RESUMO
Atrial natriuretic factor (ANF), a 28-amino-acid peptide secreted from the mammalian heart, is known to be cleared rapidly from the circulation. In vitro and in vivo studies implicate the kidney as an important site for clearance and subsequent degradation of atrial natriuretic factor. We have observed that atrial natriuretic factor is inactivated rapidly by rabbit kidney brush-border membranes. The rate of degradation of ANF measured by the loss of bioactivity followed a similar time-course to the decrease in peptide peak area measured by high-performance liquid chromatography. Interestingly, inactivation of ANF produced only a single major degradation product, which was isolated and purified. Sequence analysis revealed that the product had the same sequence of amino acids as ANF with the Cys-7-Phe-8 bond cleaved and the disulfide bridge between Cys-7 and Cys-23 remaining intact. As the renal brush border contains an abundance of proteolytic activities, it is surprising that this peptide is cleaved primarily at a single peptide bond.
Assuntos
Fator Natriurético Atrial/metabolismo , Rim/ultraestrutura , Microvilosidades/enzimologia , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Hidrólise , Cinética , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , CoelhosRESUMO
The literature reported DPP-IV substrate specificity includes oligopeptides with a penultimate Pro, Hyp or Ala residue. Bovine GRF is a substrate for DPP-IV and is rapidly degraded by the enzyme via removal of its N-terminal Tyr-Ala. Incubation of selected GRF analogs from the [X2,Ala15,Leu27]bGRF(1-29)NH2 series with a porcine-kidney-derived DPP-IV in PBS (pH 7.4) resulted in cleavage at the X2-Asp3 bond. The extent of enzymatic hydrolysis varied with X2 as reflected in the following relative cleavage rates: Ala2 (100%), Ser2 (4%), Thr2 (2.5%), Val2 (0.53%), Ile2 (0%). These cleavages were sequestered when similar experiments were performed in the presence of the DPP-IV-specific inhibitor N-epsilon-(p-NO2-benzyloxycarbonyl)-Lys-Pro-OH. A side reaction, buffer-induced deamidation of Asn8, contributed less than 5% of the total substrate degradation. Although our finding qualitatively extends the DPP-IV substrate specificity to also include N-terminal X-Ser, X-Thr and X-Val sequences, quantitatively, relatively fast cleavages of the GRFs with Ala2 make the latter preferred substrates for DPP-IV. The data presented here indicates that the observed GRF(3-29) fragment formation upon incubation of Ser2- and Thr2-substituted bGRF analogs in bovine plasma could have been DPP-IV-related.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Rim/enzimologia , Serina , Treonina , Valina , Animais , Soluções Tampão , Bovinos , Dipeptidil Peptidase 4 , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Meia-Vida , Especificidade por Substrato , SuínosRESUMO
Human prorenin, secreted from a Chinese hamster ovary cell line transfected with the cDNA for preprorenin, has been purified in mg quantities by a novel single-step procedure. The method takes advantage of reversible acid activation as a means of generating active prorenin that may be bound and eluted from an affinity column for renin. Analysis of the prorenin so purified revealed that it contained about 80% intact zymogen; the remaining 20% comprised a mixture of various prorenin derivatives truncated in the prosegment, and a small amount of renin. After exposure to 37 degrees C and pH 7.5, the refolded, partially active preparation was passed once more over the affinity column to remove renin and any truncated prorenin forms that were still active and that were, therefore, again retained by the column. Over a period of several hours, refolded and inactive prorenin not bound to the column slowly regains 5% to 10% renin activity, even when maintained under conditions that are optimal for zymogen inactivation. This activity is observed toward both model peptide substrates and natural human angiotensinogen. On the basis of these findings, we propose a model in which, under physiological conditions, a small amount of open, active prorenin is in equilibrium with a predominant, closed, and inactive form of the zymogen. Support for the model is provided by binding studies with a strong renin inhibitor that displaces the equilibrium entirely to a 1:1 zymogen:inhibitor complex. Limited cleavage of prorenin by several different proteinases has provided a number of fully active renin derivatives with varying N-terminal sequences. Results thus obtained, together with analysis of prorenin and its truncated forms bound, or not bound, to the affinity column under a variety of conditions, suggest that the propeptide region, -Arg-Ile-Phe-Leu-Lys- (positions 10P-14P), is essential for the reversible refolding of the prosegment that leads to zymogen inactivation.
Assuntos
Precursores Enzimáticos/metabolismo , Renina/metabolismo , Sítios de Ligação , Células Cultivadas , Cromatografia de Afinidade , Meios de Cultura , Precursores Enzimáticos/biossíntese , Humanos , Modelos Químicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Renina/biossíntese , Fatores de TempoRESUMO
Bovine pancreatic ribonuclease A (RNase) contains two bonds, Met29-Met30 and Tyr92-Pro93 which are representative of sites in the human immunodeficiency virus-1 (HIV-1) gag polyprotein precursors that are cleaved by the HIV-1 protease during viral maturation. Nevertheless, neither native nor performic acid-oxidized RNase is a substrate for the protease. However, RNase derivatives obtained by reduction and S-alkylation with iodoacetate or iodoacetamide undergo cleavage by the HIV-1 protease at a single site, Ala109-alkyl-Cys110, that is distinct from either of the two predicted bonds mentioned above. The neutral carboxyamido-methylcysteinyl derivative is cleaved 8 times faster than that containing the negatively charged carboxy-methyl substituent at P1'. Succinylation of these S-alkylated RNase derivatives creates a second site of cleavage by the protease between succinyl-Lys7 and Phe8. Thus, the pattern of cleavage of denatured RNase by the HIV-1 protease can be manipulated by chemical derivatization of the substrate, and the new sites of hydrolysis revealed by these studies add to our understanding of the specificity of this important enzyme.
Assuntos
Protease de HIV/metabolismo , HIV-1/enzimologia , Ribonuclease Pancreático/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/isolamento & purificação , Especificidade por SubstratoRESUMO
Two cDNAs which correspond to two very similar Class I aldolases have been isolated from a pea (Pisum sativum L.) cDNA library. With the exception of one codon they match the experimentally determined N-terminal sequence of a pea chloroplast aldolase. The deduced C-terminal sequence of one of these clones is unique among Class I aldolases. The deduced C-terminus of the other is more like the C-terminus of other eucaryotic Class I aldolases. Comparisons of sequence homology suggest that the pea chloroplast isozymes are only marginally more closely related to the anaerobically induced plant aldolases than to aldolases from animals.
Assuntos
Cloroplastos/enzimologia , Frutose-Bifosfato Aldolase/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Citoplasma/enzimologia , DNA/genética , Fabaceae , Frutose-Bifosfato Aldolase/química , Dados de Sequência Molecular , Plantas Medicinais , Mapeamento por Restrição , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Atriopeptin (AP) 24, containing amino acids Ser103-Tyr126 of the carboxy-terminal portion of the atrial natriuretic peptide prohormone, was degraded rapidly by rabbit kidney brush border membranes. The rate of degradation of AP24 measured by the loss of vasorelaxant activity followed a similar time course to the decrease in peptide peak area measured by high-performance liquid chromatography. Inactivation of AP24 produced peptide fragments which were separated by HPLC. The major products were purified individually and their peptide sequences determined. Results indicate that AP24 was proteolytically cleaved at three peptide bonds: Ser103-Ser104, Cys105-Phe106 and Ser123-Phe124. des-Ser103-AP24 had similar vasorelaxant activity to AP24, while AP24 cleaved at Cys105-Phe106 was inactive. Regarding the proteolytic cleavage at Ser123-Phe124, there was an accumulation of the C-terminal tripeptide, Phe-Arg-Tyr, only at the later time points of the incubation. Degradation experiments were repeated with an amino- and carboxy-terminal protected peptide, acetyl-AP24-amide. Peptide sequence analysis of the major degradation products of this peptide revealed that the critical peptide bond cleaved was Cys105-Phe106. We conclude that the Cys-Phe peptide bond renders atrial peptides highly susceptible to proteolysis by renal brush border membranes, resulting in inactivation.
Assuntos
Fator Natriurético Atrial/metabolismo , Rim/metabolismo , Microvilosidades/metabolismo , Sequência de Aminoácidos , Animais , Cinética , Fragmentos de Peptídeos/análise , CoelhosRESUMO
The use of yeast as an expression system for heterologous proteins offers several potential advantages with respect to industrial scale-up and genetics over other expression systems, but suffers from several drawbacks. For example, the secreted proteins of S. cerevisiae, found in the periplasm, are hyperglycosylated and the organism has a limited range of usable substrates. Other yeasts have similar disadvantages in addition to producing a variety of proteases. We have investigated the use of Schwanniomyces occidentalis as a host for developing a gene expression system in which these and several disadvantages are minimized. The present paper describes the isolation and characterization of an invertase from cell free supernatants of the yeast Schwanniomyces occidentalis grown on lactose. The enzyme is a beta-D-fructofuranoside-fructohydrolyase, composed of two identical subunits of 76,000 to 78,000 da. with a native molecular mass of 125,000 +/- 25,000 da. of which approximately 17% can be attributed to N-linked carbohydrate. The enzyme has a Vmax of 0.49 +/- 0.025 units, a Km of 21 +/- 1.5 mM, and temperature and pH optima of 55 degrees C and 3.9-4.5, respectively. The amino acid sequences of the amino terminal region and an internal tryptic peptide support an 81% identity with the invertase from Saccharomyces cerevisiae. The enzyme is induced by low glucose and is catabolite repressed.
Assuntos
Glicosídeo Hidrolases/isolamento & purificação , Saccharomycetales/enzimologia , Sequência de Aminoácidos , Aminoácidos/análise , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/metabolismo , Glicosilação , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Temperatura , beta-FrutofuranosidaseRESUMO
Limited tryptic digestion of fluorescein isothiocyanate (FITC)-labeled (H+-K+)-ATPase from rat resting light gastric membranes produced a soluble 27-kDa polypeptide which retained the fluorescence of the parent enzyme. Its production was markedly enhanced in the presence of an amphiphilic detergent, Zwittergent 3-14, which potently inhibits the ATPase activity. This increase is probably due to protection of certain tryptic cleavage sites through conformational changes of the membrane enzyme by the detergent. The NH2-terminal sequence of the 27-kDa polypeptide corresponded exactly to that beginning at Asn-369 of the cDNA-deduced primary structure of the rat ATPase. The presence of the phosphorylation site, Asp-385, and FITC-labeled Lys-517, which is known to be a part of the ATP-binding site, indicates that the 27-kDa polypeptide contains a major cytoplasmic portion of (H+-K+)-ATPase. Interestingly, the polypeptide was stained with periodate-Schiff's base, indicating its glycoprotein nature. The carbohydrate group attached to the polypeptide seems to include at least an N-linked high-mannose moiety, since the polypeptide showed Con A binding activity as detected with a Con A-biotin/avidin-peroxidase assay on nitrocellulose transblots. Also, its Con A binding activity was inhibited by excess methyl alpha-D-mannopyranoside and disappeared upon treatment of the polypeptide with endoglycosidase H and N-glycanase. Further tryptic action converted the 27-kDa polypeptide to 2 smaller FITC-labeled polypeptides of 25 and 15 kDa, which lost 18 and 96 amino acid residues, respectively, from the NH2 terminus of the parent polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Adenosina Trifosfatases/isolamento & purificação , Carboidratos/isolamento & purificação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Concanavalina A , Fluoresceína-5-Isotiocianato , Fluoresceínas , ATPase Trocadora de Hidrogênio-Potássio , Técnicas In Vitro , Masculino , Manose/isolamento & purificação , Membranas/enzimologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Ratos , Ratos Endogâmicos , Estômago/enzimologia , Tiocianatos , TripsinaRESUMO
The porcine pancreatic phospholipase A2-catalyzed hydrolysis of the water-soluble chromogenic substrate 4-nitro-3-octanoyloxybenzoate shows an initial latency phase similar to the one observed in the hydrolysis of aggregated phospholipids by the same enzyme. We report here that during the latency phase the enzyme undergoes a slow, autocatalytic, substrate-level acylation whereby in a few of the catalytic events the scissile octanoyl group of the substrate, normally transferred to water, is transferred to the epsilon-amino group of lysine 56. The N epsilon 56-octanoylphospholipase shows a strong tendency to dimerize in solution and thus may be separated from the monomeric native enzyme by gel filtration. Octanoylation of Lys-56 activates the enzyme some 180-fold toward 4-nitro-3-octanoyloxybenzoate and more than 100-fold toward monolayers of 1,2-didecanoyl-sn-glycero-3-phosphocholine. Acylation also attends the enzymatic hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with the incorporation of 1 eq of palmitate. Kinetic analysis of the early phase of reaction with 4-nitro-3-octanoyloxybenzoate shows that in this initial step the rate of activation is first order with respect to enzyme and substrate. A much more rapid, autocatalytic activation occurs in the later phases of the reaction where the activation of the enzyme is catalyzed by the activated enzyme itself. These findings with porcine pancreatic phospholipase A2, together with those relative to a snake venom enzyme monomer (Cho, W., Tomasselli, A. G., Heinrikson, R. L., and Kézdy, F. J. (1988) J. Biol. Chem. 263, 11237-11241), strongly support the proposal that interfacial activation of monomeric phospholipases is due to substrate-level autoacylation resulting in fully potentiated dimeric enzymes.
Assuntos
Lisina , Pâncreas/enzimologia , Fosfolipases A/metabolismo , Fosfolipases/metabolismo , Acilação , Animais , Ativação Enzimática , Cinética , Substâncias Macromoleculares , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , SuínosRESUMO
Extracts from human Jurkat cells or from calf thymus contain a 60-kDa protein that is bound to immobilized FK506. As expected, the NH2-terminal sequences of the 60-kDa protein from these two species were found to be nearly the same. We were surprised to discover, however, that the sequence of the human protein was identical to that of Hsp56, a heat shock protein of unknown function that has been shown to be a component of several steroid receptor complexes. Further analysis of the calf thymus protein revealed a peptide with homology to a region near the COOH terminus of both FKBP-12 and FKBP-13. It would appear, therefore, that this 60-kDa protein, or as we refer to it provisionally, "Hsp56," could have the capacity to bind FK506 directly. These observations lead us to speculate that "Hsp56" may mediate immunosuppression and inhibition of T-cell proliferation by FK506 and may do so via a cytosolic signal transduction pathway separate, but not necessarily exclusive, from that of FKBP-12 and FKBP-13.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Receptores de Esteroides/metabolismo , Tacrolimo/metabolismo , Timo/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Bovinos , Linhagem Celular , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/isolamento & purificação , Humanos , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Receptores de Esteroides/genética , Homologia de Sequência do Ácido Nucleico , Proteínas de Ligação a TacrolimoRESUMO
Recombinant human interleukin-1 beta (rIL-1 beta) was chemically modified by a 10-fold molar excess (reagent:protein) of sulfosuccinimidyl 6-(biotinamido) hexanoate (sulfo-NHS-LC-biotin) or sulfosuccinimidobiotin (sulfo-NHS-biotin) under mild conditions. The primary product was purified in each case by cation exchange high performance liquid chromatography (HPLC) and digested with endoproteinase Lys C. Peptide mapping by C18 reverse phase HPLC permitted identification of three sites of biotinylation using both reagents; N-terminal alanine, lysine 93, and lysine 94. Few additional singly modified rIL-1 beta products were obtained under these conditions, despite the presence of 15 lysine residues in this protein. These data support the view that the N terminus as well as the trilysine sequence (residues 92-94) are readily susceptible to chemical modification and are exposed on the surface of the protein. Chromatography of intact biotinylated rIL-1 beta by C4 reverse phase HPLC resolved a protein modified exclusively at the N-terminal alanine from two proteins modified singly at either lysine 93 or lysine 94. In addition, a protein product modified at lysine 103 was also obtained when rIL-1 beta was similarly modified with sulfo-NHS-biotin. Since the only difference between the two biotinylation reagents relates to spacer length and its associated hydrophobicity, these data suggest that lysine 103 is not as accessible to surface modification reagents as are lysine 93, lysine 94, or alanine 1. Initial experiments indicate that none of the modifications described above decrease thymocyte proliferation by more than one order of magnitude. Therefore, these amino acid residues are not crucial for bioactivity, and we anticipate the use of these monobiotinylated proteins in structure/function analysis of IL-1 beta.
Assuntos
Biotina/análogos & derivados , Interleucina-1/metabolismo , Succinimidas , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Humanos , Interleucina-1/isolamento & purificação , Dados de Sequência Molecular , Mapeamento de Peptídeos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The complete primary structure of a bovine-brain-derived inhibitor of protein kinase C has been established. Fragments of the purified protein were obtained by cleavage with cyanogen bromide, Staphylococcus aureus V8 protease, trypsin and chymotrypsin. Subsequent analysis of the resulting fragments by fast-atom-bombardment mass spectrometry and Edman degradation revealed a calculated molecular mass of 11,779 Da with the following 107-amino-acid sequence: [sequence: see text] This inhibitor does not share significant primary structural identity with any other known protein.
Assuntos
Inibidores Enzimáticos/química , Proteína Quinase C/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Bovinos , Quimotripsina , Brometo de Cianogênio , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , TripsinaRESUMO
We show here for the first time that actin, troponin C, Alzheimer amyloid precursor protein (AAP), and pro-interleukin 1 beta (pro-IL-1 beta), are substrates of the protease encoded by the human immunodeficiency virus (HIV) type-1. As has been seen in other non-viral protein substrates of the HIV protease, the presence of Glu residues in the P2' position appears to play an important role in substrate recognition. Three of the four bonds cleaved in actin, two of the three in troponin C, and all of the bonds hydrolyzed in AAP and pro-IL-1 beta have a P2' Glu residue. In fact, Glu residues are accommodated in all positions from P4 to P4' surrounding the scissile bond in substrates of the HIV proteases, and as many as 4 adjacent Glu residues were seen in one of the bonds cleaved in AAP. This study of non-viral protein substrates has also revealed unexpected amino acids such as Gly, Arg, and Glu in the scissile bond itself rather than the more conventional hydrophobic amino acids. The HIV-2 protease hydrolyzed actin in a manner similar to that of the HIV-1 enzyme, but its cleavage of troponin C was distinct in that it split a bond adjacent to a triplet of Glu residues in P2, P3, and P4 that was refractory to the HIV-1 enzyme. Documentation of cleavage sites in the several important cellular proteins noted above has extended our understanding of the features in a substrate that are recognized by these multi sub-site proteases of retroviral maturation. Moreover, the present work adds to an accumulating body of evidence which demonstrates that these enzymes can damage crucial structural and regulatory cellular proteins if ever their activity is expressed outside the viral particle itself.
Assuntos
Actinas/metabolismo , Peptídeos beta-Amiloides/metabolismo , Protease de HIV/metabolismo , Interleucina-1/metabolismo , Precursores de Proteínas/metabolismo , Troponina/metabolismo , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , HIV-1/enzimologia , HIV-2/enzimologia , Hidrólise , Dados de Sequência Molecular , Especificidade por Substrato , Troponina CRESUMO
The incubation of a solution of the human growth hormone releasing factor analog, [Leu27] hGRF(1-32)NH2 at pH 7.4 and 37 degrees, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [beta-Asp8, Leu27] hGRF(1-32)NH2 and [alpha-Asp8, Leu27] hGRF(1-32)NH2, produced by deamidation of the Asn8 residue. When tested as growth hormone (GH) secretagogues in cultured bovine anterior pituitary cells, [beta-Asp8, Leu27] hGRF(1-32)NH2 was estimated to be 400-500 times less potent than the parent Asn8 peptide, while [alpha-Asp8, Leu27] hGRF(1-32)NH2 was calculated to be 25 times less potent than the parent Asn8 peptide. Three additional analogs of [Leu27] hGRF(1-32)NH2 containing either Ser or Asn at positions 8 and 28 were prepared and evaluated for their GH releasing activity and stability in aqueous phosphate buffer (pH 7.4, 37 degrees). Based on disappearance kinetics, [Leu27] hGRF(1-32)NH2 had a half-life of 202 h while the other analogs had the following half-lives: [Leu27, Asn28] hGRF(1-32)NH2 (150 h); [Ser8, Leu27, Asn28] hGRF(1-32)NH2 (746 h); and [Ser8, Leu27] hGRF(1-32)NH2 (1550 h). After 14 days, incubated samples of the Asn8 analogs lost GH releasing potency, while the Ser8 analogs retained full potency. The potential for loss of biological activity brought about by deamidation of other engineered peptides and proteins should be considered in their design.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Amidas , Animais , Asparagina , Bovinos , Estabilidade de Medicamentos , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/química , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Técnicas In Vitro , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Serina , Soluções , ÁguaRESUMO
The protease from simian immunodeficiency virus (SIV) was chemically synthesized by automated solid-phase technology as an NH2-terminally extended derivative, capped with biotin. Biotin-linker-(SIV protease (1-99)): the linker segment, Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala, corresponds to the amino acid sequence preceding that of the protease in the SIV gag/pol precursor polyprotein. Accordingly, the Ala-Pro bond joining the octapeptide linker to the protease constitutes a site naturally cleaved by the protease during viral maturation. This strategy for synthesis was designed to facilitate purification of the biotinylated protein derivative from a complex mixture of reaction products by avidin/agarose-affinity chromatography and to provide the means for autocatalytic removal of the biotin-linker segment. As anticipated, folding of the full-length construct leads to activation of the enzyme and excision of the desired 99-residue SIV protease (overall yield, approximately). The specificity of the synthetic SIV protease toward a number of well characterized protein substrates was the same as observed for the nearly identical enzyme from human immunodeficiency virus type 2 (HIV-2 protease) and distinct from that of the more disparate HIV-1 protease. The same functional ordering with respect to the human retroviral proteases was reflected in Ki values observed with a number of protease inhibitors. Thus, the folded synthetic SIV protease shows patterns of specificity and susceptibility to inhibition that are in accord with what would be expected based upon its degree of structural similarity to proteases from HIV-1 and HIV-2.
Assuntos
Biotina , Endopeptidases/síntese química , Vírus da Imunodeficiência Símia/enzimologia , Sequência de Aminoácidos , Avidina , Catálise , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Protease de HIV/metabolismo , Inibidores da Protease de HIV , HIV-1/enzimologia , HIV-2/enzimologia , Dados de Sequência Molecular , Inibidores de Proteases/farmacologia , Especificidade por SubstratoRESUMO
The baculovirus expression system was used to generate recombinant Alzheimer's amyloid precursor (AAP) proteins. Recombinant baculoviruses were constructed, designed to express full-length 695-, 751-, and 770-amino acid forms. Recombinant baculoviruses designed for constitutive secretion were engineered by placing a termination codon between the beta-protein domain and cytoplasmic anchor of the full-length forms. Insect cells infected with each of these baculoviruses produced both secreted and cell-associated AAPs. Full-length constructs produced secreted derivatives which were COOH-terminally cleaved within the beta-protein domain at Gln15 or Lys16, essentially identical to previous reports utilizing mammalian cell systems. Rare secreted forms (less than 5%) appeared to extend to Lys28. Secretion constructs produced these same forms, but in different ratios. Most (approximately 60%) terminated at Gln15 or Lys16, while the remainder apparently extended to Lys28. AAPs containing the Kunitz-type serine protease inhibitory domain (AAP-751 and -770) were shown to be active inhibitors. No differences were observed in the inhibitors activities of these two forms. The similarities in AAP processing by insect and mammalian systems, together with the large amounts of recombinant protein produced by baculovirus expression, make this an attractive system for studies of AAP processing and biochemical properties.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/biossíntese , Baculoviridae/genética , Inibidores de Proteases , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Genes Virais , Vetores Genéticos , Humanos , Hidrólise , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
The 39-43 amino acid beta amyloid protein (A beta) that deposits as amyloid in the brains of patients with Alzheimer's disease (AD) is encoded as an internal sequence within a larger membrane-associated protein known as the amyloid protein precursor (APP). In cultured cells, the APP is normally cleaved within the A beta to generate a large secreted derivative and a small membrane-associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire A beta. Our study was designed to determine whether the soluble APP derivatives in human brain end within the A beta as described in cell culture or whether AD brain produces potentially amyloidogenic soluble derivatives that contain the entire A beta. We find that both AD and control brain contain nonamyloidogenic soluble derivatives that end at position 15 of the A beta. We have been unable to detect any soluble derivatives that contain the entire A beta in either the AD or control brain.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/química , Química Encefálica , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/metabolismo , Brometo de Cianogênio , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/químicaRESUMO
Active recombinant reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) with an amino-terminal extension containing a hexa-histidine sequence has been prepared in milligram quantities in a pure heterodimeric (p66/p51) form by coordinated applications of immobilized metal affinity chromatography (IMAC) and HIV-1 protease treatment. The precursor protein, isolated from extracts of recombinant Escherichia coli by IMAC in a predominantly unprocessed form (p66), migrated on sodium dodecyl sulfate-polyacrylamide gels as a 66-kDa band with minor heterogeneity at lower relative molecular mass. Incubation of this protein with recombinant HIV-1 protease produced a stable heterodimeric RT that was purified in a single step by IMAC. The purified protein retained both RT and RNase H activity, and kinetic parameters (Km and Vmax) were measured with both RNA-dependent DNA polymerization and RNase H activity assays. Carboxyl-terminal sequencing of purified heterodimeric RT indicated that one subunit is intact p66, whereas the other, p51, is a truncated form of p66 that terminates at residue Phe440. Analysis of the HIV-1 protease digest revealed two cleavage sites, at Tyr483-Leu484 and Tyr532-Leu533, in addition to the site at Phe440-Tyr441 that is cleaved to produce p51.
Assuntos
Protease de HIV/metabolismo , HIV-1/enzimologia , Processamento de Proteína Pós-Traducional , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/isolamento & purificação , Sequência de Aminoácidos , Cromatografia de Afinidade , Cromatografia em Gel , Escherichia coli/genética , Transcriptase Reversa do HIV , HIV-1/genética , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonuclease H/metabolismoRESUMO
The complete primary structure has been determined for an inhibitor protein of protein kinase C. The bovine brain-derived inhibitor has a pI of 6 and its N-terminal alanine residue is blocked by acetylation. Fragments obtained by chemical and enzymatic cleavage of the purified inhibitor were analyzed by Edman degradation, fast atom bombardment mass spectrometry, and tandem mass spectrometry. The results establish that the protein has a calculated average molecular mass of 13,690 daltons and contains 125 amino acid residues with the following sequence: (sequence: see text) The inhibitor does not show significant homology with any other known protein. Circular dichroism of the freshly prepared apoprotein indicated a secondary structural content of 23% alpha-helix, 31% beta-sheet, and 11% beta-turn. Immobilization on nitrocellulose followed by exposure to a 65Zn2(+)-containing overlay solution showed that, like protein kinase C itself, the inhibitor is a zinc-binding protein, although the sequence does not reveal a "zinc finger" structure. Competition with 10-fold molar excess Ca2+ or Mg2+ did not reduce the zinc-binding specificity of this inhibitor.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Proteína Quinase C/antagonistas & inibidores , Acetilação , Sequência de Aminoácidos , Animais , Química Encefálica , Cálcio/farmacologia , Proteínas de Transporte , Bovinos , Dicroísmo Circular , Ponto Isoelétrico , Magnésio/farmacologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos , Conformação Proteica , Serina EndopeptidasesRESUMO
Culture medium conditioned by phorbol 12-myristate 13-acetate-differentiated THP-1 cells contained interleukin 1 (IL-1) antagonist activity as measured by inhibition of both IL-1 beta binding to receptors on YT cells and inhibition of IL-1/phytohemagglutinin-stimulated IL-2 synthesis by LBRM-33-1A5 T cells. Based on their ability to compete for 125I-IL-1 beta binding to receptors on YT cells, four distinct antagonist proteins were purified from THP-1 cell conditioned medium using a combination of ion-exchange, hydrophobic interaction, and size exclusion chromatographies. The four proteins had different isoelectric points with molecular masses in the range 22-26 kDa and had similar specific activities for inhibition of IL-1 beta binding to cell surface receptors (Ki values 0.33-0.64 nM) and for inhibition of IL-1/phytohemagglutinin-stimulated IL-2 synthesis by 1A5 cells (IC50 values 25-100 pM). Amino-terminal sequence analysis of the two major forms (25 kDa/pI 5.1 and 22 kDa/pI 5.8) revealed complete identity for the first 27 residues in both forms. Based on the results of peptide mapping, amino acid compositional analysis and immune blotting, all of the forms were deduced to be variants of a common protein. Deglycosylation of the antagonist proteins with N-glycanase converted them to a common form (22 kDa/pI 5.8), indicating that the four isoforms represent glycosylation variants of a common protein and that asparagine-linked oligosaccharides are responsible for the observed size and charge heterogeneity.