High-Pressure Effects on Vacuolar H+-ATPase from Etiolated Mung Bean Seedlings

Tsai, Yuan Ru; Yang, Shu; Jiang, Shih Sheng; Ko, Shu Jin; Hung, Shu Hsien; Kuo, Soong Yu; Pan, Rong

doi:10.1023/a:1022535701279

Cited by 7 publications

References 1 publication

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External Factors Affecting Protein Aggregation

Wang

Speaker

2010

Aggregation of Therapeutic Proteins

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The behavior of biotherapeutic proteins is signifi cantly different from small molecule drugs both in liquid and solid states, especially in terms of physical stability. One such property is the high tendency of protein molecules to aggregate under a variety of conditions. The aggregation tendency is arguably the most common and troubling manifestation of protein instability during the development of protein biotherapeutics. 1 Protein aggregates usually exhibit either reduced or, in many cases, no biological activity. 2 -5 A clear correlation was established between loss of recombinant factor VIII (rFVIII ) and its degree of aggregation as shown in Fig. 4.1 . 6 To achieve effective prevention or inhibition of protein aggregation, it is of paramount importance to understand all the possible factors that affect or control the protein aggregation process.Many factors have been identifi ed and reported in the literature affecting the process and rate of protein aggregation. These factors can be roughly divided into two major categories: internal (protein structure related) and external (protein environment related). Chapter 3 in this book has focused extensively on factors that belong to the fi rst category. This chapter focuses on the external factors that affect the process and rate of protein aggregation. To facilitate discussion, these external factors are further divided into the following sections: (1) temperature; (2) solution conditions and composition (pH, buffer type and concentration, ionic strength, excipients and level, protein concentration, metal ions, denaturing and reducing agents, impurities, organic solvents, containers/closures, sources of proteins, sample treatment, and analytical methodologies); (3) processing steps (fermentation/expression,

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External Factors Affecting Protein Aggregation

Wang

Speaker

2010

Aggregation of Therapeutic Proteins

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Protein aggregation and its inhibition in biopharmaceutics

Wang

2005

International Journal of Pharmaceutics

841

581

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Inhibition of plant vacuolar H+-ATPase by diethylpyrocarbonate

Chu

Hsiao

Chen

et al. 2001

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Treatment of the tonoplast H(+)-ATPase from mung bean seedlings (Vigna radiata L.) with histidine-specific modifier, diethyl pyrocarbonate (DEP), caused a marked loss of the ATP hydrolysis activity and the proton translocation in a concentration-dependent manner. The reaction order of inhibition was calculated to be 0.98, suggesting that at least one histidine residue of vacuolar H(+)-ATPase was modified by DEP. The absorbance of the vacuolar H(+)-ATPase at 240 nm was progressively increased after incubation with DEP, suggesting that N-carbethoxyhistidine had been formed. Hydroxylamine, which could break N-carbethoxyhistidine, reversed the absorbance change and partially restored the enzymic activity. The pK(a) of modified residues of vacuolar H(+)-ATPase was kinetically determined to be 6.73, a value close to that of histidine. Thus, it is assuredly concluded that histidine residues of the vacuolar H(+)-ATPase were modified by DEP. Kinetic analysis showed that V(max) but not K(m) of vacuolar H(+)-ATPase was decreased by DEP. This result is interpreted as that the residual activity after DEP inhibition was primarily due to the unmodified enzyme molecules. Moreover, simultaneous presence of DEP and DCCD (N,N'-dicyclohexyl-carbodiimide), an inhibitor modified at proteolipid subunit of vacuolar H(+)-ATPase, did not induce synergistic inhibition, indicating their independent effects. The stoichiometry studies further demonstrate that only one out of four histidine residues modified was involved in the inhibition of vacuolar H(+)-ATPase by DEP. Mg(2+)-ATP, the physiological substrate of vacuolar H(+)-ATPase, but not its analogs, exerted preferentially partial protection against DEP, indicating that the histidine residue involved in the inhibition of enzymatic activity may locate at/or near the active site and directly participate in the binding of the substrate.

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High-Pressure Effects on Vacuolar H+-ATPase from Etiolated Mung Bean Seedlings

Cited by 7 publications

References 1 publication

External Factors Affecting Protein Aggregation

External Factors Affecting Protein Aggregation

Protein aggregation and its inhibition in biopharmaceutics

Inhibition of plant vacuolar H+-ATPase by diethylpyrocarbonate

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