同步辐射在D-甘油醛-3-磷酸脱氢酶及其系列衍生物高分辨率衍射数据收集中的应用

用气相扩散法培养出 P.Versicolor龙虾肌HOLO-D-甘油醛-3-磷酸脱氢酶(HOLO一GAPDH)、149位巯基羧甲基化的衍生物(CM—GAPDH)和紫外光激发生成的新NAD荧光衍生物(IRR—GAPDH)的晶体.用同步辐射强x光光源一磷光贮屏一Weissenberg照相机系统,选择a+b或a-b晶轴为旋转轴;收集了酶及其衍生物1.8A分辨率的三套衍射数据;用WEIS程序进行了数据处理.三套数据的Rmerge分别为4.93%、6.22%和5.77%.用CAD程序对三套数据进行了比较分析.     

蛇肌甘油醛-3-磷酸脱氢酶的研究 Ⅲ.萤光衍生物的生成

蛇肌CM-Apo-GAPDH在NAD~ 存在的条件下,经紫外光照产生萤光衍生物。其校正发射光谱峰值在420nm,校正激发光谱呈三峰形,峰值在240nm、285nm、325nm。蛇肌GAPDH萤光衍生物生成的最适条件为:在pH7.6～8.0的缓冲液中光照8分钟,NAD~ 与酶克分子浓度的比值为60。用325nm激发、410nm发射的萤光滴定测NAD~ 与CM-GAPDH的结合,表明由于蛇肌GAPDH羧甲基化使NAD~ 与酶的结合所表现的负协同性变得弱得多。萤光衍生物A_(280)/A_(260)为1.61～1.63,相当于蛇肌GAPDH萤光衍生物每分子中的四个亚基含有二分子NAD~ ,蛇肌GAPDH萤光衍生物的生成也属于半位反应。     

胰岛素及其苄基衍生物在钠-氨系统中的还原及活力恢复

(1)应用三种不同的方法制得了胰岛素苄基衍生物。胰岛素如果用钠-氨还原再与氯化苄作用,可以得到不再有完整二硫键存在的充分苄基化的产物。(2)将胰岛素、S-磺酸型A及B链混合物或者胰岛素苄基衍生物,在钠-氨内还原,然后在稀碱溶液中重氧化,都可以使胰岛素活力再生。用胰岛素或S-B磺酸型A及B链混合物时,经过此处理则活力可以恢复5—10%。用胰岛素苄基衍生物时则一般活力恢复1—2%。(3)从充分苄基化了的胰岛素衍生物能恢复活力这一事实说明可以通过人工合成的A和B链最后合成胰岛素。(4)观察了A和B键混合物巯基氧化和活力再生的过程,并进行了讨论。     

蛇肌甘油醛-3-磷酸脱氢酶的研究 Ⅰ.纯化和一些基本性质

用硫酸铵分级、DEAE-纤维素柱层析、羧甲基纤维素柱层析分离和纯化了蛇肌GAPDH。聚丙烯酰胺凝胶电泳和醋酸纤维素薄膜电泳均为一条带。用此法提纯的蛇肌GAPDH A_(280)/A_(260)为2.1,是Apo-GAPDH。在纯化的Apo-GAPDH中加入NAD~ 能够得到结晶。Sephadex G-150凝胶过滤法测得蛇肌GAPDH的分子量约为150,000。十二烷基硫酸钠凝胶电泳也为一条带,亚基分子量约为38,000。表明蛇肌GAPDH是由相同的四个亚基组成的。用Ferdinand法测得比活为100。蛇肌GAPDH的N-末端氨基酸为缬氨酸。每分子含巯基数、色氨酸和酪氨酸残基数分别为12、12和36。重量法测得蛇肌Apo-GAPDH消光系数E_(280)~(0.1%)为0.775;Holo-GAPDH的消光系数E_(280)~(0.1%)为1.02。蛇肌GAPDH对甘油醛-3-磷酸的米氏常数为1.67×10~(-3)M,对NAD~ 的米氏常数为1.11×10~(-4)M。NAD~ 与蛇肌Apo-GAPDH结合后有Racker带,每分子蛇肌GAPDH可结合4分子NAD~ ,酶与NAD~ 的结合表现为负协同性。蛇肌GAPDH的圆二色谱,近紫外区Apo-GAPDH在285nm处有一负峰,292nm、270nm处有肩。加入NAD~ 后负峰变大并且蓝移。NAD~ 的加入似乎影响了酪氨酸所处的微环境。远紫外区Apo-GAPDH在220nm处有一负峰,208nm处有一个肩;加入NAD~ 后,负峰值稍稍变大,但峰的位置和形状未变。表明蛇肌GAPDH中,含较多的β-折迭,较少的α-螺旋。NAD~ 的加入对二级结构影响不大。     

蛇肌甘油醛-3-磷酸脱氢酶的研究——Ⅲ.萤光衍生物的生成

蛇肌CM-Apo-GAPDH在NAD~ 存在的条件下,经紫外光照产生萤光衍生物。其校正发射光谱峰值在420 nm,校正激发光谱呈三峰形,峰值在240 nm、285nm、325nm。蛇肌GAPDH 萤光衍生物生成的最适条件为:在pH7.6～8.0的缓冲液中光照8分钟,NAD~ 与酶克分子浓度的比值为60。用325nm 激发、410nm 发射的萤光滴定测NAD~ 与CM-GAPDH 的结合,表明由于蛇肌GAPDH 羧甲基化使NAD 与酶的结合所表现的负协同性变得弱得多。萤光衍生物A_(280)/A260为1.61～1.63,相当于蛇肌GAPDH 萤光衍生物每分子中的四个亚基含有二分子NAD~ ,蛇肌GAPDH 萤光衍生物的生成也属于半位反应。     

蛇肌甘油醛-3-磷酸脱氢酶的研究——Ⅰ.纯化和一些基本性质

用硫酸铵分级、DEAE-纤维素柱层析、羧甲基纤维素柱层析分离和纯化了蛇肌GAPDH。聚丙烯酰胺凝胶电泳和醋酸纤维素薄膜电泳均为一条带。用此法提纯的蛇肌GAPDH A_(280)/A_(260)为2.1,是Apo-GAPDH。在纯化的Apo-GAPDH 中加入NAD~ 能够得到结晶。Sephadex G-150凝胶过滤法测得蛇肌GAPDH 的分子量约为150,000。十二烷基硫酸钠凝胶电泳也为一条带,亚基分子量约为38,000。表明蛇肌GAPDH 是由相同的四个亚基组成的。用Ferdinand 法测得比活为100。蛇肌GAPDH 的N-末端氨基酸为缬氨酸。每分子含巯基数、色氨酸和酪氨酸残基数分别为12、12和36。重量法测得蛇肌Apo-GAPDH 消光系数E_(280)~(0.1)%为0.775;Holo-GAPDH 的消光系数E_(280)~(01.)为1.02。蛇肌GAPDH 对甘油醛-3-磷酸的米氏常数为1.67×10~(-8)M,对NAD~ 的米氏常数为1.11×10~(-4)M。NAD~ 与蛇肌Apo-GAPDH 结合后有Racker 带,每分子蛇肌GAPDH 可结合4分子NAD~ ,酶与NAD~ 的结合表现为负协同性。蛇肌GA PDH 的圆二色谱,近紫外区Apo-GAPDH 在285nm 处有一负峰,292nm、270nm 处有肩。加入NAD 后负峰变大并且蓝移。NAD~ 的加入似乎影响了酪氨酸所处的微环境。远紫外区Apo-GAPDH 在220nm 处有一负峰,208nm 处有一个肩;加入NAD~ 后,负峰值稍稍变大,但峰的位置和形状未变。表明蛇肌GAPDH中,含较多的β-折迭,较少的α-螺旋。NAD~ 的加入对二级结构影响不大。     

P.versicolor龙虾D-甘油醛-3-磷酸脱氢酶的X射线晶体学研究——分子置换法结构测定

由P.versicolor龙虾尾肌提取的HOIO-D-甘油醛-3-磷酸脱氢酶(GAPDH),已长出可供Χ射线衍射用的晶体。初步Χ射线晶体学研究确定:此酶晶体属於C2空间群,不对称单位内含有半个分子,分子坐落在二重轴上。以Homarus Amercanus龙虾GAPDH结构为模型结构,应用分子置换技术进行了低分辨率Χ射线结构分析,结果表明:分子内亚基排列具有222对称性,分子Q轴平行于晶体学二重轴b,分子P和R轴分别平行于晶体学a和c轴。按分子置换法推出的结构模型算得5A分辨率的晶体学R因子为0.46。并获得了一套5A。分辨率的电子密度图。此酶的几种同晶型晶体,特别是荧光NAD衍生物晶体的较高分辨率的结构分析工作正在进行中。     

粘虫甘油醛-3-磷酸脱氢酶基因cDNA的克隆、序列分析及表达量检测

运用RT-PCR和RACE技术,以粘虫(Mythimna separata(Walker))cDNA为模板,对甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因进行克隆获得全长cDNA序列,并利用生物信息学方法,对GAPDH全长cDNA序列及推测得到的GAPDH蛋白序列进行分析.结果表明,获得的粘虫GAPDH基因cDNA序列长度为1 317 bp,其中包括80 bp的5′非编码区、238 bp的3′非编码区和999 bp的开放阅读框(Open Reading Frame,ORF),编码一个332个氨基酸蛋白,具有GAPDH蛋白家族的两个功能结构域.该GAPDH蛋白理论相对分子质量为35.498 6 kDa,等电点为7.63,富含6种类型的特定功能位点.该蛋白序列与其他动物GAPDH蛋白序列具有77.4%～92.9%高度同源性.GAPDH基因表达量检测结果显示GAPDH在粘虫6种不同组织间表达量无显著差异(P0.05),表明GAPDH可作为研究粘虫功能基因表达量分析的可靠内参基因.该基因的cDNA序列已经递交GenBank并获得登录号为HM055756.     

人类3-磷酸甘油醛脱氢酶的原核表达、纯化和鉴定

为了研究3-磷酸甘油醛脱氧酶(GAPDH)化生物学功能,以pcDNA3.1-GAPDH质粒为模板,PCR方法扩增GAPDH基因,经BamHI和SalI双酶切后插入相同酶切的pET32a(+)载体,构建His-GAPDH融合蛋白表达质粒pET32a(+)-GAPDH;转化JM109感受态细胞,并进行阳性克隆筛选,扩增目的质粒;转化大肠杆菌BL21菌株,经IPTG诱导产生融合蛋白,亲和层析柱纯化后,用SDS-PAGE和Western blotting检测GAPDH蛋白表达情况.结果表明,构建的人GAPDH基因表达载体经序列测定证实,与GenBank数据完全一致;双酶切鉴定证实,克隆基因正确插入pET32a(+)载体;表达质粒pET32a(+)-GAPDH在大肠杆菌BL21中成功地诱导表达了可溶性His-GAPDH融合蛋白,纯化后SDS-PAGE和Western blotting检测证实融合蛋白表达成功.人GAPDH原核表达载体的成功构建,及6His-GAPDH融合蛋白的正确表达,为进一步深入研究GAPDH的生物学功能奠定了基础.     

酵母GAPDH在碘化钾溶液中荧光发色团的形成

酵母GAPDH的剩余活力在碘化钾为0.1M溶液中趋近于零,内源荧光在碘化钾为0.1—0.4M时迅速被淬灭,在0.5—2.0M时基本不变,3.0M时达到最低。并在一定浓度的碘化钾溶液中形成新的荧光发色性质(Ex336nm;Em383nm),量子产率大约为0.2,但形成该荧光发色团较为缓慢。与NAD荧光衍生物(Chen-LuTsou,etal.(1983)Biochem.Soc.Trans.11,425—429)不同的是,GAPDH在碘化钾溶液中形成荧光发色团时不需要光的激发。肌酸激酶及胰岛素不形成这种荧光衍生物。     

SUBCELLULAR ANALYSIS OF THE MOLECULAR FORMS OF ACETYLCHOLINESTERASE IN RAT SKELETAL MUSCLE

Acetylcholinesterase (AChE, EC 3.1.1.7) activity of rat gastrocnemius muscle homogenized in 1 M-NaCl and 0.5% Triton X-100 was separated by velocity sedimentation in sucrose gradients into three molecular forms with sedimentation coefficients of about 4S, 10S and 16S. The distribution of homogenate AChE activity among the three peaks was 53, 34 and 13% respectively. The different molecular forms were found to be heterogeneously distributed in subcellular fractions prepared from sucrose homogenates of muscle, as follows: Subfractions of the crude sarcolemmal fraction were prepared by discontinuous sucrose gradient centrifugation. AChE was recovered in the greatest yield and with the highest specific activity in a light density subfraction (0.6/0.8 M-sucrose interface). The AChE activity in this light density subfraction was mainly (81-88%) the 10S form of the enzyme. The velocity sedimentation profiles of the AChE activity in the more dense subfractions were markedly different in that 16S AChE was a major component.     

Conformation similarities of the globular and tailed forms of acetylcholinesterase from Torpedo californica

The conformation of the globular dimer (G2), the tailed asymmetric dodecamer (A12, also containing some tailed octamer A8) and the globular tetramer (G4, prepared by removing the collagen-like tail from A12) of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) was studied by circular dichroism (CD) in the ultraviolet region. The G2 and G4 forms had similar conformation with about 40% alpha-helix, 35% beta-sheets and 4% beta-turns; the tailed form had a lower helicity (about 34%) and beta-form (about 25%) content probably because of the presence of the tail whose CD spectrum resembles that of an unordered form, but it had about the same amount of beta-turns as the other two forms. All three forms also had similar CD spectra in the near-ultraviolet region due to their non-peptide chromophores. The pH, thermal and urea denaturation of the three acetylcholinesterase forms was also similar to each other. The pH-dependency of both the enzymatic activity and CD intensity of the three forms showed bell-shaped curves with a plateau at pH 7-8. The activity was completely lost at pH below 5 or above 10, but the corresponding CD spectra retained 70-80% of the original magnitudes. Thermal denaturation of the three forms at pH 7.5 showed a conformational transition and loss of activity between 30 and 40 degrees C, but the CD intensity of the helical band at 222 nm was reduced by only 20-30%. Urea denaturation of the three forms began at 1 M urea; it was protein concentration- and time-dependent. Again, the activity disappeared faster than the decreasing CD intensity. Thus, the overall conformation of the three acetylcholinesterase forms appears to be relatively stable, but their active site is easily perturbed by changing the environment. The loss of activity correlated well with the disappearance of the CD band of tryptophan(s) in the near-ultraviolet region, suggesting that the Trp residue(s) might be at or near the active center of the enzyme.     

MULTIPLE FORMS OF β -GALACTOSIDASE AND THE CHANGES IN ITS PATTERN DURING DEVELOPMENT OF DICTYOSTELIUM DISCOIDEUM

Four forms of β-galactosidase (EC 3.2.1.23) were separated from cell extracts of Dictyostelium discoideum by polyacrylamide gel electrophoresis. Changes in the pattern of multiple forms of this enzyme during differentiation and dedifferentiation were studied. The band closest to the anode (Band 1) existed throughout development, but the other three bands appeared and disappeared at certain stages. One of those bands was specific for the vegetative stage (Band 3), and another for the morphogenetic stages (Band 2). The last one predominantly appeared during dedifferentiation of disaggregated slug cells (Band 4), although it was slightly detected during culmination. During the process of dedifferentiation, the increase in activity of Band 4 coincided with the decrease in Band 2. After the completion of dedifferentiation, Band 4 also disappeared, only Band 1 remaining. These multiple forms were not only electrophoretically separable but different in their sensitivities to various inhibitors. All forms of the enzyme were localized in subcellular particles, probably in lysosomes and phagosomes, and the relative activities in these two fractions varied during development.     

The interrelation between high- and low-molecular-weight forms of GM1-beta-galactosidase purified from porcine spleen

1) Two forms of acid beta-galactosidase [EC 3.1.23] with different molecular weights catalyzing the hydrolysis of GM1-ganglioside and p-nitrophenyl-beta-D-galactoside were separated and purified from porcine spleen. 2) The apparent molecular weights were 400,000-600,000 and 70,000-74,000 for the high (termed Am form) and low (termed A1 form) molecular weight forms, respectively. 3) On examination by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis, both forms of the enzyme had a common protein band of molecular weight 63,000, and the Am form showed three additional protein bands with molecular weights of 31,000, 21,000, and 20,000. 4) Both forms of the enzyme had similar catalytic functions with regard to pH-optimum, Km, substrate specificity and sensitivity to substrate analogues and other substances such as detergents, bovine serum albumin (BSA) and NaCl. 5) Both forms of the enzyme were fairly stable upon preincubation at 45 degrees C at acidic pH (pH 4.5), but lost their activities at neutral pH (pH 7.0). 6) The A1 form was a monomer at neutral pH (pH 7.0) and formed a dimer at acidic pH (pH 4.5). However, most of the Am form could not be converted to a dimeric form on gel filtration at acidic pH.     

Purification and Separation of A and B Forms of N-Acetyl-β-D-Hexosaminidase from Human Aorta

Human aorta has been shown to possess multiple forms of N-Acetyl-6-D-hexosaminidase (β-2-acetamido-2-deoxy-D-glucoside-acetamido-deoxyglucohydro-lase, EC 3.2, 1.30). The enzyme was separable, by gel electrophoresis, into 2 enzymatically active bands representing A and B forms. By gel electro-focussing, A and B forms were further subdivided into at least 5 and 8 bands, respectively. The B form consisted of 4 bands (B₁) and 4 bands (B₂), which were not inactivated at 50° for 3 hr. (at pH 4.4) in the presence of serum; whereas, the 5 bands found in A form were completely inactivated. All forms of the enzyme were active towards naphthol-AS-BI-N-acetyl-β-D-glucosaminide and the corresponding galactosaminide (about one-eighth of the hydrolysis rate of the former), suggesting each single enzyme acts on both substrates. The N-acetyl-hexosaminidases of bull epididymis, by comparison, were also found to be active towards both substrates and to possess 13 bands having pis more alkaline than those of the B form of the human enzyme, By heat inactivation we found that the aortic enzyme consisted of 51% of A and 49% of B (B₁ + B₂ .). Neuraminidase had no effect on either form of the aortic preparation. Both forms were partially purified and separated by conventional methods. They required BSA for their maximal activity; the A form being more dependent BSA than the B form, With PNP-N-acetyl-β-D-glucosaminide and the corresponding galactosaminide, Km of 1.04 mH and 0.54 mM, respectively, for A form and of 1.74 and 1.48 mM, respectively, for B form were obtained. While the purified B form was stable and did not transform into other species, the purified A form gradually transformed into B form as well as into other new forms during storage at -20°.     

Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics.

An extract of rat liver or human platelet displayed three cyclic 3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a continuous sucrose density gradient when assayed with millimolar adenosine 3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The three fractions obtained from each nucleotide were not superimposable. The molecular weights corresponding to the three activity peaks of cAMP phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000; and III, 140,000. In both tissues, fraction I was barely detectable when assayed with micromolar concentrations of either nucleotide, presumably because fraction I has low affinity for cAMP and cGMP. Any one of the three forms upon recentrifugation on the gradient generated the others, indicating that they were interconvertible. The multiple forms appear to represent different aggregated states of the enzyme. The ratio of the three forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the formation of fraction I. Low enzyme concentration favored the equilibrium towards fraction I, while high enzyme concentration favored fraction III. When phosphodiesterase activities in the extract of rat liver, human platelets, or bovine brain were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar cAMP or cGMP. The specific activity with millimolar cAMP was higher with low than with high protein concentrations, suggesting that the dissociated form catalyzed the hydrolysis of cAMP faster than that of the associated form. In contrast, the specific activity with millimolar cGMP was lower with low than with high protein concentrations. Supplementing the reaction mixture with bovine serum albumin to a final constant protein concentration did not affect the activity, suggesting that the concentration of the enzyme rather than that of extraneous proteins affected the enzyme activity. A change in enzyme concentration affected the kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X 10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while a high enzyme concentration yielded a nonlinear plot, and apparent Km values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X 10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low enzyme concentration favored fraction I, the dissociated form, whereas a high enzyme concentration favored fraction III, the associated form, these kinetic constants suggest that the dissociated form exhibits a high Km and the associated form exhibits a low Km. In contrast, a high enzyme concentration gave a linear kinetic plot for cGMP phosphodiesterase, while a low enzyme concentration gave a nonlinear plot...     

Isolation and characterization of mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills

A soluble F(1)-ATPase was isolated from the mitochondria of crayfish (Orconectes virilis) gill tissue. The maximal mitochondrial disruption rate (95%) was obtained by sonicating for 4 min at pH 8.6. A 15-fold purification was estimated. The properties for both soluble and membrane-bound enzyme were studied. Both enzyme forms were stable at 4 to -70 degrees C when kept in 20% glycerol. Soluble F(1)-ATPase was more stable at room temperature than membrane-bound enzyme. It displayed a narrower pH profile (pK(1) =6.58, pK(2)=7.68) and more acid pH optimum (7.13) than membrane-bound enzyme (pK(1)=6.42, pK(2)=8.55, optimum pH 7.49). The anion-stimulated activities were in the order HCO(3)(-)>SO(4)(2-)>Cl(-). The apparent K(a) values for soluble enzyme were 11.4, 11.2, and 10.9 mM, respectively, but the K(a) of HCO(3)(-) for membrane-bound enzyme (14.9 mM) was higher than for soluble enzyme. Oligomycin and DCCD inhibited membrane-bound F(1)-ATPase with I(50) of 18.6 ng/ml and 2.2 microM, respectively, but were ineffective in inhibiting soluble enzyme. Both enzyme forms shared identical sensitivity to DIDS (I(50)=12.5 microM) and vanadate (I(50)=9.0 mM). Soluble ATPase was significantly more sensitive to pCMB (I(50)=0.15 microM) and NO(3)(-) (I(50)=28.6 mM) than membrane-bound enzyme (I(50)=1.04 microM pCMB and 81.5 mM NO(3)(-)). In addition, soluble F(1)-ATPase was slightly more sensitive to azide (I(50)=91.8 microM) and NBD-Cl (I(50)=9.18 microM) than membrane-bound enzyme (I(50)=111.6 microM azide and 12.88 microM NBD-Cl). These data suggest a conformational change transmission between F(0) and F(1) sectors and slight conformational differences between soluble F(1) and membrane-bound F(1). In addition, an unmodified F(0) stabilizes F(1) and decreases F(1) sensitivities to inhibitors and modulators.     

RNA-dependent DNA polymerase of avian sarcoma virus B77. I. Isolation and partial characterization of the alpha, beta2, and alphabeta forms of the enzyme.

Three forms of the RNA-dependent DNA polymerase were isolated from highly purified avian sarcoma virus B77 grown in duck embryo fibroblasts, using sequential chromatography on DEAE-cellulose, phosphocellulose, and poly(U)-cellulose. One form, which sedimented with about 5.2 S, contained only one species of polypeptide, with a molecular weight of 63,000; a second sedimented with about 7.8 S and contained only one species of polypeptide with a molecular weight of 81,000; and a third form, which sedimented with about 7.3 S, contained two species of polypeptides with molecular weights of 63,000 and 81,000. The molecular constitution of the three enzyme forms were therefore alpha, beta2, and alphabeta. All three possessed almost the same specific activity with poly(rA)-oligo(dT) as the primer-template. Forms alpha and alphabeta of avian sarcoma virus DNA polymerase have already been described in the literature; form beta2 is a new form. All three forms possessed ribonuclease H activity, the relative specific activities of the alpha, beta2, and alphabeta forms being about 1:4:5. All three enzyme forms were inhibited by antiserum to the alphabeta form, but whereas the alpha and alphabeta forms could be inhibited about 95%, the maximum degree of inhibition of the beta2 form was about 80%. The three enzyme forms also differed with respect to heat stability at 46 degrees, the monomeric alpha form of the enzyme being only about one-half as stable as the two dimeric forms.     

Hydroxymethylglutaryl-coenzyme A reductase. Purification and properties of the enzyme from Fusarium oxysporum.

The hydroxymethylglutaryl-coenzyme A reductase (mevalonate:NADP+ oxidoreductase, EC 1.1.1.34) system in Fusarium oxysporum, a soil inhabiting plant pathogen, has been examined. Two forms of the enzyme catalyzing the conversion of hydroxymethylglutaryl-coenzyme A were obtained in the supernatant after precipitation at 75% (NH4)2SO4 saturation of the soluble culture extract which was previously separated from cell wall, mitochondria and microsomes. The two forms of the enzyme were separated electrophoretically. A third form, contained in the precipitate obtained at 35--75% (NH4)2SO4 saturation of the same extract, was further purified by Sephadex G-50 column chromatography. This purified form moved as a single band in sodium dodecyl sulphate electrophoresis and in immunological tests and has a molecular weight of 11 000. The apparent Michaelis constant for the substrate hydroxymethylglutaryl-coenzyme A is 21 micron at 2 micron NADP. NADPH is a more efficient reductant on a molar basis than NADH for the deacylation of the hydroxymethylglutaryl-coenzyme A substrate. Optimum activity of the enzyme was obtained at pH 7.4 and 37 degrees C. The enzyme demonstrated no cold sensitivity but rather was more stable at 4 degrees C than at 25 degrees C. The protection with dithiothreitol, though minimal compared to other systems, was more effective at the higher temperature.