The adsorptive immobilization of phospholipids D mediated by calcium ions

Immobilization of phospholipase D from cabbage was studied with the aim of stabilizing the enzyme for its use in synthesis of phospholipids. It was shown that phospholipase D can be immobilized by adsorption to polymeric carriers containing long chain anchor groups such as octadecyl, octyl, or other alkyl residues. Starting from the crude enzyme, phospholipase D activity is preferentially bound (up to 100%) in competition with contaminating proteins. A prerequisite of high binding rates is the presence of calcium ions, which play a mediating role in the adsorption process. The maximum activity of the carrier-enzyme complexes depends upon the calcium concentration in the immobilization process and the carrier material (>/=10mM CaCl(2) with octadecyl-Si40, >/=40 mM CaCl(2) with octyl-sepharose and butyl-fractogel). Immobilization of phospholipase D to octyl-sepharose was shown to result in a distinctly increased storage stability and an enlarged pH-optimum range for the catalytic activity. Operational stability of different phospholipase D-carrier complexes was compared. (c) 1993 Wiley & Sons, Inc.     

Culture conditions for the production of amylolytic enzymes by a thermophilic Clostridium sp.

The growth of a thermophilic Clostridium sp. and the production of α-glucosidase, α-amylase and pullulanase were studied under anaerobic conditions using different carbon and nitrogen sources and varying pH values and temperatures. Growth and enzyme activities were highest with soybean meal as the nitrogen source. The optimum concentration was 2.5% [w/v] for the production of α-amylase as well as pullulanase and 2% [w/v] for α-glucosidase. The best carbon source proved to be soluble starch for α-amylase, and pullulanase and maltose for α-glucosidase. Growth and enzyme production reached their optimum at pH 6.5 to 7.0 and 70°C. Under these conditions, the enzyme activities followed exponential growth with maximum yields of α-glucosidase, α-amylase and pullulanase at 28, 36, and 44 h.     

Identification of three phases in Onconase refolding

Onconase is an extremely stable member of the RNase A superfamily. The increase in the thermodynamic stability by 20 kJ x mol(-1) in comparison to RNase A was expected to result in altered folding behavior. Despite the lack of cis-Pro residues in native Onconase, refolding at low concentrations of guanidine hydrochloride was complex and showed three kinetic phases (fast, medium, and slow), with rate constants differing by a factor of about 10 each. None of the phases could be accelerated by peptidyl-prolyl cis-trans isomerases, pointing to the absence of kinetic phases that are limited by Pro isomerization. The detailed analysis by various probes indicates that the burial of the N-terminal Trp3, which is associated with the restoration of the active site, occurs in the slow phase, i.e. in the last step of refolding. Evidently, in contrast to the folding of RNase A, there is no catalytically active native-like intermediate in the folding of Onconase.     

Insights into the structure of plant alpha-type phospholipase D

Phospholipases D play an important role in the regulation of cellular processes in plants and mammals. Moreover, they are an essential tool in the synthesis of phospholipids and phospholipid analogs. Knowledge of phospholipase D structures, however, is widely restricted to sequence data. The only known tertiary structure of a microbial phospholipase D cannot be generalized to eukaryotic phospholipases D. In this study, the isoenzyme form of phospholipase D from white cabbage (PLDalpha2), which is the most widely used plant phospholipase D in biocatalytic applications, has been characterized by small-angle X-ray scattering, UV-absorption, CD and fluorescence spectroscopy to yield the first insights into its secondary and tertiary structure. The structural model derived from small-angle X-ray scattering measurements reveals a barrel-shaped monomer with loosely structured tops. The far-UV CD-spectroscopic data indicate the presence of alpha-helical as well as beta-structural elements, with the latter being dominant. The fluorescence and near-UV CD spectra point to tight packing of the aromatic residues in the core of the protein. From the near-UV CD signals and activity data as a function of the calcium ion concentration, two binding events characterized by dissociation constants in the ranges of 0.1 mm and 10-20 mm can be confirmed. The stability of PLDalpha2 proved to be substantially reduced in the presence of calcium ions, with salt-induced aggregation being the main reason for irreversible inactivation.     

Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazúr (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca(2+) ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn(2+) ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far.     

Identification of phospholipase D from cabbage as N-terminally acetylated PLD2

Recently, the genes of two isoenzymes of phospholipase D from white cabbage (PLD1 and PLD2) with molecular masses of 91.7 and 91.9 kDa, respectively, have been sequenced and expressed in Escherichia coli [Schäffner, I., Rücknagel, K.-P., Mansfeld, J., and Ulbrich-Hofmann, R. (2002). Eur. J. Lipid Sci. Technol. 104: 79–87]. Both enzymes are highly homologous (91% identity) and behave very similarly. Phospholipase D purified from white cabbage leaves (PLD_cab) is compared with the two recombinant enzymes in sodium dodecylsulfate and native polyacrylamide gel electrophoresis, isoelectric focusing, N-terminal sequencing, and mass spectrometry after tryptic digestion. As a result, PLD_cab clearly can be assigned to PLD2. In contrast to recombinant PLD2, however, PLD_cab is N-terminally acetylated.     

Differences in the denaturation behavior of ribonuclease A induced by temperature and guanidine hydrochloride

Moderate temperatures or low concentrations of denaturants diminish the catalytic activity of some enzymes before spectroscopic methods indicate protein unfolding. To discriminate between possible reasons for the inactivation of ribonuclease A, we investigated the influence of temperature and guanidine hydrochloride on its proteolytic susceptibility to proteinase K by determining the proteolytic rate constants and fragment patterns. The results were related to changes of activity and spectroscopic properties of ribonuclease A. With thermal denaturation, the changes in activity and in the rate constants of proteolytic degradation coincide and occur slightly before the spectroscopically observable transition. In the case of guanidine hydrochloride-induced denaturation, however, proteolytic resistance of ribonuclease A initially increases accompanied by a drastic activity decrease far before unfolding of the protein is detected by spectroscopy or proteolysis. In addition to ionic effects, a tightening of the protein structure at low guanidine hydrochloride concentrations is suggested to be responsible for ribonuclease A inactivation.     

Crystal structure of RNase A tandem enzymes and their interaction with the cytosolic ribonuclease inhibitor

Because of their ability to degrade RNA, RNases are potent cytotoxins. The cytotoxic activity of most members of the RNase A superfamily, however, is abolished by the cytosolic ribonuclease inhibitor (RI). RNase A tandem enzymes, in which two RNase A molecules are artificially connected by a peptide linker, and thus have a pseudodimeric structure, exhibit remarkable cytotoxic activity. In vitro, however, these enzymes are still inhibited by RI. Here, we present the crystal structures of three tandem enzymes with the linker sequences GPPG, SGSGSG, and SGRSGRSG, which allowed us to analyze the mode of binding of RI to the RNase A tandem enzymes. Modeling studies with the crystal structures of the RI-RNase A complex and the SGRSGRSG-RNase A tandem enzyme as templates suggested a 1 : 1 binding stoichiometry for the RI-RNase A tandem enzyme complex, with binding of the RI molecule to the N-terminal RNase A entity. These results were experimentally verified by analytical ultracentrifugation, quantitative electrophoresis, and proteolysis studies with trypsin. As other dimeric RNases, which are comparably cytotoxic, either evade RI binding or potentially even bind two RI molecules, inactivation by RI cannot be the crucial limitation to the cytotoxicity of dimeric RNases.     

Secretory phospholipase A2 from Arabidopsis thaliana: insights into the three-dimensional structure and the amino acids involved in catalysis

A low-molecular weight phospholipase A2 from Arabidopsis thaliana, isoform phospholipase A2-alpha, has been expressed in Escherichia coli in the form of inclusion bodies, refolded, and purified to homogeneity to yield the active mature enzyme. The enzyme was characterized with respect to pH, temperature optimum, and Ca2+ ion requirement. The enzyme has been shown to be a true secretory phospholipase A2 that requires Ca2+ ions in the millimolar range and belongs to group XIB. On the basis of the three-dimensional structures of secretory phospholipase A2 forms (sPLA2s) from bee venom and bovine pancreas, a homology model was generated. Analysis of this model and alignments of different plant sPLA2s showed that the common His-Asp dyad of animal sPLA2s does not exist in plant sPLA2s. In place of the aspartate residue of the dyad, the plant enzymes of group XIA contain a histidine residue, and the enzymes of group XIB contain a serine or an asparagine residue. Mutagenesis of amino acids supposed to be involved in catalysis has shown that His62, the calcium-coordinating Asp63, and the above-mentioned Ser79 residue are essential for activity.     

Proteolytic degradation of ribonuclease A in the pretransition region of thermally and urea-induced unfolding.

The method of limited proteolysis has proven to be appropriate for the determination of unfolding rate constants (k(U)) of ribonuclease A in the transition region of thermal denaturation [Arnold, U. & Ulbrich-Hofmann, R. (1997) Biochemistry 36, 2166-2172]. The aim of the present paper was to extend this procedure to the pretransition region of thermally and urea-induced denaturation where spectroscopic methods do not allow direct measurement of k(U). The results show that the approach can be applied successfully to denaturing (free energy of unfolding Delta G < 10 kJ.mol(-1)) and to marginally native conditions (Delta G = 10-25 kJ.mol(-1)). Under moderately (Delta G = 25-30 kJ.mol(-1)) and strongly native conditions (Delta G > 30 kJ.mol(-1)), however, the determination of kU was not possible in this way as the proteolytic degradation of ribonuclease A by thermolysin or trypsin was no longer determined by global unfolding. Here, proteolysis proceeds via the native RNase A. In the presence of low concentrations of urea, the rate constants of proteolysis were, surprisingly, smaller than in the absence of urea. As the protease activity has been taken into account, this result points to a local stabilization of the RNase A molecule.