predictions, the N-terminal domain of desmin is not essential for intermediate filament formation.     

The purification and characterization of a novel D(−)-specific carbamoylase enzyme from an Agrobacterium sp.

A carbamoylase enzyme was purified from a cell-free extract of Agrobacterium sp. with an overall yield of 81%. It was judged to be homogenous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight of 38,000 daltons. Further studies on the native enzyme suggested that the active enzyme was present as a dimer, with a pI of 5.5. It was able to cleave a variety of N-carbamoyl substrates, but was strictly D(−) specific. It was found to have a K_m of 0.82 m and a V_max of 31 U mg⁻¹ for D(−) N-carbamoyl hydroxyphenylglycine in the presence of 10 m dithiothreitol. It showed no metal ion requirements but was inhibited by iodoacetic acid and iodoacetamide, both thiol reagents. The N-terminal amino acid sequence of the enzyme was elucidated.     

Recognition of a 10 base pair sequence of DNA and stereochemical control of the binding affinity of chiral hairpin polyamide-Hoechst 33258 conjugates

Chiral hairpin polyamides linked to a Hoechst 33258 analogue at the -position of the hairpin turn amino acid (1, 2) were synthesized on solid phase by adopting Fmoc and ivDde techniques. The DNA-binding properties of enantiomeric conjugates 1 and 2, and N-terminal linked conjugate 3 for 8–14 bp sequences were determined by spectrofluorometric and thermal melting studies. Conjugates 1 and 2 recognize a 10 bp sequence, while conjugate 3 recognizes a 9 bp sequence. Interestingly, R-enantiomer 1 exhibited 10- to 30-fold higher binding affinities than S-enantiomer 2 for the DNA sequences studied. These binding differences were accounted for by molecular modeling studies, which revealed that the amide proton nearest to the chiral center in R-conjugate 1 is better positioned to form hydrogen bonds to the DNA bases, while S-conjugate 2 does not.     

AT4 receptor structure-binding relationship: N-terminal-modified angiotensin IV analogues

The effect of structural changes in the N-terminal amino acid of AIV, with respect to AT₄ receptor binding, was examined by competition with [¹²⁵I]AIV in bovine adrenal membranes. Analogues with modifications of the first residue -amino group possessed lower affinities than the primary amine-containing parent compound. Peptides with a residue 1 -carbon in the conformation exhibited poor affinity for the AT₄ receptor. Modifications of the residue 1 R-group demonstrate that a straight chain aliphatic moiety containing four carbons is optimal for receptor-ligand binding, as evidenced by the extremely high affinity of [Nle¹]AIV (K_i = 3.59±0.51 pM). Replacement of the 1–2 peptide bond of AIV with the methylene bond isostere Ψ (CH₂-NH), increased the K_i approximately fivefold, indicating that the peptide bond may be replaced wihle maintaining relatively high-affinity receptor binding.     

Isolation and properties of plastocyanin from Anabaena variabilis

Plastocyanin is a copper protein found in photosynethetic tissue and it exhibits the properties of a physiological redox reagent. This protein has been purified from the blue-green alga Anabaena variabilis. Plastocyanin is required for a number of partial reactions of the photosynthetic electron transfer chain. These reactions include the transfer of electrons from reduced 2,3′,6-trichlorophenolindophenol,N,N,N′,N′- tetramethyl-p-phenylenediamine and 2,3,5,6-tetramethyl-p-phenylenediamine to low potential oxidants. Reduced cytochrome c photooxidation does not appear to be dependent on plastocyanin. Cytochrome f, isolated from this alga, will partially replace plastocyanin in many of these reations. Inhibition of photosynthetic reactions by copper chelators appears to occur at some site other than the site of plastocyanin function.     

Tetrathiomolybdate inhibition of the Enterococcus hirae CopB copper ATPase.

Tetrathiomolybdate (TTM) avidly interacts with copper and has recently been employed to reduce excess copper in patients with Wilson disease. We found that TTM inhibits the purified Enterococcus hirae CopB copper ATPase with an IC(50) of 34 nM. Dithiomolybdate and trithiomolybdate, which commonly contaminate TTM, inhibited the copper ATPases with similar potency. Inhibition could be reversed by copper or silver, suggesting inhibition by substrate binding. These findings for the first time allowed an estimate of the high affinity of CopB for copper and silver. TTM is a new tool for the study of copper ATPases.     

A dicopper complex of N,N′-bis[2,2-dimethyl-4-(2-hydroxyphenyl)-3-aza-3-buten] oxamide. Structure and magnetic behaviour of the mono hydrated form

The ligand N, N′-bis[2,2-dimethyl-4-(2-hydroxyphenyl)-3-aza-3-buten] oxamide with two identical coordination sites reacts with copper ions in its tetradeprotonated form to yield the dinuclear complex [Cu₂(C₂₄H₂₆N₄O₄)]·H₂O. The structure of this compound has been determined by the X-ray diffraction method. The crystals are orthorhombic with a = 11.744(1), B = 16.369(2), C = 26.340(3) Å, V = 5064(1) ų, Z = 8, space group Pbca. The oxamide is in a trans conformation with two different environments for the copper centres, a (4 + 1) coordination mode for the first one and a square planar environment for the other one. The water molecule is not directly bound to a copper centre, but involved in hydrogen bonding with the two oxygen atoms of an N₂O₂ coordination site. Indeed, extra coordination comes from a phenolic oxygen atom belonging to an adjacent dinuclear unit. Static susceptibility measurements point to a strong intrapair antiferromagnetic exchange interaction of 2J = −520(±4) cm⁻¹ and possibly an interpair ferromagnetic exchange interaction of 10(±5) cm⁻¹.     

Hemoglobin adducts and urinary mercapturic acids in rats as biological indicators of butadiene exposure

Binding of 1,2-epoxy-3-butene, the primary metabolite of butadiene, to hemoglobin (Hb) and excretion of its mercapturic acid in urine were studies as potential indicators of butadiene exposure. Four groups of Wistar rats were exposed to butadiene at 0, 250, 500 and 1000 ppm 6 h/day, 5 days/week, during 2 weeks. Blood was collected at the end of exposure and 17 days later for analysis of hemoglobin adducts and adduct stability. Urine was collected each day during exposure (afternoon samples) and in between exposures (morning samples). Adducts of 1,2-epoxy-3-butene to N-terminal valine in Hb were measured using the N-alkyl Edman procedure and GC/MS of the thiohydantoin derivatives. The corresponding mercapturic acid was analysed, after deacetylation, through derivatization with phthaldialdehyde and HPLC with fluorescence detection. The Hb adducts proved to be stable and are therefore useful for dosimetry of long-term exposure to butadiene. The adduct levels increased linearly with exposure dose up to 1000 ppm (3 nmol/g Hb at 1000 ppm). The increase with exposure dose of the mercapturic acid concentration in urine was also compatible with a linear does response up to 1000 ppm. The sensitivity of both analytical methods needs to be improved for their application to human samples.     

An X-ray crystallographic study of the binding sites of the azide inhibitor and organic substrates to ceruloplasmin, a multi-copper oxidase in the plasma

Ceruloplasmin is a multi-copper oxidase, which contains most of the copper present in the plasma. It is an acute-phase reactant that exhibits a two- to three-fold increase over the normal concentration of 300?μg/ml in adult plasma. However, the precise physiological role(s) of ceruloplasmin has been the subject of intensive debate and it is likely that the enzyme has a multi-functional role, including iron oxidase activity and the oxidation of biogenic amines. The three-dimensional X-ray structure of the human enzyme was elucidated in 1996 and showed that the molecule was composed of six cupredoxin-type domains arranged in a triangular array. There are six integral copper atoms per molecule (mononuclear sites in domains 2, 4 and 6 and a trinuclear site between domains 1 and 6) and two labile sites with roughly 50% occupancy. Further structural studies on the binding of metal cations by the enzyme indicated a putative mechanism for ferroxidase activity. In this paper we report medium-resolution X-ray studies (3.0–3.5?Å) which locate the binding sites for an inhibitor (azide) and various substrates [aromatic diamines, biogenic amines and (+)-lysergic acid diethylamide, LSD]. The binding site of the azide moiety is topologically equivalent to one of the sites reported for ascorbate oxidase. However, there are two distinct binding sites for amine substrates: aromatic diamines bind on the bottom of domain 4 remote from the mononuclear copper site, whereas the biogenic amine series typified by serotonin, epinephrine and dopa bind in close vicinity to that utilised by cations in domain 6 and close to the mononuclear copper. These binding sites are discussed in terms of possible oxidative mechanisms. The binding site for LSD is also reported.     

Enzymatic and spectroscopic studies on the activation or inhibition effects by substituted phenolic compounds in the oxidation of aryldiamines and catechols catalyzed by Rhus vernicifera laccase

The effect of various phenolic compounds on the activity of Rhus vernicifera laccase (Lc) has been evaluated using two different substrates, N,N-dimethyl-p-phenylenediamine and p-tert-butylcatechol. The observed effect strongly depends on the phenol employed and involves either a moderate activation, by halophenols, or inhibition, by acidic phenols. The collective data are consistent with an open active site in Lc, which is capable of accommodating more than one substrate or phenol molecule. According to NMR relaxation experiments, a phenol molecule binds at an average distance from type 1 Cu of about 6 Å, while evidence from electron paramagnetic resonance (EPR) experiments shows that binding of another phenol molecule induces a change, and probably occurs close to, the type 2/type 3 cluster. The effect of phenolic compounds on Lc reactivity is related to a modification of the substrate affinity for the enzyme. This affinity can either be increased, probably through π-stacking or other types of interactions, or decreased, due to competition for the same site. In addition, the alteration induced in the trinuclear copper cluster has a marked effect on the enzyme reactivity. The inhibition observed with acidic phenols is probably due to the protonation of an enzyme intermediate produced at the trinuclear site, e.g. the peroxy intermediate, that causes the release of hydrogen peroxide and prevents the reaction of this intermediate with the substrate.     

The inhibition of bovine ceruloplasmin oxidase activity by thiomolybdates in vivo and in vitro: a reversible interaction

The administration of pharmacological doses (greater than 100 mg Mo) of trithiomolybdate or tretrathiomolydbate to ruminants causes a transient apparent decrease in the ceruloplasmin oxidase activity of plasma and a more persistent increase in copper bound to plasma albumin. Sephadex gel-filtration and/or dilution of "inhibited" samples taken from an infused animal or of plasma treated with thiomolybdate in vitro restores activity back to pretreatment levels. The increase in albumin bound copper does not appear to be related to ceruloplasmin breakdown. It is concluded that, contrary to a recent report, the inhibition of ceruloplasmin oxidase activity is reversible and thus unlikely to be of pathological relevance, since circulatory thiomolybdate concentrations in molybdenotic animals are likely to be very low. It is recommended that thiomolybdate preparations used for in vitro and in vivo studies should be carefully purified by Sephadex chromatography.     

The role of solvent exclusion in the interaction between D124 and the metal site in SOD1: implications for ALS

Structural changes in the metal site of the copper–zinc superoxide dismutase (SOD1) are involved in the various mechanisms proposed for the pathogenesis of the SOD1-linked familial form of amyotrophic lateral sclerosis (ALS). Elucidating how the metal site of SOD1 can be disrupted by ALS-linked mutations is important for a better understanding of the pathogenesis of the disease and for developing more efficient treatments. Residue D124, a second-sphere ligand of the copper and zinc ions, is known from experimental studies to be essential for the integrity of the metal-site structure. In this work, we used density functional theory calculations and molecular dynamics simulations to elucidate which factors keep D124 attached to the metal site and how structural changes may disrupt the binding between D124 and the metal first-sphere ligands. The calculations show that D124 is kept attached to the metal site in a kinetic trap. The exclusion of solvent molecules by the electrostatic loop of the protein is found to create the binding of D124 to the metal site. The calculations also indicate that changes in the structure of the electrostatic loop of the protein can weaken the D124–metal site interaction, lowering the affinity of the zinc site for the metal. Destabilization of the electrostatic loop of SOD1 has been previously shown to be a common property of ALS-linked variants of the protein, but its role in the pathogenesis of SOD1-linked ALS has not been elucidated.     

Structure of the Alzheimer's disease amyloid precursor protein copper binding domain. A regulator of neuronal copper homeostasis

A major source of free radical production in the brain derives from copper. To prevent metal-mediated oxidative stress, cells have evolved complex metal transport systems. The Alzheimer's disease amyloid precursor protein (APP) is a major regulator of neuronal copper homeostasis. APP knockout mice have elevated copper levels in the cerebral cortex, whereas APP-overexpressing transgenic mice have reduced brain copper levels. Importantly, copper binding to APP can greatly reduce amyloid beta production in vitro. To understand this interaction at the molecular level we solved the structure of the APP copper binding domain (CuBD) and found that it contains a novel copper binding site that favors Cu(I) coordination. The surface location of this site, structural homology of CuBD to copper chaperones, and the role of APP in neuronal copper homeostasis are consistent with the CuBD acting as a neuronal metallotransporter.     

Unexpected complexity in the mechanisms that target assembly of the spectrin cytoskeleton

The spectrin cytoskeleton assembles within discrete regions of the plasma membrane in a wide range of animal cell types. Although recent studies carried out in vertebrate systems indicate that spectrin assembly occurs indirectly through the adapter protein ankyrin, recent studies in Drosophila have established that spectrin can also assemble through a direct ankyrin-independent mechanism. Here we tested specific regions of the spectrin molecule for a role in polarized assembly and function. First, we tested mutant beta-spectrins lacking ankyrin binding activity and/or the COOH-terminal pleckstrin homology (PH) domain for their assembly competence in midgut, salivary gland, and larval brain. Remarkably, three different assembly mechanisms operate in these three cell types: 1) neither site was required for assembly in salivary gland; 2) only the PH domain was required in midgut copper cells; and 3) either one of the two sites was sufficient for spectrin assembly in larval brain. Further characterization of the PH domain revealed that it binds strongly to lipid mixtures containing phosphatidylinositol 4,5-bisphosphate (PIP(2)) but not phosphatidylinositol 3,4,5-trisphosphate. A K8Q mutation in the lipid binding region of the PH domain eliminated the PIP(2) interaction in vitro, yet the mutant protein retained full biological function in vivo. Reporter gene studies revealed that PIP(2) and the spectrin PH domain codistribute with one another in cells but not with authentic wild type alphabeta-spectrin. Thus, it appears that the PH domain imparts membrane targeting activity through a second mechanism that takes precedence over its PIP(2) binding activity.     

N-Ethylmaleimide inhibition of the catalytic activities of the Dunaliella salina coupling factor 1 (CF1) and the restoration of the inhibition of the CF1 ATPase activity by N-ethylmaleimide

The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF₁) to chemical modification by N-ethylmaleimide has been investigated. (i) When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF₁ ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF₁ as shown by (a) the time-course for the inhibition and (b) the maximal extent of inhibition. (ii) Treatment of the purified, latent, D. salina CF₁ with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I₅₀ ≈ 50 μM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. (iii) N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF₁ is partially reversed by either (a) prolonged incubation at low concentrations of N-ethylmaleimide or (b) short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF₁ that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original-ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF₁ are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.     

H NMR study of the interaction of N,N′,N″-triacetyl chitotriose with Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus

The interaction between Ac-AMP2, a lectin-like small protein with antimicrobial and antifungal activity isolated from Amaranthus caudatus, and N,N′,N″-triacetyl chitotriose was studied using ¹H NMR spectroscopy. Changes in chemical shift and line width upon increasing concentration of N,N′,N″-triacetyl chitotriose to Ac-AMP2 solutions at pH 6.9 and 2.4 were used to determine the interaction site and the association constant K_a. The most pronounced shifts occur mainly in the C-terminal half of the sequence. They involve the aromatic residues Phe¹⁸, Tyr²⁰ and Tyr²⁷ together with their surrounding residues, as well as the N-terminal Val-Gly-Glu segment. Several NOEs between Ac-AMP2 and the N,N′,N″-triacetyl chitotriose resonances are reported.