Efeitos de osmólitos na L- asparaginase II de Erwinia chrysanthemi em meio aquoso / Effects of omolytes in L- asparaginase II from Erwinia chrysanthemi in aqueous medium

A L- asparaginase é uma enzima aplicada no tratamento de Leucemia Linfoide Aguda, que atua na hidrólise da L- asparagina, privando a célula tumoral de um aminoácido essencial para o seu crescimento. A L- asparaginase, como outros biofármacos, deve ser estável, manter sua atividade específica e formar poucos agregados. A fim de manter a integridade do biofármaco, são utilizados adjuvantes nas formulações farmacêuticas, e dentre os mais importantes estão os osmólitos. Essas moléculas protegem a estrutura nativa da proteína, sendo capazes de interferir na formação de agregados e garantir a estabilidade proteica. O presente trabalho teve o objetivo de estudar o efeito dos osmólitos sacarose, sorbitol, arginina e glicina na atividade específica, estabilidade, cinética e caracterização de agregados na solução de L- asparaginase II de Erwinia chrysanthemi. Os resultados mostraram que a maioria dos osmólitos testados aumentou a atividade específica e a estabilidade da enzima, o que pode estar relacionado com o aumento da velocidade máxima e do kcat observados no ensaio cinético realizado com sacarose e sorbitol. Um perfil diferente de agregados foi encontrado para cada tipo de osmólito. A presença de sacarose ou sorbitol resultou na menor quantidade de agregados na faixa de, respectivamente, 100 a 200 e 200 a 300 nm em relação a enzima sem osmólito. Por outro lado, aumento no número total de agregados e presença de moléculas de alto peso molecular (300 a 500 nm) foram observados nas soluções enzimáticas contendo, respectivamente, glicina e arginina. Dessa forma, os resultados obtidos neste trabalho poderão auxiliar na produção e escolha da formulação de biofármacos, e, consequentemente, melhorar o tratamento medicamentoso de pacientes.

L L-Asparaginase is an enzyme applied in the treatment of Acute Lymphoblastic Leukemia, which acts on the hydrolysis of L- asparagine, depriving the tumor cell of an essential amino acid for its growth. L-asparaginase, as other biopharmaceuticals, must be stable, maintain its specific activity and form few aggregates. In order to maintain the integrity of the biopharmaceutical, adjuvants are used in the pharmaceutical formulations, and among the most importants adjuvants are the osmolytes. These molecules protect the native structure of the protein, being able of interfering in the formation of aggregates and guarantee protein stability. The present work had the objective of studying the effect of the osmolytes sucrose, sorbitol, arginine and glycine in the specific activity, stability, kinetic and aggregates characterization, in L- asparaginase II solution of Erwinia chrysanthemi. The results showed that the majority of the tested osmolytes increased the specific activity of the enzyme and its stability, which may be related to the augment of maximum velocity and kcat observed in the kinetic assay performed with sucrose and sorbitol. A different profile of aggregates was found for each type of osmolyte. The presence of sucrose or sorbitol resulted in the least amount of aggregates in the range of, respectively, 100-200 and 200-300nm in relation to the enzyme without osmolyte. On the other hand, increase in the total number of aggregates and the presence of high molecular weight molecules (300 to 500 nm) were observed in the enzymatic solutions containing, respectively, glycine and arginine. Thus, the results obtained in this work may help in the production and choice of the formulation of biopharmaceuticals and, consequently, improve the drug treatment of patients.

Corticoadrenal activity in rat regulates betaine-homocysteine S-methyltransferase expression with opposite effects in liver and kidney.

Betaine-homocysteine S-methyltransferase (BHMT) is an enzyme that converts homocysteine (Hcy) to methionine using betaine as a methyl donor. Betaine also acts as osmolyte in kidney medulla, protecting cells from high extracellular osmolarity. Hepatic BHMT expression is regulated by salt intake. Hormones, particularly corticosteroids, also regulate BHMT expression in rat liver. We investigated to know whether the corticoadrenal activity plays a role in kidney BHMT expression. BHMT activity in rat kidneys is several orders of magnitude lower than in rat livers and only restricted to the renal cortex. This study confirms that corticosteroids stimulate BHMT activity in the liver and, for the first time in an animal model, also up-regulate the BHMT gene expression. Besides, unlike the liver, corticosteroids in rat kidney down-regulate BHMT expression and activity. Given that the classical effect of adrenocortical activity on the kidney is associated with sodium and water re-absorption by the distal tubule leading to volume expansion, by promoting lesser use of betaine as a methyl donor, corticosteroids would preserve betaine for its other role as osmoprotectant against changes in the extracellular osmotic conditions. We conclude that corticosteroids are, at least in part, responsible for the inhibition of BHMT expression and activity in rat kidneys.