Hamilton A J, Holdom M D
St. Johns Institute of Dermatology, Guy's Hospital, London, United Kingdom.
Infect Immun. 1997 Feb;65(2):488-94. doi: 10.1128/iai.65.2.488-494.1997.
Cu,Zn superoxide dismutases (SODs) have been purified to homogeneity from the two varieties of Cryptococcus neoformans, C. neoformans var. neoformans and var. gattii. The N-terminal amino acid sequences of the two enzymes were similar, though not identical, and demonstrated homology with Cu,Zn SODs from other organisms. SOD activity was present in supernatants from stationary-phase cultures of isolates of C. neoformans var. neoformans and was also present from the mid-log phase onwards in cultures of an acapsular mutant of C. neoformans var. neoformans. SOD activity was practically undetectable in culture supernatants from isolates of C. neoformans var. gattii. The C. neoformans var. neoformans SOD had a reduced relative molecular mass of 19 kDa, and in its nonreduced form the enzyme was present as a 125-kDa species. Isoelectric focusing indicated that four species with pIs of 5.9, 6.15, 6.35, and 6.6 were present. The equivalent reduced molecular mass of the C. neoformans var. gattii enzyme was 19 kDa, with a single species present under nonreducing conditions (relative molecular mass of 145 kDa) with a pI of 7.5. The activities of the enzymes from both varieties were inhibited by KCN; however, the copper chelator diethyldithiocarbamate was inhibitory only against the C. neoformans var. gattii enzyme, as was sodium azide. The C. neoformans var. neoformans SOD was not affected by preincubation for 1 h at 70 degrees C, and it also retained most of its activity when incubated at 37 degrees C relative to its activity when incubated at 20 degrees C, in contrast to the C. neoformans var. gattii enzyme. The pronounced differences in the physical and biochemical characteristics of the Cu,Zn SODs from the two Cryptococcus varieties complement recent reports illustrating the biochemical and genetic differences between C. neoformans var. neoformans and C. neoformans var. gattii, and the successful purification of the two enzymes comprises the first step in determining what role, if any, the cryptococcal Cu,Zn SODs might have in protection against externally generated superoxide.
已从新型隐球菌的两个变种，即新型隐球菌变种新生变种和加蒂变种中，将铜锌超氧化物歧化酶（SOD）纯化至同质。两种酶的N端氨基酸序列相似但不完全相同，且与其他生物体的铜锌SOD具有同源性。新型隐球菌变种新生变种分离株的稳定期培养上清液中存在SOD活性，新型隐球菌变种新生变种的无荚膜突变体培养物从对数中期起也存在SOD活性。新型隐球菌变种加蒂变种分离株的培养上清液中几乎检测不到SOD活性。新型隐球菌变种新生变种的SOD相对分子质量降低至19 kDa，其非还原形式的酶以125 kDa的形式存在。等电聚焦表明存在四种等电点分别为5.9、6.15、6.35和6.6的同工酶。新型隐球菌变种加蒂变种酶的等效还原分子质量为19 kDa，在非还原条件下存在单一同工酶（相对分子质量为145 kDa），等电点为7.5。两种变种酶的活性均受到KCN的抑制；然而，铜螯合剂二乙基二硫代氨基甲酸盐仅对新型隐球菌变种加蒂变种的酶有抑制作用，叠氮化钠也是如此。新型隐球菌变种新生变种的SOD在70℃预孵育1小时后不受影响，并且与新型隐球菌变种加蒂变种的酶相比，在37℃孵育时相对于在20℃孵育时仍保留其大部分活性。来自两种新型隐球菌变种的铜锌SOD在物理和生化特性上的显著差异，补充了最近关于新型隐球菌变种新生变种和新型隐球菌变种加蒂变种之间生化和遗传差异的报道，而成功纯化这两种酶是确定隐球菌铜锌SOD在抵御外部产生的超氧化物中可能发挥何种作用（如果有作用的话）的第一步。
