W. Hengstenberg

Enzymology of Carbohydrate Transport in Bacteria

The structure of biological membranes is still one of the major problems of modern biochemistry which remain to be solved.

Purification of the lactose specific factor III of the staphylococcal PEP dependent phosphotransferase system.

The PEP dependent phosphotransferase system in bacteria is involved in the accumulation of certain carbohydrates into the bacterial cell [ 1,2] . The system has been investigated in detail in E. coli [ 1 ] . In S. aureus an additional component which is sugar specific like the membrane bound component designated factor III has been demonstrated [3]. A mutant missing this new cytoplasmic component was isolated and used to perform a very convenient calorimetric assay for the factor III specific for lactose [4] . The function of this new factor in the PEP dependent phosphotransferase system is not yet clear. In order to obtain information about the physiological significance of the component we extensively purified this protein. The purification procedure including some preliminary characterization of the protein will be reported in this communication.

Synthese von [32P]phosphoenolpyruvat.

Bei unseren Untersuchungen fiber das PEP-abhgngige Phosphotransferasesystem von Stuphylococcus aureus [l] bemiihten wir uns urn eine einfache Synthese von 32P-markiertem Phosphoenolpyruvat (32PEP). Die gebrluchlichen Darstellungsverfahren von PEP erfordern fliichtige Phosphorylierungsmittel wie poC13 [2,3] oder Trimethylphosphit [4] , die als 32P-markierte Verbindungen unangenehm zu handhaben und wesentlich teurer als 32P-Orthophosphat sind. Das biologische Darstellungsverfahren [ 51, das die Isolierung von Mitochondrien mit intakter oxydativer Phosphorylierung erfordert, benutzt als Phosphatdonor zwar 32POqH3, ist jedoch umstindlich und liefert 32PEP von gering er spezifischer Aktivitiit. Im folgenden beschreiben wir eine einfache chemische Synthese von 32PEP unter Verwendung von 32~03 4 /%Chlormilchslure wird mit Orthophosphat in Gegenwart von Trichloracetonitril und iiberschiissigem Dilthylamin in DMSO phosphoryliert analog dem Phosphorylierungsverfahren fiir Nukleoside [6]. Unter diesen Bedingungen erfolgt offenbar die Eliminierung von HCl aus phosphorylierter P-Chlormilchshre unter Bildung von 32PEP. Das gebildete Produkt (Ausbeute 70% bezogen auf eingesetztes 32P03-) hat dieselbe elektrophoretische Beweglichkeit wie P%P, phosphoryliert ADP zu ATP in Gegenwart von Pyruvatkinase, und ist Phosphatdonor im PEPabhtigigen Phosphotransferasesystem von S. aureus. Die beiden enzymatischen Reaktionen identifzieren das Phosphorylierungsprodukt als PEP.

The staphylococcal PEP dependent phosphotransferase system, proton magnetic resonance (PMR) studies on the phosphoryl carrier protein HPr: Evidence for a phosphohistidine residue in the intact phospho-HPr molecule

The phosphorylated forms of the P-carrier proteins HPr and F III can be demonstrated using 32P PEP as phosphoryl donor. After alkaline hydrolysis in 3 M NaOH lOO”C, 3 hr 1-[32P]histidine was isolated from 32 P-HPr whereas 3-[32 P] histidine was isolated from 32P-F III, leading to the conclusion that the phosphoryl group in HPr was linked to the N-l of the imidazol ring of the histidine residue in the protein

Solubilization of the membrane bound lactose specific component of the staphylococcal PEP dependant phosphotransferase system

The membrane bound lactose specific component of the PEP dependant phosphotransferase system of Staphylococcus aureus has been solubilized using the non ionic detergent Triton X‐100. Some properties of the crude soluble enzyme are reported.

Structure determination of polypeptides and proteins by two-dimensional nuclear magnetic resonance spectroscopy

Determination of the three-dimensional structure of small, soluble proteins by two-dimensional nuclear magnetic resonance (NMR) spectroscopy represents an alternative method of X-ray crystallography and is an important application of high magnetic fields in science. The basic methodology is introduced and shortly reviewed, limitations and possible future developments are discussed.