Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer.

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3'-ends of the tRNAs at the peptidyl-transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.

Histidine 229 in protein L2 is apparently essential for 50S peptidyl transferase activity.

It has recently been suggested that peptidyl transferase activity is primarily a property of ribosomal RNA and that ribosomal proteins may act only as scaffolding. On the other hand, evidence from both photoaffinity labeling studies and reconstitution studies suggest that protein L2 may be functionally important for peptidyl transferase. In the work reported here, we reconstitute 50S subunits in which the H229Q variant of L2 replaces L2, with all other ribosomal components remaining unchanged, and determine the catalytic and structural properties of the reconstituted subunits. We observe that mutation of the highly conserved His 229 to Gin results in a complete loss of peptidyl transferase activity in the reconstituted 50S subunit. This is strong evidence for the direct involvement of L2 in ribosomal peptidyl transferase activity. Control experiments show that, though lacking peptidyl transferase activity, 50S subunits reconstituted with H229Q-L2 appear to be identical with 50S subunits reconstituted with wild-type L2 with respect to protein composition and 70S formation in the presence of added 30S subunits. Furthermore, as shown by chemical footprinting analysis, H229Q-L2 appears to bind 23S RNA in the same manner as wild-type L2. Thus, the effect of H229 mutation appears to be confined to an effect on peptidyl transferase activity, providing the most direct evidence for protein involvement in this function to date.

Comparison of cerebrospinal fluid pressure in propofol- and thiopental-anesthetized eucapnic dogs.

Cerebrospinal fluid pressure (CSFp) was measured as part of the neurologic assessment of dogs with suspected intracranial disease. Because propofol has not been shown to cause an increase in intracranial pressure in humans, the authors examined its effect on (CSFp) in dogs to determine if it would be an appropriate substitute for thiopental as an anesthetic agent for the measurement of CSFp. The CSF pressure in eucapnic propofol-anesthetized dogs (105 +/- 5.6 mm H2O) was not significantly different (p < .05) from CSFp in eucapnic thiopental-anesthetized dogs (103.8 +/- 6.6 mm H2O).

Storage of platelet concentrates after high-dose ultraviolet B irradiation.

Ultraviolet B (UVB) irradiation of platelet concentrates (PCs) may prevent the development of posttransfusion HLA alloimmunization. As irradiation performed in a blood center or a hospital will probably be associated with a variable postirradiation delay before transfusion, the ability to store PCs after UVB irradiation becomes important. The effects have been studied of a UVB dose of 10,000 mJ per cm2, the dose used in our institution for UVB clinical trials, on PCs pooled and stored for up to 96 hours after irradiation. Results showed that after 96 hours of storage, though there were no changes in pH, platelet count, white cell count, percent discharge of lactate dehydrogenase, or beta-thromboglobulin, there were significant decreases in morphology score and osmotic recovery. These changes, however, were not evident after 24 hours of storage. Similarly, there was a 60-percent decrease in immunoreactive membrane glycoprotein (GP) Ib after 96 hours of storage, but these changes were not seen after 48 hours of storage. No changes were seen in levels of GPIIb/IIIa in either group during the 96 hours of storage. On computer-analyzed two-dimensional polyacrylamide gel electrophoresis, PCs irradiated at 10,000 mJ per cm2 and stored for 72 hours had changes in over 50 platelet proteins as compared to those proteins in nonirradiated age-matched control PCs. It can be concluded that UVB irradiation of PCs at 10,000 mJ per cm2 does not lead to significant platelet deterioration after short-term storage (24-48 hours) but is likely to be deleterious after long-term (72-96 hours) storage.(ABSTRACT TRUNCATED AT 250 WORDS)