Spectrophotometric determination of protein concentration in cell extracts containing tRNA's and rRNA's

Ehresmann, Bernard; Imbault, P.; Weil, Jacques-Henry

doi:10.1016/0003-2697(73)90374-6

Cited by 246 publications

(78 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The final purity obtained was 95% for EcoL1 and at least 97% for the other L1 proteins as estimated from Coomassie-blue-stained polyacrylamide/SDS gels [23]. The concentration of proteins was determined spectrophotometrically as described by Ehresmann et al [24] and by a Bio-Rad protein assay using BSA as a standard.…”

Section: Methodsmentioning

confidence: 99%

Interaction of ribosomal L1 proteins from mesophilic and thermophilic Archaea and Bacteria with specific L1‐binding sites on 23S rRNA and mRNA

Köhrer

Mayer

Neumair

et al. 1998

European Journal of Biochemistry

View full text Add to dashboard Cite

In Bacteria and Archaea (formerly Archaebacteria) ribosomal protein L1 has a dual function, as a primary rRNA-binding protein and as a translational repressor which binds to its own mRNA. The L1-binding site on the mRNA exhibits high similarity in both sequence and secondary structure to the binding site for L1 on the 23 S rRNA. A sensitive membrane-filter-binding assay has been used to examine the interactions between ribosomal L1 proteins from different archaeal and bacterial species, and 23S rRNA and mRNA fragments from Methanococcus vannielii containing the MvaL1-binding site. Under standard conditions (0°C, pH 7.5, 20 mM Mg 2ϩ , 500 mM KCl), the apparent dissociation constant Kd of the homologous MvaL1-23S rRNA complex is 5 nM, the apparent dissociation constant K d of the MvaL1-mRNA complex is 0.15 µM. L1 proteins from Escherichia coli (EcoL1) and from the thermophilic Bacterium Thermus thermophilus (TthL1), and from the thermophilic Archaea Methanococcus thermolithotrophicus (MthL1), Methanococcus jannaschii (MjaL1), and Sulfolobus solfataricus (SsoL1) were tested for their affinity to the specific L1-binding sites on the 23 S rRNA and mRNA. In general, the affinity of L1 proteins from thermophilic species to the binding sites on both 23 S rRNA and mRNA is about one order of magnitude higher than that of their mesophilic counterparts. This stronger protein-RNA interaction might make a substantial contribution to the thermal tolerance of ribosomes in thermophilic organisms.Keywords : RNA-ribosomal protein L1 interaction ; filter-binding assay; Archaea ; mesophilic and thermophilic Methanococcus.Over the last two decades, a growing number of organisms living at temperatures close to or above the boiling point of water have been discovered. With a few (bacterial) exceptions, they have been classified as Archaea [1]. The adaption of these thermophilic organisms requires, amongst a number of modifications at the molecular level, a structural response of the ribosomes to ensure both high efficiency and high accuracy of the translational machinery at high temperatures. To date, very little is known about the molecular mechanisms regulating the thermal stabilization of ribosomes. Only for the hyperthermophilic Crenarchaeon, Sulfolobus solfataricus, have the mechanisms involved in the thermal stabilization of ribosomes been investigated in some detail. The heat stability of both 50S and 30S ribosomal subunits appears to be accounted for by the intrinsic stabilization of the helical rRNA domains (by a higher number of GϩC base pairs) and by a more extensive interaction between ribosomal proteins and rRNA [2Ϫ4].Ribosomal protein L1 from mesophilic and (hyper)thermophilic organisms is particularly suitable to study the specific interaction with RNA. In Bacteria and Archaea, ribosomal protein L1 has a dual function as a primary rRNA-binding protein and as a translational repressor. The primary and secondary structure of the L1-binding region on the 23S and 28S rRNA is highly conserved in Bacteria, Archaea and Eukarya ...

show abstract

Section: Methodsmentioning

confidence: 99%

Interaction of ribosomal L1 proteins from mesophilic and thermophilic Archaea and Bacteria with specific L1‐binding sites on 23S rRNA and mRNA

Köhrer

Mayer

Neumair

et al. 1998

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The laccase preparation (12.5 mg ml − 1 , 315 U mg − 1 ) was stored in 50 mM phosphate buffer pH 6.5, at −18°C. The laccase concentration was measured spectrophotometrically at 228.5 nm and 234.5 nm using BSA as standard [29].…”

Section: Isolation and Purification Of Laccasementioning

confidence: 99%

Design of a bioelectrocatalytic electrode interface for oxygen reduction in biofuel cells based on a specifically adapted Os-complex containing redox polymer with entrapped Trametes hirsuta laccase

Ackermann

Guschin

Eckhard

et al. 2010

Electrochemistry Communications

View full text Add to dashboard Cite

The design of the coordination shell of an Os-complex and its integration within an electrodeposition polymer enables fast electron transfer between an electrode and a polymer entrapped high-potential laccase from the basidiomycete Trametes hirsuta. The redox potential of the Os 3+/2+ -centre tethered to the polymer backbone (+ 720 mV vs. NHE) is perfectly matching the potential of the enzyme (+ 780 mV vs. NHE at pH 6.5). The laccase and the Os-complex modified anodic electrodeposition polymer were simultaneously precipitated on the surface of a glassy carbon electrode by means of a pH-shift to 2.5. The modified electrode was investigated with respect to biocatalytic O 2 reduction to H 2 O. The proposed modified electrode has potential applications as biofuel cell cathode.

show abstract

“…Specific activity was expressed as pmol cofactor consumed min-l (mg protein)-'. Protein concentration was assayed by measurement of UV absorbance at 228.5 nm and 234-5 nm (Ehresmann, 1973) using bovine serum albumin as standard. All enzyme assays and protein concentration determinations were done in duplicate on two independently prepared cell-free extracts.…”

Section: Methodsmentioning

confidence: 99%

A Hexokinase Associated with Catabolite Repression in Pachysolen tannophilus

Wedlock

Thornton

1989

Microbiology

View full text Add to dashboard Cite

~ ~ ~~A hexokinase (ATP : D-hexose 6-phosphotransferase; EC 2.7.1.. 1) associated with catabolite repression was isolated and purified from the yeast Pachysolen tannophilus. The enzyme phosphorylated D-fructose at a rate 1.5 times greater than that for D-glUCOSe. The K , values for D-glucose and D-fructose were 0.36 and 2.28 mM, respectively. Neither xylose reductase nor xylitol dehydrogenase were subject to catabolite repression in mutants defective in this enzyme.Two of the enzymes associated with xylose catabolism, aldose (xylose) reductase and xylitol dehydrogenase, have been purified from Pachysolen tannophilus and examined (Ditzelmuller et al., 1984a(Ditzelmuller et al., , b, 1985 Verduyn et al., 1985;Bolen et al., 1986;Morimoto et al., 1986 Morimoto et al., , 1987. However, little is known about other enzymes in this yeast. We have recently demonstrated the existence of three hexose-phosphorylating enzymes in P . tannophilus; hexokinases A and B and a glucokinase specific for D-glucose (Wedlock et al., 1989; accompanying paper). In this paper, we report the isolation and partial purification of a hexokinase (ATP : D-hexose 6-phosphotransferase; EC 2.7.1 , I), designated A, from a mutant which is defective in the glucokinase. The abcPg1ce of the glucokinase, which co-elutes with hexokinase A in the wild-type strain of P . tannophilus, aids the isolation of the hexokinase without contamination by the glucokinase. The hexokinase PI1 enzyme of Saccharomyces cerevisiae has been implicated in catabolite repression (Kopetzki & Entian, 1985;Hong & Botstein, 1986) as has a hexokinase isolated from Schwanniomyces occidentalis (McCann et al., 1987). Xylose utilization by P. tannophilus is subject to hexose-sugar catabolite repression (Slininger et al., 1987; Bicho et al., 1988). The effect of the P . tannophilus hexokinase A on the activities of xylose reductase and xylitol dehydrogenase and on D-xylose utilization is described here. METHODSYeast strains and growth conditions. The Pachysolen tannophilus strains used in enzyme analyses and sugar utilization experiments were P444-3 the wild-type strain, P5 10-5A defective in a glucokinase enzyme (glul), P509-3C defective in hexokinase A (hxk2) and P509-1B defective in both hexokinase A and the glucokinase (hxk2 glul). Strain P510-5A was also used in the purification of hexokinase A.Cells were grown in 1 litre flasks containing 250 ml of YEP-glucose [ 1 %, w/v, yeast extract (Difco), 2%, w/v, peptone (Difco) and 2%, w/v, ~-glucose]. The culture was incubated for 42 h at 30 "C and 180 r.p.m. on a gyratory shaker. Cells grown for 48 h in YEP-glucose were used as the inoculum. For the measurement of xylose reductase and xylitol dehydrogenase activities, cells were grown for 16 h with either Dglucose, Dxylose, or D-glucose and Dxylose as carbon source.Growth and sugar utilization were followed in YNB [0.67% yeast nitrogen base (Difco), 2%, w/v, sugar] liquid media. A 48 h YNB-xylose culture (100 ml) was washed with deionized water and resuspended in water to an optical...

show abstract

Spectrophotometric determination of protein concentration in cell extracts containing tRNA's and rRNA's

Cited by 246 publications

References 7 publications

Interaction of ribosomal L1 proteins from mesophilic and thermophilic Archaea and Bacteria with specific L1‐binding sites on 23S rRNA and mRNA

Interaction of ribosomal L1 proteins from mesophilic and thermophilic Archaea and Bacteria with specific L1‐binding sites on 23S rRNA and mRNA

Design of a bioelectrocatalytic electrode interface for oxygen reduction in biofuel cells based on a specifically adapted Os-complex containing redox polymer with entrapped Trametes hirsuta laccase

A Hexokinase Associated with Catabolite Repression in Pachysolen tannophilus

Contact Info

Product

Resources

About