In vivo target of benzylpenicillin in Gaffkya homari.
In vivo target of benzylpenicillin in Gaffkya homari.
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Gaffkya homari 中苄青霉素的体内靶点。
DOI：
10.1128/aac.29.3.432
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发表时间：
1986
影响因子：
4.9
通讯作者：
Neuhaus,FC
中科院分区：
文献类型：
--
作者：
Wrezel,PW;Ellis,LF;Neuhaus,FC
已确定DD-羧肽酶是Gaffkya homari（W. P. Hammes，Eur. 70：107-113，1976）。为了确定这种酶是否也是苄青霉素在体内的主要靶点，我们比较了这种β-内酰胺、头孢甲肟、头孢噻吩和头孢西丁对生长的影响及其对青霉素结合蛋白（PBP）9（DD-羧肽酶）的酰化作用。三种类型的膜壁实验的结果表明，PBP 9是这种酶，它是在十二烷基硫酸钠（SDS）不溶性肽聚糖的合成这些β-内酰胺的主要体外目标。首先，这些β-内酰胺对PBP 9的酰化抑制DD-羧肽酶和SDS-不溶性肽聚糖的合成。第二，青霉素从PBP 9释放的速率与DD-羧肽酶的回收率相关。第三，DD-羧肽酶活性检测在蛋白质具有相同的表观分子量的PBP 9从SDS-聚丙烯酰胺凝胶洗脱后。当用苄青霉素处理完整细胞时，最小生长抑制浓度（MGIC）与使PBPs 6和9酰化50%所需的[35 S]苄青霉素浓度相关。当用头孢甲肟、头孢噻吩或头孢西丁处理完整细胞时，MGIC与未标记β-内酰胺的浓度相关，所述未标记β-内酰胺的浓度是将随后的[35 S]苄青霉素与PBP 6的结合降低50%（ED 50）所需的。相比之下，这些β-内酰胺的MGIC与PBP 9的ED 50不相关。PBP 9在其MGIC处不被头孢甲肟或头孢噻吩酰化，而该PBP在其MGIC的十分之一处被头孢西丁完全酰化。研究表明，PBP 6可能是苄青霉素、头孢甲肟、头孢菌素和头孢西丁抑制生长的主要靶点; PBP 9（DD-羧肽酶）对于实验室条件下的生长是不可或缺的; PBP 9似乎不是这些β-内酰胺类药物的主要体内靶点，尽管这种PBP是它们的主要体外靶点。
It has been established that the DD-carboxypeptidase is the primary in vitro target of benzylpenicillin in Gaffkya homari (W. P. Hammes, Eur. J. Biochem. 70:107-113, 1976). To determine whether this enzyme is also the primary target of benzylpenicillin in vivo, we compared the effects of this beta-lactam, cefmenoxime, cephalothin, and cefoxitin on growth with their acylation of penicillin-binding protein (PBP) 9, the DD-carboxypeptidase. Results of three types of experiments with membrane-walls indicated that PBP 9 is this enzyme and that it is the primary in vitro target of these beta-lactams in the synthesis of sodium dodecyl sulfate (SDS)-insoluble peptidoglycan. First, the acylation of PBP 9 by these beta-lactams paralleled the inhibition of DD-carboxypeptidase and the inhibition of SDS-insoluble peptidoglycan synthesis. Second, the rate of benzylpenicillin release from PBP 9 correlated with the recovery of DD-carboxypeptidase. Third, DD-carboxypeptidase activity was detected in a protein with the same apparent molecular weight as PBP 9 after elution from an SDS-polyacrylamide gel. When intact cells were treated with benzylpenicillin, the minimum growth inhibitory concentration (MGIC) correlated with the concentration of [35S]benzylpenicillin required to acylate PBPs 6 and 9 by 50%. When intact cells were treated with cefmenoxime, cephalothin, or cefoxitin, the MGICs correlated with the concentration of unlabeled beta-lactam required to reduce the subsequent binding of [35S]benzylpenicillin by 50% (ED50) for PBP 6. In contrast, the MGICs of these beta-lactams did not correlate with the ED50s for PBP 9. PBP 9 was not acylated by cefmenoxime or cephalothin at their MGICs, whereas this PBP was fully acylated by cefoxitin at one-tenth of its MGIC. It is suggested that PBP 6 may be a primary target of growth inhibition by benzylpenicillin, cefmenoxime, cephalothin, and cefoxitin; PBP 9, the DD-carboxypeptidase, is dispensable for growth under laboratory conditions; and PBP 9 does not appear to be a primary in vivo target of these beta-lactams, even though this PBP is their primary target in vitro.