Free NH2 Terminus Research Articles

β-cleavage of the human prion protein impacts Cu(II) coordination at its non-octarepeat region

The cellular prion protein (PrPC) is a membrane-anchored copper binding protein that undergoes proteolytic processing. β-cleavage of PrPC is associated with a pathogenic condition and it yields two fragments: N2 with residues 23–89, and C2 including residues 90–231. The membrane-bound C2 fragment retains the Cu binding sites at His96 and His111, but it also has a free N-terminal NH2 group. In this study, the impact of β-cleavage of PrPC in its Cu(II) binding properties was evaluated, using the peptide of the human prion protein hPrP(90–115) as a model for the C2 fragment. The Cu(II) coordination properties of hPrP(90–115) were studied using circular dichroism (CD) and electron paramagnetic resonance (EPR); while the H96A and H111A substitutions and its acetylated variants were also studied. Cu binding to hPrP(90–115) is dependent on metal ion concentration: At low copper concentrations the participation of His96 and free NH2-terminus is evident, while at high copper concentrations the His111 site is populated without participation of the N-terminal NH2 group. The presence of a free NH2-terminal group in the C2 fragment significantly impacts the Cu(II) coordination properties of the His96 site, where the NH2 group also anchors the metal ion. This study provides further insights into the impact of proteolytic processing of PrPC in the Cu binding properties of this important neuronal protein.

Influence of the N-terminus acetylation of Semax, a synthetic analog of ACTH(4-10), on copper(II) and zinc(II) coordination and biological properties

Semax is a heptapeptide (Met-Glu-His-Phe-Pro-Gly-Pro) that encompasses the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone and a C-terminal Pro-Gly-Pro tripeptide. N-terminal amino group acetylation (Ac-Semax) modulates the chemical and biological properties of parental peptide, modifying the ability of Semax to form complex species with Cu(II) ion. At physiological pH, the main complex species formed by Ac-Semax, [CuLH−2]2−, consists in a distorted CuN3O chromophore with a weak apical interaction of the methionine sulphur. Such a complex differs from the Cu(II)-Semax complex system, which exhibits a CuN4 chromophore. The reduced ligand field affects the [CuLH−2]2− formal redox potential, which is more positive than that of Cu(II)-Semax corresponding species.In the amino-free form, the resulting complex species is redox-stable and unreactive against ascorbic acid, unlike the acetylated form. Semax acetylation did not protect from Cu(II) induced toxicity on a SH-SY5Y neuroblastoma cell line, thus demonstrating the crucial role played by the free NH2 terminus in the cell protection. Since several brain diseases are associated either to Cu(II) or Zn(II) dyshomeostasis, here we characterized also the complex species formed by Zn(II) with Semax and Ac-Semax. Both peptides were able to form Zn(II) complex species with comparable strength. Confocal microscopy imaging confirmed that peptide group acetylation does not affect the Zn(II) influx in neuroblastoma cells. Moreover, a punctuate distribution of Zn(II) within the cells suggests a preferred subcellular localization that might explain the zinc toxic effect. A future perspective can be the use of Ac-Semax as ionophore in antibody drug conjugates to produce a dysmetallostasis in tumor cells.

A signal-on electrochemical strategy for protease detection based on the formation of ATCUN-Cu(II)

Amino terminal Cu(II)- and Ni(II)-binding (ATCUN) motif is included in a protein or peptide with a structural characterization comprising of a free NH2-terminus, a histidine residue in the third position and two intervening peptide nitrogens. We reported a signal-on electrochemical strategy for detection of protease based on the formation of ATCUN-Cu(II) on electrode surface. In the method, an amino residue in the native peptide substrate was replaced with a histidine. Cleavage of the sequence-specific peptide pre-immobilized on an electrode by the protease led to the presence of ATCUN motif and the formation of ATCUN-Cu(II) in the presence of Cu(II). Consequently, a voltammetric signal was observed. Contrarily, inhibition of protease by its potential inhibitor decreased the voltammetric response. To demonstrate the feasibility and sensitivity of our strategy, β-secretase was tested as a model protease. As a result, a detection limit of 0.08nmolL−1 was achieved. The half-maximum inhibition value (IC50) of a well-known β-secretase inhibitor was found to be 22.6nmolL−1, which is in agreement with the reported value.

N-Terminal ubiquitination of extracellular signal-regulated kinase 3 and p21 directs their degradation by the proteasome.

Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH(2) terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized.

Library of Sequence-specific Radioimmunoassays for Human Chromogranin A

Human chromogranin A (CgA) is an acidic protein widely expressed in neuroendocrine tissue and tumors. The extensive tissue- and tumor-specific cleavages of CgA at basic cleavage sites produce multiple peptides. We have developed a library of RIAs specific for different epitopes, including the NH2 and COOH termini and three sequences adjacent to dibasic sites in the remaining part of CgA. The antisera raised against CgA(210-222) and CgA(340-348) required a free NH2 terminus for binding. All antisera displayed high titers, high indexes of heterogeneity ( approximately 1.0), and high binding affinities (Keff0 approximately 0.1 x 10(12) to 1.0 x 10(12) L/mol), implying that the RIAs were monospecific and sensitive. The concentration of CgA in different tissues varied with the assay used. Hence, in a carcinoid tumor the concentration varied from 0.5 to 34.0 nmol/g tissue depending on the specificity of the CgA assay. The lowest concentration in all tumors was measured with the assay specific for the NH2 terminus of CgA. This is consistent with the relatively low concentrations measured in plasma from carcinoid tumor patients by the N-terminal assay, whereas the assays using antisera raised against CgA(210-222) and CgA(340-348) measured increased concentrations. Only some CgA assays appear useful for diagnosis of neuroendocrine tumors, but the entire library is valuable for studies of the expression and processing of human CgA.

Continuous Spectrophotometric Assay of Peptide Deformylase

A continuous assay for peptide deformylase has been developed using a formylated dipeptide, formyl-Met-Leu-p-nitroanilide, as substrate. Removal of the formyl group by a peptide deformylase renders the dipeptide product, which contains a free NH2terminus, a substrate for an aminopeptidase fromAeromonas proteolytica.Sequential hydrolysis of the dipeptide by the aminopeptidase releases ap-nitroaniline, which is monitored spectrophotometrically at 405 nm. This assay was applied to determine the pH optimum and the catalytic activity of a peptide deformylase fromEscherichia coli.TheE. colienzyme is most active near neutral pH (pH 7.0) and displays Michaelis–Menten kinetics toward the formylated dipeptide, withKM= 20.3 ± 1.3 μm,kcat= 38 ± 2 s−1, andkcat/KM= 1.9 × 106m−1s−1. It also exhibits an acylase activity, capable of deacylatingN-acetyl-Met-Leu-p-nitroanilide andN-trifluoroacetyl-Met-Leu-p-nitroanilide, albeit at drastically reduced rates. These results demonstrate that the current assay is a convenient, rapid, and sensitive method for kinetic studies of peptide deformylase. The strategy employed in this work should also be generally applicable to the characterization of other acylases.

Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration.

Macrophages are typically stimulated by components of microbial cell walls. Surprisingly, cell wall-less mycoplasmas can also very efficiently stimulate macrophages. We showed recently that mycoplasma-derived lipopeptides constitute the active principle. We have now isolated a clone of Mycoplasma fermentans expressing mainly one macrophage-stimulating lipopeptide. This lipopeptide was detergent-extracted and isolated by reversed-phase high-performance liquid chromotography, using nitric oxide release from C3H/HeJ mouse macrophages as bioassay for detection. In contrast to "conventional" bacterial lipoproteins, this lipopeptide had a free NH2 terminus. Amino acid composition, sequence, and the molecular weight of 2,163. 3 are consistent with the following structure: S-(2, 3-bisacyloxypropyl)cysteine-GNNDESNISFKEK with one mole C16:0, and a further mole of a mixture of C18:0 and C18:1 fatty acid per lipopeptide molecule. The sequence could not be found in either the protein identification resource nor the Swiss Prot data bank. We named this 2-kD lipopeptide, macrophage-activating lipopeptide-2 (MALP-2). Synthetic dipalmitoyl MALP-2 and mycoplasma-derived MALP-2 were compared with the bioassay. Both lipopeptides showed an identical dose dependency with a half-maximal response at 10(-11) M concentration. MALP-2 may be one of the most potent natural macrophage stimulators besides endotoxin.

Structure and Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Glutamine Site and Communication with the Phosphoribosylpyrophosphate Site

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli exhibits a basal PRPP-independent glutaminase activity having a kcat/Km that is 0.3% of fully active enzyme. Binding of PRPP activates the enzyme by a structural change that lowers the Km for glutamine 100-fold and couples glutamine hydrolysis to synthesis of 5-phosphoribosylamine. By analysis of the x-ray structure of the glutamine site containing bound 6-diazo-5-oxonorleucine, a glutamine affinity analog, and by site-directed mutagenesis we have identified residues important for glutamine binding, catalysis, and coupling with PRPP. Tyr74 is a key residue in the coupling between the sites for glutamine in the NH2-terminal domain and PRPP in the COOH-terminal domain. Arg73 and Asp127 have roles in glutamine binding. The x-ray structure indicates that there are no amino acid side chains sufficiently close to Cys1 to participate as a proton acceptor in formation of the thiolate needed for nucleophilic attack on the carboxamide of glutamine, nor as a general acid for amide nitrogen transfer. Based on the x-ray model of the glutamine site and analysis of a mutant enzyme we propose that the free NH2 terminus of Cys1 functions as the proton acceptor and donor. The results indicate that the side chain of Asn101 and the backbone nitrogen of Gly102 function to stabilize a tetrahedral oxyanion resulting from attack of Cys1 on the glutamine carboxamide. Cys1, Arg73, Asn101, Gly102, and Asp127 are conserved in the NH2-terminal domain of a subfamily of amidotransferases that includes asparagine synthetase, glucosamine 6-phosphate synthase, and glutamate synthase, implying a common function in the four enzymes. Tyr74, on the other hand, is conserved only in glutamine PRPP amidotransferase sequences consistent with a specific role in interdomain coupling. The catalytic framework of key glutamine site residues supports the assignment of glutamine PRPP amidotransferase to a recently described Ntn (NH2-terminal nucleophile) hydrolase family of enzymes.

Cathepsin G: Localization in human cerebral cortex and generation of amyloidogenic fragments from the β-amyloid precursor protein

Amyloid deposits in Alzheimer's disease, Down's syndrome and aged brain are composed largely of Aβ protein, which is generated by proteolytic processing of β-amyloid precursor protein. Proteases responsible for liberating the Aβ protein from the precursor have not yet been identified. Here, we examined the ability of cathepsin G, a chymotrypsin-like protease, to cleave two protease substrates: (i) a fluorogenic hexapeptide, whose sequence spans the cleavage site in the precursor for generating the Aβ NH 2-terminus, and (ii) recombinant human β-amyloid precursor protein purified from a baculovirus expression system. Unlike two other members of the chymotrypsin family, cathepsin G readily degraded the hexapeptide. Furthermore, cathepsin G cleaved the β-amyloid precursor protein to generate several breakdown products, including a prominent 11,500 mol. wt fragment immunoreactive with antibodies directed against the COOH-terminus of the protein. This COOH-terminal fragment co-migrated using two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with C-100, a recombinant COOH-terminal segment of the β -amyloid precursor, whose NH 2-terminus is one residue upstream of the NH 2-terminus of the Aβ domain. We also examined the localization of cathepsin G in human brain. The distribution of cathepsin G-containing cells was examined by immunohistochemistry in the temporal cortex of both Alzheimer's and aged control samples. Cathepsin G-like immunoreactivity was contained specifically within neutrophils. As visualized by double-labeling with antibodies to cathepsin G and Factor VIII, neutrophils were most frequently found within meningeal or cortical blood vessels. In addition, occasional neutrophils could be identified without an apparent vascular surround, in the brain parenchyma. By simultaneous labeling with antibodies to cathepsin G and Aβ protein, neutrophils were also sometimes found associated with both parenchymal and vessel amyloid deposits; however, these associations were rare. These findings indicate that cathepsin G is capable of cleaving the β-amyloid precursor protein to liberate the free NH 2-terminus of the Aβ protein and may have access to areas where this material is deposited in Alzheimer's disease. However, since there is no physical association between neutrophils and deposited amyloid and no increase in the number of neutrophils in an Alzheimer's brain, cathepsin G seems to be an unlikely mediator of amyloid deposition in this disease.

Pegylated peptides II

General procedures are presented for the site-specific pegylation of peptides at the NH2-terminus, side-chain positions (Lys or Asp/Glu) or COOH-terminus using solid-phase Fmoc/tBu methodologies. A model tridecapeptide fragment of interleukin-2, IL-2(44-56)-NH2, was chosen for this study since it possesses several trifunctional amino acids which serve as potential sites for pegylation. The pegylation reagents were designed to contain either Nle or Orn, which served as diagnostic amino acids for confirming the presence of 1 PEG unit per mole of peptide. NH2-Terminal pegylation was carried out by coupling PEG-CH2CO-Nle-OH to the free NH2-terminus of the peptide-resin. Side-chain pegylation of Lys or Asp was achieved by one of two pathways. Direct side-chain pegylation was accomplished by coupling with Fmoc-Lys(PEG-CH2CO-Nle)-OH or Fmoc-Asp(Nle-NH-CH2CH2-PEG)-OH, followed by solid-phase assemblage of the pegylated peptide-resin and TFA cleavage. Alternatively, allylic protective groups were introduced via Fmoc-Lys(Alloc)-OH or Fmoc-Asp(O-Allyl)-OH, and selectively removed by palladium-catalyzed deprotection after assemblage of the peptide-resin. Solid-phase pegylation of the side-chain of Lys or Asp was then carried out in the final stage with PEG-CH2CO-Nle-OH or H-Nle-NH-(CH2)2-PEG, respectively. COOH-Terminal pegylation was achieved through the initial attachment of Fmoc-Orn(PEG-CH2CO)-OH to the solid support, followed by solid-phase peptide synthesis using the Fmoc/tBu strategy. The pegylated peptides were purified by dialysis and preparative HPLC and were fully characterized by analytical HPLC, amino acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry.

Removal of the amino-terminal acidic residues of yeast actin. Studies in vitro and in vivo.

We have examined the role of the acidic residues Asp2 and Glu4 at the NH2 terminus of Saccharomyces cerevisiae actin through site-directed mutagenesis. In DNEQ actin, these residues have been changed to Asn2 and Gln4, whereas in delta DSE actin, the Asp2-Ser-Glu tripeptide has been deleted. Both mutant actins can replace wild type yeast actin. Peptide mapping studies reveal that DNEQ, like wild type actin, retains the initiator Met and is NH2 terminally acetylated, whereas delta DSE has a free NH2 terminus and has lost the initiator Met. Interestingly, microscopic examination of filaments of these two actins reveal the appearance of bundled filaments. The DNEQ bundles are smaller and more ordered, whereas the delta DSE bundles are larger and more loosely organized. Additionally, both mutant actins activate the ATPase activity of rabbit muscle myosin S1 fragment to a lesser extent than wild type. We have also developed a sensitive assay for actin function in vivo that enabled us to detect a slight defect in the ability of these mutant actins to support secretion, an important function in yeast. Thus, although the mutant actins resulted in no gross phenotypic changes, we were able to detect a defect in actin function through this assay. From these studies we can conclude that 1) although NH2-terminal negative charges are not essential to yeast life, the loss of such charges does result in a slight defect in the actins' ability to support secretion, 2) removal of the NH2-terminal negative charges promotes the bundling of actin filaments, and 3) actins lacking NH2-terminal negative charges are unable to activate the myosin S1 ATPase activity as well as wild type actin.

Degradation of Proteins with Acetylated Amino Termini by the Ubiquitin System

A free NH2-terminal group has been previously shown to be an obligatory signal for recognition and subsequent degradation of proteins in a partially fractionated and reconstituted ubiquitin proteolytic system. Naturally occurring proteins with acetylated NH2-termini--most cellular proteins fall in this category--were not degraded by this system. Other studies have suggested that the identity of the NH2-terminal residue is important in determining the metabolic stability of a protein in vivo (N-end rule). Whole reticulocyte lysate and antibodies directed against the ubiquitin-activating enzyme (E1) have now been used to show that such acetylated proteins are degraded in a ubiquitin-dependent mode. Although fractionation of lysate does not affect its proteolytic activity toward substrates with free NH2-termini, it completely abolishes the activity toward the blocked substrates, indicating that an important component of the system was either removed or inactivated during fractionation. An NH2-terminal "unblocking" activity that removes the blocking group, thus exposing a free NH2-terminus for recognition according to the N-end rule, does not seem to participate in this pathway. Incubation of whole lysate with labeled histone H2A results in the formation of multiple ubiquitin conjugates. In contrast, the fractionated system is devoid of any significant conjugating activity. These results suggest that a novel conjugating enzyme (possibly a ubiquitin-protein ligase) may be responsible for the degradation of these acetylated proteins by recognizing structural features of the substrate that are downstream and distinct from the NH2-terminal residue.

Opioid and other peptides as inhibitors of leumorphin (dynorphin B-29) converting activity

A thiolprotease from rat brain membranes was shown to convert synthetic dynorphin B-29 (Dyn B-29, “leumorphin”) to the tridecapeptide dynorphin B (Dyn B, “rimorphin”). This represents a “single-arginine cleavage” between threonine-13 and arginine-14 of the substrate. The dynorphin converting activity displayed typical Michaelis-Menten kinetics with an apparent K m for the substrate of 0.58 μM. Surprisingly, a synthetic peptide, Dyn B-29-(9–22), which contains the cleavage site, did not inhibit the activity. Dyn A inhibited the activity competitively with an apparent K i of 3.7 μM. The converting activity was also inhibited by Dyn A-(6–17) but not by Dyn A-(8–17), suggesting a role of Arg 6-Arg 7 in the inhibition of converting activity. Bovine adrenal medulla Peptide E inhibited the converting activity substantially whereas metorphamide did not, suggesting the importance of COOH-terminal residues in recognition. β-Endorphin was an effective inhibitor of converting activity, and [α-N-acetyl]β-endorphin was not, indicating a crucial role of the free NH 2-terminus in recognition by the enzyme. ACTH inhibited the activity competitively with an apparent K i of 39 nM. The converting activity was also inhibited substantially by ACTH-(1–13) but not by α-MSH, again indicating a requirement of the free NH 2-terminus for recognition. The above results suggest that the converting enzyme recognizes peptides of the three known opioid gene families.

Role of the alpha-amino group of protein in ubiquitin-mediated protein breakdown.

Previous studies suggest that the conjugation of ubiquitin to NH2 groups of proteins is required for protein breakdown. We now show that the selective modification of NH2-terminal alpha-NH2 groups of globin and lysozyme prevents their degradation by the ubiquitin proteolytic system from reticulocytes. The conjugation by ubiquitin of epsilon-NH2 groups of lysine residues, usually seen in multiples, was also inhibited in alpha-NH2-blocked proteins. Naturally occurring N alpha-acetylated proteins are not degraded by the ubiquitin system at a significant rate, while their nonacetylated counterparts from other species are good substrates. This suggests that one function of N alpha-acetylation of cellular proteins is to prevent their degradation by the ubiquitin system. alpha-NH2-blocked proteins can have their activity as substrates for degradation increased by incorporation of alpha-NH2 groups through the introduction of polyalanine side chains. Proteins in which most epsilon-NH2 groups are blocked but the alpha-NH2 group is free are degraded by the ubiquitin system, but at a reduced rate. It is therefore suggested that the exposure of a free NH2 terminus of proteins is required for degradation and probably initiates the formation of ubiquitin conjugates committed for degradation.

Tripeptidyl aminopeptidase in the extralysosomal fraction of rat liver.

An enzyme which removes tripeptides from the free, NH2-terminal end of oligopeptides has been detected in the extralysosomal fraction of rat liver. The enzyme was partially purified by Sepharose CL-4B and DEAE-cellulose chromatography. The pH optimum was in the neutral range and the apparent native molecular weight was above 10(6), as judged by the Sepharose chromatography. The enzyme cleaved the phosphopeptide Gly-Val-Leu-Arg-Arg-Ala-Ser(P)-Val-Ala (I), first at the Leu-Arg bond and then at the Ala-Ser(P) bond. The cleavage of the former bond was inhibited by Arg-Arg-Ala-Ser(P)-Val-Ala (II), which indicated that both bonds were cleaved by the same enzyme. Km for II was 0.01 mM at pH 6.5-7.5. Val-Leu-Arg-Arg-Ala-Ser(P)-Val-Ala (III) and Leu - Arg - Arg - Ala - Ser(P)-Val-Ala were poor substrates. III was, however, found to be an efficient inhibitor. The Ala-Ser(P) bond of Arg-Arg-Ala-Ser(P)-Val (IV) was cleaved at the same rate as that of II. The enzyme was active also with the unphosphorylated peptides corresponding to II and IV and tolerated the substitution of lysine for the NH2-terminal arginine of the latter peptide. Substitution of guanidovaleric acid for the NH2-terminal arginine of IV and of guanidovaleric acid or epsilon-amino-hexanoic acid for the NH2-terminal arginine of unphosphorylated IV reduced the rate of hydrolysis to insignificant levels, demonstrating the importance of a free NH2 terminus. The results provide evidence of a unique tripeptidyl aminopeptidase.

Measurement of total opioid peptides in rat brain and pituitary by radioimmunoassay directed at the alpha-N-acetyl derivative.

A sensitive assay, which cross-reacts with and is specific for diverse opioid peptides, is described. This is based on the prior acetylation of samples and subsequent radioimmunoassay with an antiserum highly specific for the acetylated NH2 terminus of opioid peptides. The result is a procedure that can be used to investigate multiple forms of opioid peptides in extracts of biological material. The sensitivity of the assay is approximately 15 fmol of beta-endorphin per incubation tube, i.e., approximately 100-fold greater sensitivity than the radioreceptor assay used in our laboratory. The peptide concentration required for 50% displacement of trace ranged from 0.65 nM (beta-endorphin) to 1.6 nM (Met-enkephalin). The assay apparently shows an absolute requirement for a free (or acetylated) NH2 terminus corresponding to either a Leu- or Met-enkephalin sequence. Use of the assay with and without prior acetylation of sample provides a method for estimation of the ratio of acetylated:nonacetylated opioid peptides in crude or fractionated extracts. The procedure is used to investigate the forms of opioid peptide found in rat brain and pituitary.

Purification and characterization of multiplication-stimulating activity. Insulin-like growth factors purified from rat-liver-cell-conditioned medium.

Multiplication-stimulating activity (MSA) refers to a family of insulin-like growth factors that have been purified from serum-free medium conditioned by a Buffalo rat liver cell line (BRL-3A). Using Dowex ion-exchange chromatography, gel chromatography on Sephadex G-75, and preparative disc acrylamide gel electrophoresis, several polypeptides with the full biological multiplication-stimulating activity have been isolated. One of these polypeptides, designated MSA II-1, previously has been used to study the relationship of the activity to the insulin-like growth factors (somatomedins) purified from human plasma. Polypeptide II-1 is a single chain polypeptide of molecular weight 8700. Glycine is the COOH-terminal amino acid. Edman degradation of carboxymethylated MSA II-1 did not reveal a free NH2-terminus. A polypeptide of lower molecular weight than MSA II-1 has also been purified. This polypeptide (MSA III-2) has been shown to be more potent than MSA II-1 in the rat-liver-membrane radioreceptor assay and in a competitive binding assay utilizing the rat-serum somatomedin-binding protein(s). The relationship of these various polypeptides has been investigated by gel filtration in guanidine hydrochloride and by acrylamide gel electrophoresis of the reduced and native polypeptides.

Rat muscle 5'-adenylic acid aminohydrolase. I. Purification and subunit structure.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) has been purified to apparent homogeneity from rat muscle. The preparation exhibits a single polypeptide band with a molecular weight of 60,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a sedimentation coefficient of 11.3 S. Analysis by sedimentation equilibrium techniques showed the nat-ive enzyme to have a molecular weight of 238,000, whereas the enzyme, when analyzed in 6 M guanidine hydrochloride and 10 mM 2-mercaptoethanol, had a molecular weight of only 59,500. The amino acid composition of the enzyme was determined and peptide mapping was performed on a tryptic digest of S-carboxymethylated enzyme. NH2-terminal analysis by both the dansylation and cyanate procedures failed to identify a free NH2 terminus. Treatment of the enzyme with carboxypeptidase A resulted in the release of approximately 0.5 mol each of valine and leucine per 60,000 g of enzyme. The data presented indicate that hte native enzyme has a tetrameric structure consisting of four polypeptide chains each having a molecular weight of 60,000. The COOH-terminal analysis can be interpreted either as an indication of subunit heterogeneity or as a result of incomplete digestion of a -X-Leu-Val sequence at the end of a single type of polypeptide chain. Tryptic peptide maps strongly support the latter interpretation and suggest that the subunits are essentially identical.

The Control of Pyruvate Kinases of Escherichia coli: I. PHYSICOCHEMICAL AND REGULATORY PROPERTIES OF THE ENZYME ACTIVATED BY FRUCTOSE 1,6-DIPHOSPHATE

Abstract The pyruvate kinase of Escherichia coli activated by fructose 1,6-diphosphate has been purified to homogeneity. It is a tetramer of molecular weight about 240,000. By electrophoresis on sodium dodecyl sulfate polyacrylamide gels, Sephadex gel filtration in denaturing solvents, and ultracentrifugational experiments, the monomer molecular weight is found to be 60,000. Tryptophan is absent from the enzyme and no free NH2 terminus can be found. Of several nucleoside diphosphates which act as phosphate acceptors in the reaction, GDP, judging by its Km value of only 0.05 mm, is the best substrate for the enzyme. Magnesium or manganese is required as a divalent cation, but the kinetics of activation by fructose 1,6-diphosphate is different, depending upon which cation is used in assay mixtures. The enzyme is inhibited by GTP at concentrations much lower than any other nucleoside triphosphate, especially when GDP is used as the substrate. Succinyl-CoA by itself inhibits the enzyme to a small extent, but in the presence of ATP it exerts a cooperative inhibition.

The Isolation of a New Hypotensive Peptide, Neurotensin, from Bovine Hypothalami

Abstract A hypotensive peptide, designated neurotensin, has been discovered and isolated in pure form from acid-acetone extracts of bovine hypothalami by column chromatography and paper electrophoresis. The results of amino acid analyses of material recovered after paper electrophoresis at pH 3.5, 6.5, and 8.9 and chromatographic analyses of the dansylated material indicate that the peptide isolated by this procedure is homogenous. Its amino acid composition and apparent molecular weight estimated by chromatography on Sephadex G-25 indicate that neurotensin is a tridecapeptide composed of Lys, Arg2, Asx, Glx2, Pro2, Ileu, Leu2, Tyr2. Neurotensin lacks a free NH2-terminus; however, it possesses a free COOH terminus which can be acted upon by carboxypeptidase A. Neurotensin induces hypotension in the rat and can stimulate the contraction of guinea pig ileum and rat uterus; however, it produces relaxation of the rat duodenum. These pharmacological properties classify it as a kinin, yet its chemical composition distinguishes it from any known peptide.