Effect of chemical glycosylation of RNase A on the protein stability and surface histidines accessibility in immobilized metal ion affinity electrophoresis (IMAGE) system

Baek, Won-Ok; Vijayalakshmi, M.A.

doi:10.1016/s0304-4165(97)00050-0

Cited by 41 publications

(31 citation statements)

References 26 publications

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“…Dentre as várias técnicas utilizando quelatos metálicos imobilizados, pode-se citar, como exemplos de separação de células, proteínas e ácidos nucléicos, a partição em duas fases aquosas por afinidade, 96,97 a precipitação por afinidade 62,95,98 e a técnica de eletroforese de afinidade. 99,100 …”

Section: Exemplos De Aplicações De Imacunclassified

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

Bresolin¹,

Miranda²,

Bueno³

2009

Quím. Nova

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Recebido em 16/7/08; aceito em 16/12/08; publicado na web em 11/5/09 IMMOBILIZED METAL-ION AFFINITY CHROMATOGRAPHY (IMAC) OF BIOMOLECULES: FUNDAMENTAL ASPECTS AND TECHNOLOGICAL APPLICATIONS. Immobilized Metal Ion Affinity Cromatography -IMAC -is a group-specific based adsorption applied to the purification and structure-function studies of proteins and nucleic acids. The adsorption is based on coordination between a metal ion chelated on the surface of a solid matrix and electron donor groups at the surface of the biomolecule. IMAC is a highly selective, low cost, and easily scaled-up technique being used in research and commercial operations. A separation process can be designed for a specific molecule by just selecting an appropriate metal ion, chelating agent, and operational conditions such as pH, ionic strength, and buffer type.Keywords: immobilized metal-ion affinity chromatography; purification; biomolecules. INTRODUÇÃOOs princípios fundamentais da afinidade de biomoléculas por íons metálicos são conhecidos desde o início do século passado. Em 1974, Everson e Parker demonstraram que íons metálicos presentes em metaloproteínas são os principais responsáveis pela adsorção dessas em resinas contendo quelantes imobilizados e exploraram esta afinidade na separação deste tipo de proteínas. 1 A técnica proposta por Everson e Parker popularizou-se com o trabalho publicado por Porath e colaboradores em 1975, quando os autores introduziram o termo cromatografia de afinidade por íons metálicos imobilizados (Immobilized Metal-Ion Affinity Chromatography -IMAC). 2 IMAC explora a interação entre espécies doadoras de elétrons presentes na superfície de biomoléculas em solução e íons metálicos quelatados imobilizados em um suporte sólido. Inúmeras são as aplicações analíticas, preparativas e industriais de IMAC, como a separação e purificação de diferentes biomoléculas (peptídeos, proteínas e ácidos nucléicos), a separação de células a partir de extratos biológicos e estudos de estrutura-função de proteínas.Inicialmente a técnica de IMAC foi extensamente utilizada para purificação de biomoléculas contendo naturalmente grupos doadores de elétrons em resíduos de aminoácidos expostos na superfície (tais como o anel imidazol de histidina), os quais são primariamente responsáveis pela interação biomolécula-íon metálico. Com o advento da tecnologia do DNA recombinante, foi possível a incorporação de caudas (tag) em proteínas que não contêm naturalmente espécies doadoras de elétrons, tal como a fusão de sequência de seis histidinas na porção C ou N-terminal da proteína alvo, conferindo à mesma a possibilidade de purificação por IMAC. 3,4 Apesar de na literatura existirem excelentes artigos de revisão em inglês sobre IMAC 5-14 , nesta revisão, devido à intensa atividade neste campo, pretende-se abordar aspectos fundamentais, aplicações tecnológicas (escala de laboratório e industrial) e científicas com ênfase na purificação de proteínas nativas e recombinantes (com cauda de poli(histidina)). Dentre os aspectos fundamentais do mé...

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Section: Exemplos De Aplicações De Imacunclassified

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

Bresolin¹,

Miranda²,

Bueno³

2009

Quím. Nova

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“…Several mechanisms may account for the glycation-induced diminished sensitivity of K Ca channels to CO. Glycation of K Ca channels may produce steric hindrance for the exposure of the CO acting sites on the K Ca channel proteins (33). Although the major glycation sites on proteins are asparagine, glutamine, and lysine (34,35), the glycation process will also affect the microenvironment of other amino acid residues in proteins, including histidine. An altered accessibility to the surface histidines has been shown in the glycated RNase A protein (34).…”

Section: Vascular Effect Of Co In Diabetesmentioning

confidence: 99%

“…Although the major glycation sites on proteins are asparagine, glutamine, and lysine (34,35), the glycation process will also affect the microenvironment of other amino acid residues in proteins, including histidine. An altered accessibility to the surface histidines has been shown in the glycated RNase A protein (34). CO mainly interacts with a histidine residue located on the extracellular domain of K Ca channels (3).…”

Section: Vascular Effect Of Co In Diabetesmentioning

confidence: 99%

Reduced Vasorelaxant Effect of Carbon Monoxide in Diabetes and the Underlying Mechanisms

Wang

et al. 2001

Diabetes

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Carbon monoxide (CO) is an endogenous gaseous factor that relaxes vascular tissues by acting on both the cGMP pathway and calcium-activated K + (K Ca ) channels. Whether the vascular effect of CO is altered in diabetes had been unknown. It was found that the CO-induced relaxation of tail artery tissues from streptozotocininduced diabetic rats was significantly decreased as compared with that of nondiabetic control rats. The blockade of the cGMP pathway with ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one) completely abolished the CO-induced relaxation of diabetic tissues but only partially inhibited the CO effect in normal tissues. Single-channel conductance of K Ca channels in diabetic smooth muscle cells (SMCs) was not different from that of normal SMCs. However, the sensitivity of K Ca channels to CO in diabetic SMCs was significantly reduced. CO (10 µmol/l) induced an 81 ± 24% increase in the mean open probability of single K Ca channels in normal SMCs but had no effect in diabetic SMCs. Longterm culture of normal vascular SMCs with 25 mmol/l glucose or 25 mmol/l 3-OMG (3-O-methylglucose) but not 25 mmol/l mannitol significantly reduced the sensitivity of K Ca channels to CO. On the other hand, the sensitivity of K Ca channels to CO was regained in diabetic SMCs that were cultured with 5 mmol/l glucose for a prolonged period. The decreased vasorelaxant effect of CO in diabetes represents a novel mechanism for the vascular complications of diabetes, which could be closely related to the glycation of K Ca channels in diabetic vascular SMCs. Diabetes 50: [166][167][168][169][170][171][172][173][174] 2001 T he vasorelaxant effects of carbon monoxide (CO) have been demonstrated (1-4), and heme oxygenase (HO) that cleaves the heme ring to form biliverdin and CO has been located in many different types of vascular smooth muscles (5,6). The production of CO from vascular tissues has also been directly measured (7). These studies emphasize the importance of CO as an endogenous vasorelaxant factor under physiological (8) or pathophysiological (9,10) conditions. The elevation of cellular cGMP levels and the opening of plasma membrane K + channels are the main mechanisms that have been proposed to explain the vascular effects of CO. CO may increase cGMP content via its stimulatory interaction with the heme in the regulatory subunit of guanylyl cyclase. Increased cGMP would consequently decrease the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in smooth muscle cells (SMCs) through the inhibition of inositol triphosphate formation, the activation of Ca 2+ -ATPase, and the inhibition of Ca 2+ channels. The opening of K + channels leads to membrane hyperpolarization, which in turn inhibits the agonist-induced increase in inositol triphosphate, reduces Ca 2+ sensitivity and resting Ca 2+ level, and relaxes SMCs (11). Our studies have demonstrated that CO directly enhanced the activity of the big-conductance calcium-activated K + (K Ca ) channels in rat tail artery SMCs via a cGMP-independent mechanism (2,3). Whethe...

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“…Supplementing the succinimide ester was deemed to be important to obtain high degree of glycation, as examples from the literature reveal failed attempts to obtain a satisfactionary degree of glycation when using solely a WSC [8,13,14].…”

Section: Introductionmentioning

confidence: 99%

Conjugation of d-glucosamine to bovine trypsin increases thermal stability and alters functional properties

Gizurarson¹,

Filippusson²

2015

Enzyme and Microbial Technology

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Effect of chemical glycosylation of RNase A on the protein stability and surface histidines accessibility in immobilized metal ion affinity electrophoresis (IMAGE) system

Cited by 41 publications

References 26 publications

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

Reduced Vasorelaxant Effect of Carbon Monoxide in Diabetes and the Underlying Mechanisms

Conjugation of d-glucosamine to bovine trypsin increases thermal stability and alters functional properties

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