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1.
Med Hypotheses ; 99: 1-14, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28110688

RESUMO

The CK/PCr-system, with creatine (Cr) as an energy precursor, plays a crucial role in cellular physiology. In the kidney, as in other organs and cells with high and fluctuating energy requirements, energy-charged phospho-creatine (PCr) acts as an immediate high-energy source and energy buffer, and as an intracellular energy transport vehicle. A maximally filled total Cr (Cr plus PCr) pool is a prerequisite for optimal functioning of the body and its organs, and health. Skeletal- and cardiac muscles of dialysis patients with chronic kidney disease (CKD) are depleted of Cr in parallel with the duration of dialysis. The accompanying accumulation of cellular damage seen in CKD patients lead to a deterioration of musculo-skeletal and neurological functioning and poor quality of life (QOL). Therefore, to counteract Cr depletion, it is proposed to supplement CKD patients with Cr. The anticipated benefits include previously documented improvements in the musculo-skeletal system, brain and peripheral nervous system, as well as improvements in the common comorbidities of CKD patients (see below). Thus, with a relatively simple, safe and inexpensive Cr supplementation marked improvements in quality of life (QOL) and life span are likely reached. To avoid Cr and fluid overload by oral Cr administration, we propose intradialytic Cr supplementation, whereby a relatively small amount of Cr is added to the large volume of dialysis solution to a final concentration of 1-10mM. From there, Cr enters the patient's circulation by back diffusion during dialysis. Because of the high affinity of the Cr transporter (CRT) for Cr affinity for Cr (Vmax of CRT for Cr=20-40µM Cr), Cr is actively transported from the blood stream into the target cells and organs, including skeletal and cardiac muscle, brain, proximal tubules of kidney epithelial cells, neurons, and leukocytes and erythrocytes, which all express CRT and depend on the CK/PCr system. By this intradialytic strategy, only as much Cr is taken up by the body as is needed to fill the tissue Cr pools and no excess Cr has to be excreted, as is the case with oral Cr. Because aqueous solutions of Cr are not very stable, Cr must be added immediately before dialysis either as solid Cr powder or from a frozen Cr stock solution to the dialysate, or alternatively, Cr could become an additional component of a novel dry dialysate mixture in a cartridge device.


Assuntos
Creatinina/administração & dosagem , Diálise Renal/métodos , Insuficiência Renal/terapia , Administração Oral , Animais , Apoptose , Densidade Óssea , Creatina , Creatinina/metabolismo , Citosol/metabolismo , Feminino , Taxa de Filtração Glomerular , Humanos , Mucosa Intestinal/metabolismo , Isquemia , Rim/metabolismo , Transplante de Rim , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Qualidade de Vida , Insuficiência Renal/psicologia
2.
Biotechniques ; 46(6): ix-xii, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19480643

RESUMO

The standard Akta Explorer high-performance liquid chromatography (HPLC) system has limitations for the automation of multidimensional protein purification. Here, we describe simple modifications that allow for automated multidimensional purification protocols to extend the possibilities of the Akta three-dimensional purification kit in terms of column number, flexibility of volumes stocked for re-injection of samples, and available choice of buffers. These modifications do not preclude the use of standard one-dimensional purification protocols. Additionally, we demonstrate a technology for encrypted full remote control of the machine over the Internet by cost-effective use of standard asymmetric digital subscriber line (ADSL) that enables direct remote interaction with the machine without preventing local control. A 4-column purification scheme, including equilibration and cleaning in place (CIP) procedures, was implemented on such a system. It significantly increased reproducibility and shortened processing time by 85%, as compared with manual operation, thus allowing for automated protein purification overnight.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Internet , Proteínas/isolamento & purificação , Robótica/métodos , Quinases Proteína-Quinases Ativadas por AMP , Cromatografia Líquida de Alta Pressão/normas , Computadores , Proteínas Quinases/isolamento & purificação
3.
Biotechniques ; 45(2): 187-9, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18687068

RESUMO

We have developed an automated fermentation system for cost-efficient upscaling of protein expression in bacteria. The system, built for use by nonbiotechnologists, can be assembled mostly from standard laboratory equipment and allows a largely unattended growth of bacteria to OD 25 (at 600 nm) in a 12 L vessel. The typical yield of 250-350 g of wet weight cell pellet per run, which is equivalent to the biomass obtained from 250 shake flask cultures containing 400 mL Luria-Broth medium each, facilitates the production of large amounts of purified recombinant protein without the laborious need for optimization of expression and purification conditions.


Assuntos
Escherichia coli/metabolismo , Fermentação , Proteínas Recombinantes/biossíntese , Biomassa , Concentração de Íons de Hidrogênio
4.
J Biol Chem ; 283(26): 18331-43, 2008 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-18372250

RESUMO

Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing gamma(1), (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (~1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by approximately 5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr(172), thus positively affecting AMPK activity.


Assuntos
Complexos Multienzimáticos/química , Proteínas Serina-Treonina Quinases/química , Proteínas Quinases Ativadas por AMP , Animais , Dimerização , Humanos , Ligantes , Luz , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Conformação Molecular , Complexos Multienzimáticos/fisiologia , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/fisiologia , Saccharomyces cerevisiae/enzimologia , Schizosaccharomyces/enzimologia
5.
Mol Biotechnol ; 36(3): 220-31, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17873408

RESUMO

The tumour suppressor LKB1 plays a critical role in cell proliferation, polarity and energy metabolism. LKB1 is a Ser/Thr protein kinase that is associated with STRAD and MO25 in vivo. Here, we describe the individual expression of the three components of the LKB1 complex using monocistronic vectors and their co-expression using tricistronic vectors that were constructed from monocistronic vectors using a fully modular cloning approach. The data show that among the three individually expressed components of the LKB1 complex, only MO25alpha can be expressed in soluble form, whereas the other two, LKB1 and STRADalpha are found almost exclusively in inclusion bodies. However, using the tricistronic vector system, functional LKB1-MO25alpha-STRADalpha complex was expressed and purified from soluble extracts by sequential immobilized-metal affinity and heparin chromatography, as shown by Western blotting using specific antibodies. In size exclusion chromatography, MO25alpha and STRADalpha exactly co-elute with LKB1 with an apparent molecular weight of the heterotrimeric complex of 160 kDa. The specific activity in the peak fraction of the size exclusion chromatography was 250 U/mg at approximately 25% purity. As shown by autoradiography, LKB1 and STRADalpha, both strongly autophosphorylate in vitro. Moreover, recombinant LKB1 complex activates AMPK by phosphorylation of the alpha-subunit at the Thr-172 site as shown (i) by Western blotting using phospho-specific antibodies after LKB1-dependent phosphorylation, (ii) by LKB1-dependent incorporation of radioactive phosphate into the alpha-subunit of kinase dead AMPK heterotrimer, and (iii) by activity determination of AMPK. Functional mammalian LKB1 complex is constitutively active, and when enriched from bacteria should prove to be a valuable tool for studying its molecular function and regulation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transporte Vesicular/biossíntese , Escherichia coli/genética , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Recombinantes/biossíntese , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Proteínas de Ligação ao Cálcio , Cromatografia em Gel , Vetores Genéticos/genética , Humanos , Corpos de Inclusão/química , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade
6.
J Biol Chem ; 281(43): 32207-16, 2006 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-16943194

RESUMO

AMP-activated protein kinase (AMPK) is a heterotrimeric protein kinase that is crucial for cellular energy homeostasis of eukaryotic cells and organisms. Here we report on the activation of AMPK alpha1beta1gamma1 and alpha2beta2gamma1 by their upstream kinases (Ca(2+)/calmodulin-dependent protein kinase kinase-beta and LKB1-MO25alpha-STRADalpha), the deactivation by protein phosphatase 2Calpha, and on the extent of stimulation of AMPK by its allosteric activator AMP, using purified recombinant enzyme preparations. An accurate high pressure liquid chromatography-based method for AMPK activity measurements was established, which allowed for direct quantitation of the unphosphorylated and phosphorylated artificial peptide substrate, as well as the adenine nucleotides. Our results show a 1000-fold activation of AMPK by the combined effects of upstream kinase and saturating concentrations of AMP. The two AMPK isoforms exhibit similar specific activities (6 mumol/min/mg) and do not differ significantly by their responsiveness to AMP. Due to the inherent instability of ATP and ADP, it proved impossible to assay AMPK activity in the absolute absence of AMP. However, the half-maximal stimulatory effect of AMP is reached below 2 microm. AMP does not appear to augment phosphorylation by upstream kinases in the purified in vitro system, but deactivation by dephosphorylation of AMPK alpha-subunits at Thr-172 by protein phosphatase 2Calpha is attenuated by AMP. Furthermore, it is shown that neither purified NAD(+) nor NADH alters the activity of AMPK in a concentration range of 0-300 microm, respectively. Finally, evidence is provided that ZMP, a compound formed in 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside-treated cells to activate AMPK in vivo, allosterically activates purified AMPK in vitro, but compared with AMP, maximal activity is not reached. These data shed new light on physiologically important aspects of AMPK regulation.


Assuntos
Regulação Alostérica/fisiologia , Complexos Multienzimáticos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Ativação Enzimática , Glutationa Transferase/metabolismo , Técnicas In Vitro , Camundongos , Complexos Multienzimáticos/análise , Complexos Multienzimáticos/genética , Mutação , NAD/farmacologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleotídeos/farmacologia
7.
J Biol Chem ; 281(10): 6366-75, 2006 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-16407220

RESUMO

We previously reported the phosphoinositide 3-kinase-dependent activation of the 5'-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway. Exposure of bovine aortic endothelial cells to ONOO- significantly increased the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- was accompanied by increased phosphorylation of protein kinase Czeta (PKCzeta) (Thr410/403) and translocation of cytosolic PKCzeta into the membrane. Further, inhibition of PKCzeta abrogated ONOO- -induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCzeta mutant enhanced the phosphorylation of AMPK-Thr172, suggesting that PKCzeta is upstream of AMPK activation. In contrast, ONOO- activated PKCzeta in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCzeta-enhanced AMPK activation. In vitro, recombinant PKCzeta phosphorylated LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolished ONOO- -induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCzeta significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO-. Taken together, we conclude that PKCzeta can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1.


Assuntos
Endotélio Vascular/enzimologia , Complexos Multienzimáticos/metabolismo , Ácido Peroxinitroso/fisiologia , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Animais , Bovinos , Diferenciação Celular/fisiologia , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Endotélio Vascular/citologia , Metabolismo Energético , Ativação Enzimática/fisiologia , Células HeLa , Humanos , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Ratos
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