The gene yvpA from Bacillus subtilis was cloned and expressed in Escherichia coli. It encoded a pectate lyase of 221 amino acids that was denominated PelC. The heterologously expressed enzyme was purified by His-tag affinity chromatography and characterized. PelC depolymerized polygalacturonate and pectins of methyl esterification degree from 22 % to 89 %, exhibiting maximum activity on 22 % esterified citrus pectin. It showed an absolute Ca 2+ requirement and the optimum temperature and pH were 65 6C and pH 10, respectively. The deduced amino acid sequence of PelC showed 53 % identity to pectate lyase PelA from Paenibacillus barcinonensis, which was also characterized. Similarly to PelC, purified PelA showed activity on polygalacturonate and pectins with a high degree of methyl esterification. The two enzymes cleaved pectic polymers to a mixture of oligogalacturonates, indicating an endo mode of action. Analysis of activity on trigalacturonate showed that PelC cleaved it to galacturonic acid and unsaturated digalacturonate, whereas PelA did not show activity on this substrate. PelC and PelA showed high homology to a few recently identified pectate lyases of family 3 and form with them a cluster of small-sized pectate lyases from non-pathogenic micro-organisms.

Strains Paenibacillus sp. BP-23 and Bacillus sp. BP-7, previously isolated from soil from a rice field, secreted high levels of pectinase activity in media supplemented with pectin. Production of pectinases in strain Paenibacillus sp. BP-23 showed catabolite repression, while in Bacillus sp. BP-7 production of pectin degrading enzymes was not negatively affected by glucose. The two strains showed lyase activities as the predominant pectinases, while hydrolase activity was very low. Analysis of Paenibacillus sp. BP-23 in SDS-polyacrylamide gels and zymograms showed five pectinase activity bands. The strict requirement of Ca(2+) for lyase activity of the strain indicates that correspond to pectate lyases. For Bacillus sp. BP-7, zymograms showed four bands of different size. The strain showed a Ca(2+) requirement for lyase activity on pectate but not on pectin, indicating that the pectinolytic system of Bacillus sp. BP-7 is comprised of pectate lyases and pectin lyases. The results show differences in pectin degrading systems between the two aerobic sporogenous bacterial strains studied.

The gene pelA encoding a pectate lyase from the strain Bacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1214 bp DNA fragment containing pelA gene was determined, revealing an ORF of 666 nucleotides that encoded a protein of 23 233 Da. The deduced amino acid sequence of the encoded enzyme showed homology to pectate lyases A, B, C and D from Fusarium solani, Pel-3 and PelB from Erwinia carotovora and PelI from Erwinia chrysanthemi. Homology was also found to the protein deduced from the Bacillus subtilis yvpA gene, the function of which is unknown. The heterologous expressed enzyme depolymerized polygalacturonate and pectins of methyl esterification degree from 22 to 89 %, and exhibited similar activity on polygalacturonate and on 89 % esterified citrus pectin. Optimum temperature and pH for enzymic activity were 50 mC and pH 10, respectively. Ca 2M was required for activity on pectic substrates, while the enzyme was strongly inhibited by Ba 2M .

Plasma-membrane-associated protein kinase(s) from normal rat liver phosphorylates exogenous bovine brain calmodulin in the absence of Ca2+ and in the presence of histone or poly(L-lysine). Maximum levels of calmodulin phosphorylation are obtained at a poly(L-lysine)/calmodulin molar ratio of 0.4. Phosphoamino acid analysis revealed that calmodulin is phosphorylated on serine, threonine and tyrosine residues. Endogenous plasma-membrane-associated calmodulin was also phosphorylated by plasma-membrane-associated protein kinase(s) in the absence of added cationic protein or polypeptide. The identity of endogenous phosphocalmodulin was confirmed by immunoprecipitation with a specific anti-calmodulin monoclonal antibody. Ehrlich ascites tumor cell plasma membranes do not contain endogenous calmodulin. However, membrane-associated protein kinase(s) from these tumor cells phosphorylates bovine brain calmodulin in the presence of poly(L-lysine). These data demonstrate that phosphocalmodulin is present in liver plasma membranes and suggest that this post-translational modification could have a physiological role in this location.