棉铃虫卵内蛋白酶性质研究

在棉铃虫Helicoverpa armigera卵母细胞内检测到蛋白酶活性,其作用Ph在酸性范围,酶活性受E-64、Pepstatin和iPr₂P-F等多种抑制剂抑制。在Ph4.0时蛋白酶对牛血红蛋白有较高水解率。抗蓖麻蚕Philosamia cynthia ricini卵半胱氨酸蛋白酶血清和抗蓖麻蚕卵天冬氨酸蛋白酶血清可以识别棉铃虫卵内成分。实验结果表明;棉铃虫卵内可能存在半胱氨酸蛋白酶类、丝氨酸蛋白酶类和天冬氨酸蛋白酶类,并且与蓖麻蚕卵内蛋白酶有一定的相似性。     

棉铃虫组织蛋白酶B酶原在毕赤酵母中的表达

棉铃虫组织蛋白酶B（ Helicoverpa armigera Cathepsin B ,HCB）属于半胱氨酸蛋白酶类,参与胚胎发育中卵黄蛋白水解供给胚胎发育的氨基酸。本研究将HCB基因克隆到pPIC9K载体并转化毕赤酵母KM71菌株,经甲醇诱导,HCB表达并分泌到培养上清中。表达产物经SDS-PAGE测定分子量为38 kD, 与HCB基因编码的蛋白质分子量一致。用HCB的特异性抗体检测表明重组表达产物为棉铃虫组织蛋白酶B,原位水解实验显示重组表达的蛋白酶具有蛋白水解活性,表明在毕赤酵母中表达了有活性的棉铃虫组织蛋白酶B, 可用于组织蛋白酶B酶原活化机理研究及开发新蛋白酶产品。     

柞蚕卵组织蛋白酶B纯化及性质

昆虫卵内蛋白酶在胚胎发育中水解卵黄蛋白,为胚胎发育提供氨基酸。昆虫中已报道过几类卵蛋白酶,如家蚕中半胱氨酸蛋白酶和丝氨酸蛋白等。但是,目前尚不清楚这些蛋白酶是否存在于其他鳞翅目昆虫。了解这些蛋白酶的作用机理可以为我们提供害虫防治的新方法,并且,由于蛋白水解在许多生理过程中具有重要作用。如蛋白质的成熟和转运、受精、萌芽、肿瘤转移和其他形态发生等。因此,阐明这些蛋白酶的生物功能具有重要意义。由于Oi蚕卵粒大,产卵量也很大,因此被选作研究鳞翅目昆虫卵蛋白酶的材料,我们希望通过对数种昆虫卵内蛋白酶的研究,找出卵黄蛋白水解的一般规律。在我们前一篇文章中报道了oi蚕组织蛋白酶B的鉴定。该蛋白酶属于半胱酸蛋白酶类的组织蛋白,最适pH为3．5,可被E－64抑制。本文报道蛋白酶的纯化和蛋白质。经过5步纯化过程,从oi蚕卵母细胞中纯化出组织蛋白酶B,用SDS聚丙烯酰胺凝胶电泳测得蛋白酶的亚基分子量在47kDa左右。纯化的蛋白酶活性可被E－64和Leupeptin抑制。因此,该蛋白酶属于半胱氨酸蛋白酶。天冬氨酸蛋白酶特异性抑制Pepstatin不抑制其活性。其活性可被DFP和PMSF部分抑制制。这两种抑制剂通常抑制丝氨酸蛋白酶活性,但在家蚕中有报道,半氨酸蛋白也可被这两种抑制剂抑制。推测该知性中心除含有半胱氨酸残基外,可能还含有丝氨酸残基。由牛血红蛋白测得蛋白酶的最适pH为3．5。在pH3．5条件下对胚胎发育中蛋白酶活性变化进行了研究,并用纯化的蛋白酶制备了抗血清,采用单向免疫扩散对胚胎发育中组织蛋白酶B的含量进行了测定,结果表明这种蛋白酶在胚胎发育中含量较高,是胚肥发育中蛋白酶活性来源之一。     

柞蚕卵组织蛋白酶B纯化及性质

昆虫卵内蛋白酶在胚胎发育中水解卵黄蛋白,为胚胎发育提供氨基酸,昆虫中已报道过几类卵蛋白酶,如家蚕中半胱氨酸蛋白酶和丝氨酸蛋白酶等。但是,目前尚不清楚这些蛋白酶是否存在于其他鳞翅目昆虫。了解这些蛋白酶的作用机理可以为我们提供害虫防治的新方法。并且,由于蛋白水解在许多生理过程中具有重要作用,如蛋白质的成熟和转运、受精、萌芽、肿瘤转移和其他形态发生等。因此,阐明这些蛋白酶的生物功能具有重要意义。由于（ｉ蚕卵粒大产卵量也很大,因此被选作研究鳞翅目昆虫卵蛋白酶的材料,我们希望通过对数种昆虫卵内蛋白酶的研究、找出卵黄蛋白水解的一般规律。在我们前一篇文章中报道了（ｉ蚕组织蛋白酶Ｂ的鉴定,该蛋白酶属于半胱氨酸蛋白酶类的组织蛋或最适ｐＨ为３．５,可被Ｅ－６４抑制。本文报道蛋白酶的纯化和性质。经过５步纯化过程,从（ｉ蚕卵母细胞中纯化出组织蛋白酶Ｂ,用ＳＤＳ聚丙烯酰胺凝胶电泳测得蛋白酶的亚基分子量在４７ｋＤａ左右。纯化的蛋白酶活性可被Ｅ－６４和Ｌｅｕｐｅｐｔｉｎ抑制。因此,该蛋白酶属于半胱氨酸蛋白酶。天冬氨酸蛋白酶特异性抑制剂ｐｅｐｓｔａｔｉｎ不抑制其活性。其活性可被ＤＦＰ和ＰＭＳＦ部分抑制。这两种抑制剂通常抑制丝氨酸蛋白酶活性     

猕猴脑胱天蛋白酶-3活化及其靶蛋白的体外研究

凋亡的主要生化过程包括胱天蛋白酶的活化及其对细胞内蛋白质的选择性切割.在已知的胱天蛋白酶中,可被多种凋亡刺激信号激活的胱天蛋白酶-3备受注目.为进一步揭示灵长类动物神经组织中未知的胱天蛋白酶-3靶蛋白,采用成年猕猴脑组织粗提物作为无细胞体系,通过加入granzyme B引发凋亡途径的部分反应,如胱天蛋白酶-3的活化及随后发生的蛋白质水解.经蛋白质印迹分析发现,与granzyme B共孵育后,猕猴脑胱天蛋白酶-3以两步方式从酶原转化为活性酶.对猕猴脑组织自身蛋白质的进一步分析显示,多聚ADP-核糖聚合酶(PARP)被水解为长85 ku的片段,此片段提示胱天蛋白酶-3的特异切割活性.此外,神经元凋亡抑制蛋白(NAIP)也被切割,产生长约40 ku的小片段,但是它的出现不被胱天蛋白酶-3特异性抑制剂Ac-DEVD-CHO阻断,因此可能是granzyme B直接作用于NAIP所致.以上结果提示,凋亡相关酶切反应可在成年猕猴脑组织提取物中得到重现;NAIP可能是granzyme B而非胱天蛋白酶-3的作用靶点.     

猕猴脑胱天蛋白酶-3活化及其靶蛋白的体外研究(英)

凋亡的主要生化过程包括胱天蛋白酶的活化及其对细胞内蛋白质的选择性切割.在已知的胱天蛋白酶中,可被多种凋亡刺激信号激活的胱天蛋白酶-3备受注目.为进一步揭示灵长类动物神经组织中未知的胱天蛋白酶-3靶蛋白,采用成年猕猴脑组织粗提物作为无细胞体系,通过加入granzyme B引发凋亡途径的部分反应,如胱天蛋白酶-3的活化及随后发生的蛋白质水解.经蛋白质印迹分析发现,与granzyme B共孵育后,猕猴脑胱天蛋白酶-3以两步方式从酶原转化为活性酶.对猕猴脑组织自身蛋白质的进一步分析显示,多聚ADP-核糖聚合酶（PARP）被水解为长85 ku的片段,此片段提示胱天蛋白酶-3的特异切割活性.此外,神经元凋亡抑制蛋白（NAIP）也被切割,产生长约40 ku的小片段,但是它的出现不被胱天蛋白酶-3特异性抑制剂Ac-DEVD-CHO阻断,因此可能是granzyme B直接作用于NAIP所致.以上结果提示,凋亡相关酶切反应可在成年猕猴脑组织提取物中得到重现;NAIP可能是granzyme B而非胱天蛋白酶-3的作用靶点.     

家蚕卵半胱氨酸蛋白酶纯化及抗血清制备

据报道,家蚕卵中存在半胱氨酸蛋白酶(Cysteine proteinase,CP),其性质与哺乳类溶酶体半胱氨酸蛋白酶类的组织蛋白酶L相似,最佳作用pH为3.5,体外最适作用底物为牛血红蛋白,体内最适作用底物为卵黄磷蛋白。经SDS—PAGE分析,分子量47KD。其主要作用是在胚胎发育过程中降解卵黄蛋白质,供胚胎发育之需要。 在成熟卵中具很高含量的半胱氨酸蛋白酶,在胚胎发育开始前,并不发生卵黄蛋白质的水解,其作用机制尚待阐明。为了进一步研究半胱氨酸蛋白酶的组织分布、合成位点、及cDNA克隆等,作者从家蚕卵中纯化了半胱氨酸蛋白酶,并制备了抗血清。     

棉铃虫卵内半胱氨酸蛋白酶的分离纯化、氨基酸序列分析及蛋白酶鉴定

采用阴离子交换层析法,从棉铃虫Helicoverpa armigera卵母细胞中分离纯化到一种半胱氨酸蛋白酶,SDS-PAGE电泳显示为一条带,分子量约为29 kD,原位水解电泳表明其具有蛋白水解活性。对其进行了部分氨基酸序列测定,初步确定这种蛋白酶属于半胱氨酸蛋白酶类中的组织蛋白酶B类。     

棉铃虫组织蛋白酶B组织分布与合成部位的研究

蛋白酶是指裂解肽链的所有酶类 ,根据作用位点的催化基团将蛋白酶分为 4大类 ,即丝氨酸蛋白酶、半胱氨酸蛋白酶 (ＣｙｓｔｅｉｎｅＰｒｏｔｅｉｎａｓｅｓ ,ＣＰ)、天冬氨酸蛋白酶和金属蛋白酶。每一大类又包括多种不同的蛋白酶 ,其中半胱氨酸蛋白酶是一类细胞内蛋白酶 ,包括组织蛋白酶Ｂ、Ｌ、Ｈ、Ｎ、Ｓ、Ｔ等 ,其活性中心含有活性必需的半胱氨酸残基 ,细胞内高度的还原环境对它们的作用非常重要 (Ｔｕｒｋ＆Ｂｏｂｔ,1991)。蛋白酶参与多种生理、病理性蛋白水解 ,在昆虫中的分布和功能也有报道 ,如蚊子卵中含有组织蛋白酶Ｂ ,参与胚胎发…     

海枣曲霉地衣多糖酶和木聚糖酶的底物特异性

海枣曲霉木聚糖酶x—I、x—u和x一III作用于不同底物对,x_I对地衣多糖的水解活性最强,对麦麸半纤维素H和B也有一定的水解活性,因而该酶为具有木聚糖酶活性的地衣多糖酶(LichⅢe,l,3一l,4一卢一D—Glucan 4一glucnohydrolasc,Ec 3．2．1．73)。 X—II对燕麦木聚糖、麦麸半纤维素B和H均有很高的水解活性,对其他木聚糖及地衣多糖的水解活性也较高,因而为具有地衣多糖酶话性的木聚糖酶o x—Iil对落叶松木聚糖的水解活性最高,对其他木聚糖也有较高的水解话性,但不能水解地衣多糖等β一葡聚糖,故为一种专一的木聚糖酶。X一1水解麦麸半纤维素B、x一Ⅱ水解燕麦术聚糖及x—Iu承解落叶松木聚糖的Km值分别为9·9、2．1和1．8mg／ml。酶水解产物的纸层析分析结果表明,x—I水解不同木聚糖后的产物主要为分子量较大的寡聚木糖,未发现木二糖、木糖及阿拉伯糖。X_Il的水解产物主要为木二糖 及木二糖以上的寡糖,并有少量木糖和阿拉伯糖,且阿拉伯糖远多于木糖。X-III的水解产物中以木二糖为最多,也有较多的木二糖以上的寡聚木糖,木糖和阿拉伯糖的量较少,且阿拉伯糖远少于木糖。     

MECHANISM OF ACTIVATION AND POSSIBLE ROLES OF THE CATHEPSIN B-LIKE AND D-LIKE PROTEINASES IN THE EGGS OF PHILOSAMIA CYNTHIA RICINI*

Abstract The mechanism of activation and possible roles in the yolk protein's degradation of the cathepsin B-like and cathepsin D-like proteinases in eggs of Philosamia cynthia ricini were studied. The results showed that acidification could not obviously change the molecular masses of the proteinases. The two proteinases were not likely to modify with each other. The results suggested that the two proteinases might hydrolyze different yolk proteins, or hydrolyze the yolk proteins at different stages during embryogenesis. The results of double immunodiffusion showed that the cathepsin B-like and cathepsin D-like proteinases might exist in the eggs of some different species in Lepidoptera.     

A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases

Protein engineering approaches are currently being devised to improve the inhibitory properties of plant proteinase inhibitors against digestive proteinases of herbivorous insects. Here we engineered a potent hybrid inhibitor of aspartate and cysteine digestive proteinases found in the Colorado potato beetle, Leptinotarsa decemlineata Say. Three cathepsin D inhibitors (CDIs) from stressed potato and tomato were first compared in their potency to inhibit digestive cathepsin D-like activity of the insect. After showing the high inhibitory potency of tomato CDI (M(r) approximately 21 kDa), an approximately 33-kDa hybrid inhibitor was generated by fusing this inhibitor to the N terminus of corn cystatin II (CCII), a potent inhibitor of cysteine proteinases. Inhibitory assays with recombinant forms of CDI, CCII, and CDI-CCII expressed in Escherichia coli showed the CDI-CCII fusion to exhibit a dual inhibitory effect against cystatin-sensitive and cathepsin D-like enzymes of the potato beetle, resulting in detrimental effects against 3rd-instar larvae fed the hybrid inhibitor. The inhibitory potency of CDI and CCII was not altered after their fusion, as suggested by IC(50) values for the interaction of CDI-CCII with target proteinases similar to those measured for each inhibitor. These observations suggest the potential of plant CDIs and cystatins as functional inhibitory modules for the design of effective broad-spectrum, hybrid inhibitors of herbivorous insect cysteine and aspartate digestive proteinases.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: molecular cloning and gene expression during vitellogenesis and oocyte maturation

The cysteine proteinases cathepsins B and L are members of the multigene family of lysosomal proteases that have been implicated in the processing of yolk proteins (YPs) in teleost oocytes. However, the full identification of the type of cathepsins expressed in fish ovarian follicles and embryos, as well as their regulatory mechanisms and specific function(s), are not yet elucidated. In this study, cDNAs encoding cathepsins B, L, F, K, S, Z, C, and H have been isolated from the teleost Fundulus heteroclitus, and the analysis of their deduced amino acid sequences revealed highly similar structural features to vertebrate orthologs, and confirmed in this species the existence of cathepsin L-like, cathepsin B-like, and cathepsin F-like subfamilies of cysteine proteinases. While all identified cathepsins were expressed in ovarian follicles, the corresponding mRNAs showed different temporal expression patterns. Thus, similar mRNA levels of cathepsins L, F, S, B, C, and Z were found throughout the oocyte growth or vitellogenesis period, whereas those for cathepsin H and K appeared to decrease as vitellogenesis advanced. During oocyte maturation, a transient accumulation of cathepsins L, S, H, and F mRNAs, approximately a 3-, 1.5-, 1.6-, and 6-fold increase, respectively, was detected in ovarian follicles within the 20-25 hr after hormone stimulation, coincident with the maximum proteolysis of the oocyte major YPs. The specific temporal pattern of expression of these genes may indicate a potential role of cathepsin L-like and cathepsin F proteases in the YP processing events occurring during fish oocyte maturation and/or early embryogenesis.     

Cysteine proteinase plays a key role for the initiation of yolk digestion during development of Xenopus laevis

In electrophoretic analyses, extracts of Xenopus laevis neurulae exhibited activities digesting yolk proteins maximally at pH4.8. These activities were completely inhibited by a mixture of pepstatin A and Z-Phe-Phe-CHN₂, thus being identifiable as cathepsin D and cysteine proteinase. The electrophoretic profiles of yolk proteins cleaved by embryonic extracts changed at gastrula stages; the profile before stage 13 was the same as that given by cathepsin D treatment and the profile at stage 13 was a combination of the profile given by cathepsin D treatment and that given by cysteine proteinase treatment. Quantitative measurement of enzyme activities showed that the cathepsin D activity that was preserved from the beginning of development increased from stages 13 to 25 and decreased thereafter, whereas the cysteine proteinase activity appeared at stage 13, gradually increased until stage 35 and strongly increased thereafter. Immunoblot analyses showed that the 43 kDa form of cathepsin D was processed to its 36 kDa form, presumably by cysteine proteinase. This change can explain the increase of cathepsin D activity at stage 13 and thereafter. Immunofluorescent staining with the antibody against cysteine proteinase occurred in mesodermal and ectodermal cells other than neural ones at stages 13–24, and in the endodermal cells at stages 24–36. Faint staining in the neural ectoderm persisted from stages 18 to 36. Immunoelectron microscope observation showed that what stained was the superficial layer of yolk platelets. All these results indicate that cysteine proteinase plays a key role in the initiation of yolk digestion during embryonic development.     

Multiple acid proteinases in the cellular slime mould Dictyostelium discoideum.

Proteinase activity in the cellular slime mould Dictyostelium discoideum has been analyzed by electrophoresis on polyacrylamide gels containing denatured hemoglobin. At least eight bands due to acid proteinases have been defined using extracts of myxamoebae, four bands A-D which move faster than the fifth and major band E, a minor band E' which moves just behind E and two slow bands G and H. Fruiting body formation was accompanied by the appearance of one new proteinase band F. The proteinases were present in extracts of both axenically-grown and bacterially-grown cells. Differences between the pH dependence and stability of the individual proteinases were detected. Inhibitor studies suggested that the faster proteinases A-D may be cathepsin B-like, whilst the slower enzymes E, E' and F do not fit readily into any known group of proteinases since they were sensitive to HgCl2 but not to other inhibitors of cathepsin B and not to inhibitors of cathepsin D-like proteinases under standard conditions. None of the proteinases was apparently formed during or after preparation of extracts and the proteinases could be re-run on polyacrylamide gels to give only the band expected from the first run. The bands are believed to reflect multiple proteinase activities within the cell.     

Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris

Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z-phe-tyr-DMK, specific for cathepsin L. A cDNA library representing larval gut tissue mRNA contained cysteine proteinase-encoding clones at high frequency. Sequence analysis of 11 cysteine proteinase cDNAs showed that 9 encoded cathepsin L-like enzymes, and 2 encoded cathepsin B-like enzymes. Three enzymes (two cathepsin L-like, DvRS5 and DvRS30, and one cathepsin B-like, DvRS40) were expressed as recombinant proteins in culture supernatants of the yeast Pichia pastoris. The cathepsin L-like enzymes were active proteinases, whereas the cathepsin B-like enzyme was inactive until treated with bovine trypsin. The amino acid residue in the S2 binding pocket, the major determinant of substrate specificity in cathepsin cysteine proteinases, predicted that the two cathepsin L-like enzymes, DvRS5 and DvRS30, should differ in substrate specificity, with the latter resembling cathepsin B in hydrolysing substrates with a positively charged residue at P2. This prediction was confirmed; DvRS5 only hydrolysed Z-phe-arg-AMC and not Z-arg-arg-AMC, whereas DvRS30 hydrolysed both substrates. The enzymes showed similar proteolytic activity towards peptide substrates.     

Expression of the Helicoverpa cathepsin B-like proteinase during embryonic development

Cathepsin B-like proteinase from Helicoverpa armigera (HCB) was proposed as being involved in the degradation of yolk proteins during embryonic development. Recombinant HCB was expressed as a fusion protein with GST in Escherichia coli BL21 on the basis of its cDNA and purified to homogeneity. The fusion protein was cleaved with thrombin to generate a soluble protease with a mass of 37 kDa. A polyclonal antiserum against this recombinant protein, raised in the rabbit, recognized three isoforms of HCB in an ovary homogenate of this insect. Expression of this enzyme during embryonic development was studied using immunoblotting, immunohistochemistry and activity assay. It was found that HCB was expressed during embryonic development and that its proteolytic activity was detected from embryonic developmental eggs. The fact that HCB activity is observed in ovaries and developing eggs suggested that the enzyme had already been activated before embryonic development. Immunohistochemistry indicated that the enzyme was located in follicular cells, the sphere of yolk granules, and the fat bodies of female adult. These lines of evidence suggested strongly that HCB takes part in the degradation of yolk proteins during the development of embryo.     

Bombyx acid cysteine proteinase

Summary

Three kinds of yolk proteins (vitellin, egg-specific protein and 30 k-proteins) are found in silkmoth eggs and have been well characterized. Essentially these proteins are considered to be amino acid reserves for developing embryos. Since at an early stage of egg development the cysteine proteinase accounts for the majority of the total proteinase activity, it may be involved in the degradation of yolk proteins. The enzyme is stored in the eggs as an inactive pro-form, indicating that the activation of the enzyme might be one of the key steps in yolk protein degradation. To investigate at the molecular level how yolk proteins degradation takes place, we have studied Bombyx acid cysteine proteinase (BCP) during an early period of embryonic development. We summarize how proteinases are regulated and are involved in the degradation of Bombyx yolk proteins during embryogenesis. These will be discussed mainly in light of recent results obtained from eggs of the silkmoth, Bombyx mori.     

Expression of proteolytic enzymes during Dictyostelium discoideum spore germination

The specific activity of cathepsin B-like, cathepsin D-like, and leucine aminopeptidase enzymes was measured in dormant, aging, and germinating spores of wild-type and mutant Dictyostelium discoideum.The activity of leucine aminopeptidase was relatively constant during spore aging and spore germination. The level of cathepsin D-like activity was highest in young dormant spores but decreased during germination or aging.The level of cathepsin B-like activity remained constant in wild-type spores which were aged for 13 days. The dormant spores of spontaneous germination mutants initially contained low levels of cathepsin B-like activity which increased during aging. Thus, there was no correlation between the level of endogenous cathepsin B activity and the ability to be autoactivated or heat-activated. The level of cathepsin B-like activity does not have a role in the generation of energy for the swelling stage of germination. Finally, the combined level of endogenous and exogenous cathepsin B activity increased more than 20-fold during the emergence of myxamoebae suggesting that the enzyme(s) may play a role at this development stage of germination.