UDP-acetyl-mannosamine dehydrogenase is an endogenous protein substrate of Staphylococcus aureus protein-tyrosine kinase activity.

The in silico analysis of the amino acid sequences deduced from the complete genome sequence of Staphylococcus aureus suggested the presence of two protein tyrosine kinase activities, each split into two distinct polypeptides, respectively Cap5A1/Cap5B1 and Cap5A2/Cap5B2, like in some other Gram-positive bacteria. To check this prediction, the corresponding genes were cloned and overexpressed, and the four corresponding proteins were purified by affinity chromatography and assayed for phosphorylating activity in vitro. Individually, none of them was found to autophosphorylate. However, when Cap5B2 was incubated in the presence of Cap5A2 or, with a larger efficiency, in the presence of Cap5A1, this protein exhibited intensive autokinase activity, occurring selectively at tyrosine residues. On the other hand, whatever the protein combination assayed, Cap5B1 did not present any phosphorylating activity. In search of a possible role for the phosphorylation reaction mediated by Cap5B2, an endogenous substrate of this kinase was characterized. This substrate, termed Cap5O, is the enzyme UDP-acetyl-mannosamine dehydrogenase involved in the cascade of reactions leading to the synthesis of the bacterial capsule. It represents the first endogenous substrate for a tyrosine kinase activity so far identified in S. aureus. The analysis of its dehydrogenase activity showed that it was positively controlled by its phosphorylation at tyrosine.

Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb.

Two proteins of Escherichia coli, termed Wzc and Wzb, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, Wzc was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive ATP, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, Wzb was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Wzb was able to dephosphorylate previously autophosphorylated Wzc. Together, these data demonstrate, for the first time, that E. coli cells contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. From comparative analysis of amino acid sequences, Wzc was found to be similar to a number of proteins present in other bacterial species which are all involved in the synthesis or export of exopolysaccharides. Since these polymers are considered important virulence factors, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.

Autophosphorylation of a bacterial protein at tyrosine.

Autophosphorylation at tyrosine is a common process in eukaryotic kinases, which is generally modulated by regulatory ligands and affects the properties of these enzymes. We report that this type of modification occurs also in bacteria, namely in an 81 kDa protein from Acinetobacter johnsonii. This protein is phosphorylated at the expense of ATP exclusively at tyrosine residues. It is located in the inner-membrane fraction of cells and can be totally solubilized by detergents. It has been purified to homogeneity by antiphosphotyrosine immunochromatography. Analysis of the peptides released under trypsin proteolysis of the protein has shown that it autophosphorylates at several tyrosine residues. The discovery of protein autophosphorylation in bacteria seems of special interest for studying the regulatory aspects of this modification when considering the relative simplicity of the bacterial systems, as compared with most eukaryotic systems, namely in terms of physiology and genetics.

Use of Percoll gradients for isolation of human placenta mitochondria suitable for investigating outer membrane proteins.

Human mitochondria were isolated from placenta by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation as assessed by marker enzyme analysis and electron microscopy. The advantages over previous methods are the rapidity of the procedure and the excellent resolution of mitochondria and lysosomes. Moreover, the high extent of intactness of the mitochondria so obtained made them particularly well suited for investigating outer membrane proteins. Taking advantage of this method, we have purified human mitochondrial porin. The purified protein consists of a single unglycosylated polypeptide of molecular mass 33 kDa.

Mitochondrial dolichyl-phosphate mannose synthase. Purification and immunogold localization by electron microscopy.

Mitochondrial dolichyl-phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step. The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band. A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30-kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl-phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria.

Investigation of glycosylation processes in mitochondria and microsomal membranes from human skeletal muscle.

Glycoconjugates are directly involved in major skeletal muscle functions. As little is known about glycosylation processes in muscle, we investigated glycoconjugate synthesis in subcellular fractions from human skeletal muscle tissue. Mitochondria and microsomal membranes were prepared from muscle biopsies by thorough mechanical disruption and differential centrifugations. This procedure resulted in the isolation of intact mitochondria (1 mg protein/g muscle) and of a microsomal fraction (1.5 mg protein/g muscle). Glycosyltransferases were studied in both subcellular fractions using either dolichylmonophosphate as a polyprenic acceptor or chemically modified fetuin as a glycoprotein substrate. Our results provide evidence for high rates of glycosylation in muscle. The highest activities were obtained with GDP-mannose: dilichylmonophosphate mannosyltransferase, a key enzyme in glycosylation process (220 pmol/mg per h in mitochondria and 1,550 pmol/mg per h in microsomal membranes). Substantial individual variations were observed for dolichol pathway glycosyltransferases but low individual variations were found for glycosyltransferases involved in maturation of glycoproteins. The role which glycosylation defects may play in muscle dysfunction has yet to be defined.