scholarly journals Unfolding and refolding of phospholipase C from Bacillus cereus in solutions of guanidinium chloride

1979 ◽  
Vol 179 (3) ◽  
pp. 509-514 ◽  
Author(s):  
C Little ◽  
S Johansen
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Native Enzyme ◽  
Guanidinium Chloride ◽  
Protein Fluorescence ◽  
Reversible Inactivation ◽  
Chloride Solutions ◽  
Biphasic Kinetics ◽  
Histidine Residues ◽  
Fluorescence Studies

1. Protein-fluorescence studies indicated that phospholipase C from Bacillus cereus is denatured in solutions of guanidinium chloride. The denaturation was not thermodynamically reversible and followed biphasic kinetics. 2. Guanidinium chloride solutions released the structural Zn2+ from the enzyme and rendered all histidine residues chemically reactive. In the presence of free Zn1+ the enzyme was much more resistant to denaturation. Also, the addition for free Zn2+ to the denatured enzyme induced refolding. 3. The Zn2+-free apoenzyme was much more sensitive to guanidinium chloride than was the native enzyme and the denaturation appeared to be thermodynamically reversible. 4. Guanidinium chloride denaturation was associated with a reversible inactivation of the enzyme. Heat-inactivated, coagulated enzyme was substantially re-activated on dissolution in guanidinium chloride solutions followed by dialysis against a Zn2+-containing buffer.

scholarly journals The histidine residues of phospholipase C from Bacillus cereus

1977 ◽  
Vol 167 (2) ◽  
pp. 399-404 ◽  
Author(s):  
C Little
Keyword(s):  
Catalytic Activity ◽  
Bacillus Cereus ◽  
Phospholipase C ◽  
Maximum Velocity ◽  
Native Enzyme ◽  
Histidine Residue ◽  
Histidine Residues ◽  
Residue Removal ◽  
Diethyl Pyrocarbonate ◽  
Km Value

The inactivation of phospholipase C from Bacillus cereus at pH6 by diethyl pyrocarbonate parallelled the N-ethoxyformylation of a single histidine residue in the enzyme. The inactivation arose from a decrease in the maximum velocity of the enzymic reaction with no effect on the Km value. The inactivation did not apparently alter the ability of the enzyme to bind to a substrate-based affinity gel. The native enzyme contained only one reactive histidine residue. Removal of the two zinc atoms from the enzyme increased the number of reactive histidine residues to five, whereas in the totally denatured enzyme nearly eight such residues were available for reaction with diethyl pyrocarbonate. The enzyme thus appears to contain one histidine residue that is essential for catalytic activity and four that may be involved in co-ordinating the zinc atoms in the structure.

scholarly journals Conformational studies on phospholipase C from Bacillus cereus. The effect of urea on the enzyme

1978 ◽  
Vol 175 (3) ◽  
pp. 977-986 ◽  
Author(s):  
C Little
Keyword(s):  
Circular Dichroism ◽  
Bacillus Cereus ◽  
Phospholipase C ◽  
Thermal Inactivation ◽  
Elevated Temperatures ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Protein Fluorescence ◽  
Fluorescence Emission Spectra ◽  
Circular Dichroïsm

1. When heated in 8 M-urea, phospholipase C(EC 3.1.4.3) from Bacillus cereus undergoes conformational transitions depending on the temperatures used. These transitions were studied by examining protein fluorescence, iodide quenching of protein fluorescence, u.v. difference spectroscopy, chemical availability of histidine residues in the enzyme, circular dichroism and catalytic activity. 2. Unless simultaneously exposed to elevated temperatures the enzyme appears to be unaffected by 8 M-urea. Removal of the two zinc atoms from the enzyme renders phospholipase C very sensitive to denaturation by 8 M-urea as indicated by fluorescence emission spectra and circular dichroism. 3. Both the native and the zinc-free enzymes are markedly more resistant to irreversible thermal inactivation in the presence of 8 M-urea than in its absence. 4. The response of the enzyme to 8 M-urea and the role of zinc in stabilizing the enzyme are discussed.

Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus

1976 ◽  
Vol 450 (2) ◽  
pp. 154-164 ◽  
Author(s):  
Ikezawa Hiroh ◽  
Yamanegi Masato ◽  
Taguchi Ryo ◽  
Miyashita Tomoyuki ◽  
Ohyabu Tetsuo
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C

Effect of Phospholipase C on Human Platelets

1975 ◽  
Author(s):  
A.-B. Otnœss
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Electron Micrographs ◽  
Gel Filtration ◽  
Outer Part ◽  
Human Platelets ◽  
Scanning Electron Micrographs ◽  
Platelet Factor ◽  
Asymmetrical Distribution ◽  
Scanning Electron

The effect on human platelets of phospholipase C (Bacillus cereus) has been studied. Platelets prepared by gel filtration lost 20-30% of their phospholipids when incubated with phospholipase C for 20 min. Phosphatidylethanolamine (PE) was reduced by about 50%, whereas phosphatidylcholine and phosphatidylserine were reduced each by about 20%. Sphingomyelin was not reduced.These data suggest an asymmetrical distribution of phospholipids in the platelet membrane, PE being more accessible and therefore probably mainly located in the outer part of the membrane.The loss of phospholipids was not accompanied by aggregation, nor did the platelets lose their ability to aggregate with thrombin or ADP. Data on release of serotonin, platelet factor 3 and 4 and scanning electron micrographs of treated platelets will be given.

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