Purification of isoenzyme I of phospholipase C from Clostridium perfringens

V. I. Lenin Tashkent State University. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 888–890, November–December, 1988.     

Stabilization of phospholipase C fromClostridium perfringens by conjugation with a trypsin inhibitor

A new approach for the stabilization of phospholipase C by conjugation with a proteinase inhibitor is proposed. A heat-stable fraction obtained from a conjugate retained its initial activity for an hour at an incubation temperature of 50°C. An appropriate method of conjugation using an affinity adsorbent preventing the appearance of inactive forms of the conjugate has been developed.Mirzo Ulugbek Tashkent State University, fax (3712) 46 24 72. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 115–117, January–February, 1998.     

Mode of action of Clostridium perfringens initiation protein (spore-lytic enzyme)

The extracellular initiation protein (IP; spore germination enzyme) produced by Clostridium perfringens was further purified and characterized. IP hydrolysed spore cortical fragments with the release of free amino groups. End group analysis of hydrolysed fragments indicated the presence of N-terminal alanine but no reducing sugars. Molecular weight analysis of IP- and lysozyme-treated fluorescamine-labelled cortical fragments indicated that IP acts only on peptidoglycan chains containing cross-linked peptide subunits. IP failed to hydrolyse a number of nitrophenyl-conjugated glucopyranosides and galactopyranosides. The results indicate that IP is an N-acetylmuramyl-L-alanine amidase.     

Purification and some properties of phospholipase C from Achromobacter xylosoxidans.

A non-haemolytic phospholipase C (EC 3.1.4.3) was purified from the culture medium of Achromobacter xylosoxidans with a 5% yield and a purification factor of 330. A combination of ultrafiltration, acetone precipitation and two subsequent affinity chromatographic steps was used. The affinity chromatography is a new application of 2-(4-aminophenylsulphonyl)ethyl-cellulose, a sorbent that has previously been used for the purification of phospholipase C from Bacillus cereus. The purified enzyme gave four distinct bands on polyacrylamide gel electrophoresis, and each band was catalytically active. Under our experimental conditions, the phospholipids examined were hydrolysed in the following order: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin. Neither the synthetic substrate p-nitrophenylphosphorylcholine nor phosphatidylinositol was hydrolysed under different experimental conditions. For maximal hydrolytic activity toward phosphatidylcholine, the enzyme required Triton X-100 and Ca2+ ions. EDTA was inhibitory, but the enzyme activity was almost completely restored by Zn2+. The molecular mass of the phospholipase C, estimated by gel permeation, was 34,000 daltons.     

Effect of some divalent metal cations on phospholipase C from Bacillus cereus

Incubation of phospholipase C from Bacillus cereus with certain divalent metal cations caused enzyme inactivation with Cu(II) being particularly effective. The inactivation arose from the reversible exchange of Zn(II) in the enzyme with the metal cations. Both zinc atoms in the enzyme exchanged rapidly with Cu(II) whereas only one exchanged spontaneously with Co(II). With lecithin substrates, CoZn-phospholipase C had a specific activity of 3.6-11.3% of that of ZnZn-phospholipase C, whereas the CoCo-enzyme was less than 1% active relative to the native enzyme. The CoZn-enzyme had the same Km value for dihexanoyllecithin as had the native enzyme, but the Vm value was markedly lower. ZnZn-, CoZn- and CoCo-phospholipase C all had very low activities towards sphingomyelin micelles, although for the CoCo-enzyme, the sphingomyelinase activity was 4-7-fold greater than for the native enzyme.     

Synthesis and evaluation of fluorogenic substrates for phospholipase D and phospholipase C

Fluorogenic analogues of phosphatidylcholine and lysophosphatidylcholine, DDPB and lysoDDPB, were synthesized by an enzyme-assisted strategy. The analogues were evaluated as substrates for phospholipases C and D and lysophospholipase D. DDPB was cleaved by bacterial and plant phospholipase D (PLD) enzymes and represents the first direct fluorogenic substrate for real-time measurement of PLD activity. Both fluorogenic substrates have potential in screening for PLD and PC-PLC inhibitors and for monitoring spatiotemporal changes in PLD activity in cells. [structure: see text]     

Inactivation of phospholipase C from Bacillus cereus by a carboxyl group modifying reagent.

Phospholipase C from Bacillus cereus was inactivated by incubation with either of the carboxyl reagents, a water-soluble carbodimide plus a nucleophile or Woodward's reagent K. With the former reagent, the incorporation into the enzyme of the first mol of nucleophile caused a 4-5-fold increase in the Km for dihexanoyllecithin with no significant effect on the Vm. The second mol of nucleophile incorporated caused no further change in Km but destroyed most of the catalytic activity. Modification of the enzyme by carbodiimide plus nucleophile did not alter the relative activity of the enzyme towards micelles and monomolecularly dispersed solutions of diheptanoyllecithin. Furthermore, inactivation by this reagent did not significantly decrease the ability of the enzyme to bind to a substrate-based affinity gel. It was concluded that phospholipase C contains a single carboxyl group that is essential for catalytic activity. The enzyme also contains a total of 4-5 reactive/exposed carboxyl groups.     

A novel class of zinc-binding inhibitors for the phosphatidylcholine-preferring phospholipase C from Bacillus cereus

The phospholipase C (PLC) isozymes catalyze the hydrolysis of phospholipids to provide diacylglycerol (DAG) and a phosphorylated headgroup. Because DAG has been implicated in cellular signal transduction cascades in mammalian systems, there has been considerable interest in the development of inhibitors of these enzymes. Toward this end, we have discovered that the cyclic N,N'-dihydroxyureas 6-10 inhibit the phosphatidylcholine preferring PLC from Bacillus cereus (PLCBc). This class of inhibitors is believed to function by the bidentate chelation of the N,N'-dihydroxyurea array to one or more of the zinc ions at the active site of the enzyme. Because the affinities of these compounds correlate with the pKaS of the N-OH hydroxyl groups, it is apparent that one or both of the hydroxyl groups must be ionized for effective coordination to the zinc ions. It is also apparent that there may be rather strict steric requirements for these inhibitors.     

Si/C Phases from the IR Laser-induced Decomposition of Silacyclobutane and 1,3-Disilacyclobutane

CO₂ laser-induced infrared multiphoton decomposition (IRMPD) and SF₆ photosensitized decomposition (LPD) of silacyclobutane (SCB) and 1,3-disilacyclobutane (DSCB) in the gas phase results in the very efficient deposition of Si/C/H and SiC materials, and it is inferred that the process is dominated by formation of transient silene; silene rearrangement to methylsilylene; silene and methylsilylene dehydrogenation; and polymerization of SiCH_n (n < 4) species. The deposits are sensitive to oxygen. Decomposition and SiC formation are favoured with IRMPD experiments conducted with high-energy fluxes. The laser technique is promising for low-temperature chemical vapour deposition of amorphous SiC.     

Methane Decomposition and C2 Hydrocarbon Formation under the Condition of DC Discharge Plasma

The infrared emission spectra of methane, H, CH and C2 hydrocarbons in natural gas were measured. The processes of methane decomposition and formation of C2 hydrocarbons were studied. The experiment shows that methane decomposition can be divided into three periods as the reaction proceeds.In the first period, a large number of free radicals were formed. While in the last period, the formation of C2 hydrocarbons and the decrease of free radicals were observed. The time and conditions of methane decomposition and formation of C2 hydrocarbons are different.     

Quantitative determination of phosphocholine and the activity of phospholipase C

Summary 1. A quantitative method for determining phosphocholine by means of thin-layer chromatography has been proposed.2. In the enzymatic cleavage of lecithins with phospholipase C, this method permits the amount of diglycerides formed to be determined as well as the phosphocholine.3. The agreement of the results of the analysis of phosphocholine and the diglycerides is a reliable criterion of the activity of the phospholipase C.Khimiya Prirodnykh Soedinenii, Vol. 2, No. 4, pp. 233–235, 1966     

Quantitative determination of phosphocholine and the activity of phospholipase C

Summary 1. The ester of dextran and pelentanic acid with a maximum value of 140 has been synthesized.2. The influence of some reaction conditions on the composition of the esters obtained has been investigated.Khimiya Prirodnykh Soedinenii, Vol. 1, No. 4, pp. 245–246, 1965     

Quantitation of phospholipase A2 and phospholipase C activity using alkaline phosphatase impregnated liposomes

Phospholipase A₂ and C activity was quantitated using liposomes impregnated with alkaline phosphatase. Release of alkaline phosphatase was dependent on phospholipase related hydrolysis of intact vesicles. Released alkaline phosphatase was quantitated after addition of its chromogenic substrate p-nitrophenyl phosphate. The lower limit of detectability for phospholipase A₂ and C activity was 0.5 unit/ml. These limits were 10-fold lower than a titrimetric method. Liposome destruction as measured by alkaline phosphatase release was calcium dependent and inhibited by 1 mM EDTA and 1 mM ZnSO₄. The assay was technically simple, generated same day results, and used automated enzyme-linked immunosorbent assay instrumentation.     

Purification of phospholipase C by hydrophobic interaction affinity chromatography.

A simple procedure is described for the purification of phosphatidylcholine-hydrolyzing phospholipase C(PLC). Lecithin, the substrate for PLC, was ligated hydrophobically to octyl-Sepharose in 2 M (NH4)2SO4. The washed lecithin-conjugated resin was then used to purify PLC from crude preparations by affinity chromatography. PLC binds to the lecithin moiety in the presence of Zn2+ and is eluted with an acidic buffer containing EDTA. PLC activity was recovered in the eluate. Both sodium dodecyl sulphate polyacrylamide gel electrophoresis and pI electrofocusing showed that the eluate contained a single monomeric protein with an apparent molecular mass of 66 kDa and a pI of 5.5.