Karl-Heinz Scheit

Localization and structural characterization of an oligosaccharide O-linked to bovine PDC-109 Quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa

PDC‐109 (13 kDa) is the most abundant component, and the major heparin‐binding protein, of bovine (Bos taurus) seminal plasma. Here, we show that PDC‐109 contains a single O‐linked oligosaccharide (NeuNAcα(2–6)‐Galβ(1–3)‐GalNAc‐) attached to Thr11. Immunoquantitation of PDC‐109 indicates that its concentration in seminal plasma is 15–20 mg/ml. Though PDC‐109 is not present on epididymal sperm, ejaculated spermatozoa on average are coated with (9.5 ± 0.3) × 106 molecules of PDC‐109/cell. This value remained constant in swim‐up sperm and decreased to (7.7 ± 0.4) × 106/spermatozoon after incubation for 24 h in capacitation medium at 39°C. These data substantiate the hypothesis that PDC‐109 may be one of the seminal plasma components that enhance the fertilizing capacity of bull spermatozoa upon interaction with heparin‐like glycosaminoglycans present in the female genital tract.

In vitro incorporation of 2′-deoxyadenosine and 3′-deoxyadenosine into yeast tRNAPhe using tRNA nucleotidyl transferase, and properties of tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA

Abstract 2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNA Phe by tRNA nucleotidyl transferase. tRNA Phe -C-C-2′dA and tRNA Phe -C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNA Phe -C-C-3′dA proved to be chargeable.

Die Methylierung von Dinucleosidphosphaten mit Diazomethan

Abstract The methylation of dinucleoside phosphates with diazomethane The methylation of diribonucleoside phosphates with diazomethane is accompanied by the fission of certain phosphodiester bonds. A larger excess of diazomethane leads to a higher degree of fission. The 2′-OH group adjacent to the internucleotide linkage is responsible for this hydrolysis caused by diazomethane.

Die Methylierung von Inosin und uridylyl-(3′–5′)Inosin durch dimethylsulfat

Abstract Methylation of inosine and uridylyl-(3′–5′)inosine with dimethylsulfate Methylation of inosine with diazomethane at pH 7 yields 1-methylinosine, 7-methylinosine, 9-(β, d -ribofuranosyl)-6-methoxypurine, 5-(N- formyl , N- methyl)-amino -6-( d -ribofuranosyl)aminopyrimidine -4- on and 3- methyl -5-(N- formyl , N- methyl)amino -6-( d -ribofuranosyl)aminopyrimidine -4- on . The reaction of 2′, 3′- O -isopropylideneinosine with dimethylsulfate at pH 7 yielded 3- methyl -5-(N- formyl , N- methyl)amino -6-( d -2′,3′-O- isopropylideneribofuranosyl)aminopyrimidine -4- on as the sole reaction product. The ultraviolet- and NMR spectra of these compounds are discussed. The methylation of uridylyl-(3′–5′)inosine with dimethylsulfate at pH 7 occurred without fission of the phosphodiester linkage selectively at the hypoxanthine moiety with the formation of 3-methyl-5-( N -formyl, N -methyl)-6-aminopyrimidine-4-on and 5-( N -formyl, N -methyl)-6-aminopyrimidine-4-on.

Binding of Rifampicin to Escherichia coli RNA Polymerase: Thermodynamic and Kinetic Studies

INTRODUCTION The antibiotic rifampicin is a specific inhibitor of bacterial RNA polymerases (Hartmann et al. 1967) which, unlike many other compounds that interfere with transcription (Kersten and Kersten 1974), acts by direct interaction with the enzyme. The existence of a stoichiometric and remarkably stable complex of rifampicin and RNA polymerase has been established in many ways (Wehrli et al. 1968; Wehrli and Staehelin 1970, 1971; Neuhoff, Schill and Sternbach 1970; Eilen and Krakow 1973; Scheit and Stutz 1975; Stender, Stutz and Scheit 1975). An actively transcribing ternary complex consisting of enzyme, DNA and RNA, however, does not bind the drug (Eilen and Krakow 1973) and is consequently resistant to its inhibitory action (Sippel and Hartmann 1968). The precise step in the sequence of reactions involved in transcription (Goldthwait, Anthony and Wu 1970; Chamberlin 1974b) beyond which sensitivity is no longer manifested remains to be established. Thus in recent years it has been postulated that rifampicin prevents either (a) the binding and stabilization effect of the initiating purine triphosphate (di Mauro et al. 1969); (b) the formation of an active state of the binary complex required for initiation (Sippel and Hartmann 1970); (c) the formation of the first phosphodiester bond (So and Downey 1970); or (d) the formation of the second phosphodiester bond (Johnston and McClure, this volume). Although bound rifampicin does not prevent the specific recognition of promoter sequences directed by the σ subunit (Hinkle, Mangel and Chamberlin 1972; Bordier 1974), physical studies have demonstrated interference with the binding of...

The affinity of E. coli RNA polymerase to matrix bound rifamycin.

The antibiotic rifamycin and its various derivatives strongly inhibit procaryotic DNA dependent RNA polymerases [ 11. Presently it is believed that the antibiotic complexes with free RNA polymerase molecules which then become unable to bind the initial nucleoside triphosphate thus preventing transcription [2-51. Studies with E. coli RNA polymerase revealed that both core (p’pol?) and holo enzyme (fl’& u) are inhibited by rifamycin derivatives [ 1 ] . Although the mechanism of action of rifamycin has been the subject of many studies, there remained open questions. We were interested in the problem, whether certain protein subunits, like u or w are displaced from holo enzyme upon binding of rifamycin. The experimental approach which we employed, was to investigate the binding of RNA polymerase from E. coli to matrix bound rifamycin [6].