RESUMO
Carbohydrate mimics have been studied for a long time as useful sugar substitutes, both in the investigation of biological events and in the treatment of sugar-related diseases. Here we report further evaluation of the capabilities of inositols as carbohydrate substitutes. The conformational features of an inositol-model of a simplified repeating unit corresponding to the capsular polysaccharide of Streptococcus pneumoniae 19F has been evaluated by computational analysis, and compared to the native repeating unit. The inositol mimic was synthesized, and its experimental spectroscopic data allowed for verification of the theoretical results.
Assuntos
Cápsulas Bacterianas/química , Inositol/química , Inositol/síntese química , Modelos Moleculares , Somatomedinas/química , Somatomedinas/síntese química , Streptococcus pneumoniae/química , Configuração de Carboidratos , Técnicas de Química SintéticaRESUMO
The fourth member of Ca2+-dependent mammalian secretory subtilase, PC4 (proprotein convertase 4), is primarily expressed in testicular germ cell and ovarian macrophage. Its role in sperm fertilization and in early embryonic development has been demonstrated earlier through several studies, including those with PC4 null mice. A number of physiological substrates found in reproductive tissues have been postulated or identified for PC4 by various biochemical studies. These include growth factors IGF-1 (insulin-like growth factor-1) and IGF-2, hormonal polypeptide proPACAP (where PACAP stands for pituitary adenylate cyclase-activating polypeptide) and a number of surface proteins of ADAM (ADisintegrin And Metalloproteinase-like) family such as ADAM-1 (fertilin a), ADAM-2 (fertilin b), ADAM-3 (procyritestin) and ADAM-5. To provide further evidence in support of this notion and also to study the substrate specificity and bioassay of PC4, a series of intramolecularly quenched fluorogenic peptides containing the cleavage sites and several mutants were prepared. A comparative kinetic analysis and measurement of Vmax (app)/Km (app) ratio of these fluorogenic substrates against PC4 and PC7 revealed that the mutant variants of h (human) proPACAP and m (mouse) ADAM-5 derived peptides Q-PACAP141-151-mutant [Abz-141RVKNKGRRI150P151SY(NO2)-A-CONH2] (150A151Y replaced by PS) and Q-ADAM-5380-388-mutant [Abz-380E381PKPARRP388RY(NO2)A-CONH2] (381R replaced by P) are most efficiently and selectively cleaved by PC4. Using these two and Q-IGF-263-71 peptides, we showed that the sperm extract of normal adult mice is much higher when compared with that of PC4-null mice. This suggests that these fluorogenic peptides are useful for specific bioassay of PC4 activity. In addition, kinetic studies with various peptidyl-MCA indicate that the hexapeptide Ac-KTKQLR-MCA (where MCA stands for 4-methyl coumaryl-7-amide) is most efficiently and selectively cleaved by PC4 at RMCA, making it another effective agent for bioassay of PC4 activity. The study concludes that the most probable sequence motif for recognition by PC4 is KXKXXR or KXXR, where X is any amino acid other than cysteine and that it prefers proline at P3, P5 and/or P2' positions. It was also revealed that PC4 is a good candidate processing enzyme for growth factors IGF-1 and -2, neuropeptide proPACAP and several ADAM proteins such as ADAM-1, -2, -3 and -5.
Assuntos
Corantes Fluorescentes/metabolismo , Peptídeos/metabolismo , Serina Endopeptidases/metabolismo , Animais , Bioensaio/métodos , Cumarínicos/metabolismo , Corantes Fluorescentes/síntese química , Humanos , Masculino , Camundongos , Camundongos Knockout , Peptídeos/síntese química , Pró-Proteína Convertases , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/deficiência , Somatomedinas/síntese química , Somatomedinas/química , Somatomedinas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Especificidade por Substrato/fisiologia , SubtilisinasRESUMO
The primary structure of human IGF-I, except for the disulfide bond system, has been reported by Rinderknecht and Humbel. IGF-I afforded the corresponding characteristic peptide fragment on V8 protease digestion, which contained Cys6, Cys47, Cys48, and Cys52. Two possible fragments, Type I with Cys6-Cys47 and Cys48-Cys52, and Type II with Cys6-Cys48 and Cys47-Cys52, were synthesized. The disulfide bond system of IGF-I was unequivocally determined to be the Type II form along with Cys18-Cys61. Interestingly, the Type I system was included in the disulfide bond isomer produced as the main by-product in the refolding step on IGF-I synthesis by the recombinant DNA method.
Assuntos
Fator de Crescimento Insulin-Like I/síntese química , Somatomedinas/síntese química , Cisteína , Dissulfetos/síntese química , Humanos , Estrutura Molecular , Fragmentos de Peptídeos/síntese química , Proteínas Recombinantes/biossínteseAssuntos
Fator de Crescimento Insulin-Like I/síntese química , Somatomedinas/síntese química , Sequência de Aminoácidos , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Replicação do DNA/efeitos dos fármacos , Indicadores e Reagentes , Fator de Crescimento Insulin-Like I/isolamento & purificação , Fator de Crescimento Insulin-Like I/farmacologia , Oxirredução , Resinas VegetaisRESUMO
Human insulin-like growth factor II (IGF-II) with 67 amino acids and three disulfide bridges has been synthesized by the solid-phase method. The synthetic hormone is shown to be homogeneous in high performance liquid chromatography (HPLC), high performance partition chromatography (HPPC), and chromatofocusing. It is indistinguishable from natural hormone in HPLC, peptide map of thermolysin digests, amino acid composition and radioreceptor binding assay. Thus, synthetic and natural IGF-II are identical.
Assuntos
Fator de Crescimento Insulin-Like II/síntese química , Somatomedinas/síntese química , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Indicadores e Reagentes , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Fígado/metabolismo , Coelhos , Receptores de Superfície Celular/metabolismo , Receptores de SomatomedinaRESUMO
The photoactive insulin-like growth factor (IGF)-II analogue 4-azidobenzoyl-125I-IGF-II was synthesized and used to label specifically and covalently the Mr = 250,000 Type II IGF receptor. When rat adipocytes are irradiated after a 10-min incubation with 4-azidobenzoyl-125I-IGF-II at 10 degrees C and immediately homogenized, most of the labeled IGF-II receptors are associated with the plasma membrane fraction, indicating that receptors accessible to the labeling reagent at low temperature are on the cell surface. However, when the photolabeled cells are incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors are rapidly internalized with a half-time of 3.5 min as evidenced by a loss from the plasma membrane fraction and a concomitant appearance in the low density microsome fraction. The low density microsomes were previously shown to contain intracellular membranes (Oka, Y., and Czech, M.P. (1984) J. Biol. Chem. 259, 8125-8133). The steady state level of cell surface IGF-II receptors in the presence or absence of IGF-II, measured by the binding of anti-IGF-II receptor antibody to cells, remains constant under these conditions, demonstrating that IGF-II receptors rapidly recycle back to the cell surface at the same rate as receptor internalization. Using the above methodology, it is shown that acute insulin action: 1) increases the steady state number of cell surface IGF-II receptors; 2) increases the number of ligand-bound IGF-II receptors that are internalized per unit of time, as evidenced by a large increase in the photolabeling of intracellular membrane IGF-II receptors when cells are incubated at 37 degrees C with insulin and 4-azidobenzoyl-125I-IGF-II prior to photoactivation; and 3) increases the rate of cellular 125I-IGF-II degradation by a process that is blocked by anti-IGF-II receptor antibody. The results indicate that the action of insulin to elevate the steady state number of cell surface IGF-II receptors leads to an increased internalization flux of IGF-II-bound receptors, mediating increased IGF-II uptake and degradation.
Assuntos
Tecido Adiposo/metabolismo , Fator de Crescimento Insulin-Like II/análogos & derivados , Insulina/metabolismo , Insulina/farmacologia , Peptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Somatomedinas/metabolismo , Marcadores de Afinidade/síntese química , Animais , Azidas/síntese química , Membrana Celular/metabolismo , Feminino , Radioisótopos do Iodo , Cinética , Masculino , Peso Molecular , Placenta/metabolismo , Gravidez , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Somatomedina , Somatomedinas/síntese químicaRESUMO
An insulin-like hybrid molecule consisting of the A chain of insulin and a B chain corresponding to the B domain of human insulin-like growth factor I (growth factor I sequence 1-30) has been synthesized essentially by the procedures developed in this laboratory for the synthesis of insulin and analogues. The hybrid competed with 125I-insulin for insulin receptors in rat liver plasma membranes and was a full agonist in stimulating incorporation of [3(-3)H]glucose into lipids in rat adipocytes. In both assays, the compound displayed ca. 2% of the potency of insulin. The compound was recognized by anti-insulin antibodies but was only ca. 0.25% as potent as insulin in this activity. The hybrid exhibited growth-promoting activity in fibroblasts, displaying 3-8% of the activity of insulin. In contrast, the compound was recognized by insulin-like growth factor carrier proteins, a property not associated with insulin. Two points of nonhomology between the B chain of insulin and the B domain of insulin-like growth factor I are considered in connection with these observations.
Assuntos
Fator de Crescimento Insulin-Like I/síntese química , Insulina/síntese química , Multimerização Proteica , Somatomedinas/síntese química , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Bovinos , Membrana Celular/metabolismo , Humanos , Insulina/farmacologia , Lipídeos/biossíntese , Fígado/metabolismo , Substâncias Macromoleculares , Fragmentos de Peptídeos , Ratos , Receptor de Insulina/metabolismoRESUMO
Human insulin-like growth factor II with 67 amino acid residues and three disulfide bridges has been synthesized by the solid-phase method. Homogeneity of the synthetic product is ascertained by chromatofocusing, high performance liquid chromatography and amino acid analysis. In both radioimmunoassay and radioreceptor assay, the synthetic product is indistinguishable from the natural hormone.
Assuntos
Insulina/síntese química , Peptídeos/síntese química , Somatomedinas/síntese química , Aminoácidos/análise , Fenômenos Químicos , Química , Cromatografia Líquida de Alta Pressão , Humanos , Focalização Isoelétrica , Radioimunoensaio , Ensaio RadioliganteRESUMO
The biological and immunological properties of a chemically synthesized preparation of somatomedin C (Sm-C) were compared with those of the natural product isolated from human plasma. The two preparations produced identical curves in the radioimmunoassay and radioreceptor assay for Sm-C and in the radioreceptor assay for insulin. They were identical in their ability to stimulate DNA synthesis in confluent BALB/c 3T3 cells previously exposed to platelet-derived growth factor, and the biological activities of both preparations were completely neutralized by a monoclonal antibody raised against native Sm-C. These studies demonstrate that the chemically synthesized product is equivalent to the native molecule in all important respects and that it can be used interchangeably with the natural product for any studies that are contemplated.
Assuntos
Somatomedinas/farmacologia , Animais , Anticorpos Monoclonais , Bioensaio , Células Cultivadas , Replicação do DNA/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I , Camundongos , Camundongos Endogâmicos BALB C , Radioimunoensaio/métodos , Somatomedinas/sangue , Somatomedinas/síntese químicaRESUMO
Human insulin-like growth factor I (somatomedin C) with 70 amino acid residues and three disulfide bridges has been synthesized by the solid-phase method. The synthetic product behaves as a pure polypeptide in paper electrophoresis, isoelectric focusing, and high-performance liquid chromatography, Its elution behavior in Sephadex G-50, isoelectric point, amino acid composition, and growth-stimulating activity in bovine adrenal cortical cells or granulosa cells are comparable to those reported for the natural product. In radioimmunoassay, the synthetic product is indistinguishable from the natural hormone when either synthetic or natural product is used as the labeled ligand.