Acylation of l-asparaginase with total retention of enzymatic activity

Martins, M.B.F.; Jorge, J.; Cruz, M.E.M.

doi:10.1016/0300-9084(90)90050-q

Cited by 26 publications

(15 citation statements)

References 11 publications

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“…The dispersion obtained was shaken for 30 min at 250 rpm at room temperature. Then the precipitated palmitic acid (5) was separated from the cystatin containing supernatant by 30 min centrifugation at 15557 g and 7 °C.…”

Section: Methodsmentioning

confidence: 99%

Improved Acylation Method Enables Efficient Delivery of Functional Palmitoylated Cystatin into Epithelial Cells

et al. 2007

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The effective delivery of therapeutic proteins to the site of action is of great importance in achieving an effective therapy. Due to hydrophilicity, proteins are generally poorly transported across biological membranes. Chemical acylation represents one of the basic methods for improving their membrane permeability. A novel method for acylation is presented, based on the formation of palmitoylchloride dispersion in aqueous acetonitrile solution, using chicken cystatin as a model protein. We examined the effects of palmitoylchloride/cystatin molar ratio, reaction pH and introduction of successive palmitoylation cycles on the protein modification degree. The reaction products were analysed by capillary electrophoresis and SDS-PAGE, and the in vitro inhibitory activity was determined by N-benzoyl-D,L-arginine-beta-naphthylamide assay. Using cell culture-based assays, we examined the transport properties of unmodified and palmitoylated cystatin, its efficiency to inhibit intracellular enzymes, and its cytotoxicity. We demonstrated that palmitoylated cystatin rapidly internalized into the cell and caused a complete loss of cathepsin B activity. In contrast, the unmodified control cystatin was unable to inhibit the intracellular enzymes. These results strongly suggest protein palmitoylation to be a very effective strategy for improving cell internalization.

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Section: Methodsmentioning

confidence: 99%

Improved Acylation Method Enables Efficient Delivery of Functional Palmitoylated Cystatin into Epithelial Cells

et al. 2007

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“…In this case also in vivo studies are still awaited. Acylation has also been applied as a method for l-asparaginase modification [68] but the limitation of this approach is that enzyme becomes hydrophobic after modification. l-Asparaginase entrapped in red blood cells was quite stable and had markedly prolonged in vivo half-life [69,70].…”

Section: Modified Enzymementioning

confidence: 99%

Pharmacological and clinical evaluation of l-asparaginase in the treatment of leukemia

Narta

Kanwar

Azmi

2007

Critical Reviews in Oncology/Hematology

332

229

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“…The dispersion obtained was shaken for 30 min at 250 rpm at room temperature. Then, the reaction mixture was centrifuged at 11 000 rpm and 7 °C to separate the precipitated fatty acyl chloride (21) from the cystatin containing supernatant.…”

Section: Methodsmentioning

confidence: 99%

Membrane Permeability of Acylated Cystatin Depends on the Fatty Acyl Chain Length

Kočevar¹,

Obermajer

Kreft

2008

Chem Biol Drug Des

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Hydrophobization of proteins, such as chemical acylation, has been recognized as an efficient method for improving their membrane permeability. In this research, chicken cystatin, a model protein inhibitor of cysteine proteinases, was acylated with fatty acyl residues of 6-18 carbon atoms. The chemical modification was performed using fatty acyl chloride dispersion in aqueous acetonitrile solution. The reaction products were analyzed by capillary electrophoresis, SDS-PAGE and isoelectric focusing. In vitro inhibitory activity was determined by N-benzoyl-D,L-arginine-beta-naphthylamide assay and membrane permeability properties of non-acylated and acylated cystatin by measuring its efficiency to inhibit intracellular cathepsin B in MCF-10A neo T cells. The experiments showed that acylated cystatin quickly internalized into the cells and effectively inhibited cathepsin B. In contrast, non-acylated cystatin did not cause inhibition as it was unable to enter the cell. The permeability enhancement effect was shown to depend on the length of the attached fatty acyl chain as the strongest inhibition was caused by cystatin acylated with stearoyl chloride. In addition, chemical modification did not influence the protein's immunogenicity.

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Acylation of l-asparaginase with total retention of enzymatic activity

Cited by 26 publications

References 11 publications

Improved Acylation Method Enables Efficient Delivery of Functional Palmitoylated Cystatin into Epithelial Cells

Improved Acylation Method Enables Efficient Delivery of Functional Palmitoylated Cystatin into Epithelial Cells

Pharmacological and clinical evaluation of l-asparaginase in the treatment of leukemia

Membrane Permeability of Acylated Cystatin Depends on the Fatty Acyl Chain Length

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