David H. Malarek

[25] Affinity labeling of tRNA-binding sites on ribosomes

During the past several years a variety of chemically reactive derivatives of tRNA have been synthesized for use as affinity labels for amino acid-tRNA ligases, ribosomes, and factors involved in protein biosynthesis. Particularly in the study of Escherichia coli ribosomes, progress has been rather rapid. Besides affinity-label derivatives of tRNA, reactive derivatives of oligo- and polynucleotides have been constructed to label the ribosomal site for mRNA binding. Furthermore, a number of small-molecular-weight substances, such as antibiotics and GTP, have been chemically modified to obtain specific reagents for their respective ribosomal binding sites. The field of affinity labeling of ribosomes has been reviewed before with emphasis on various aspects of this problem (Cantor et al. 1974; Pellegrini and Cantor 1977; Zamir 1977; Cooperman 1978; Kuechler 1978). Here we want to concentrate primarily on affinity-labeling studies dealing with the ribosomal binding site for tRNA and on the interaction of tRNA with mRNA. The results will be correlated with the existing data on the structural organization of the ribosome as obtained by electron and immuno-electron microscopy and used to outline the functional domains on the ribosome for different regions of the tRNA. AFFINITY- AND PHOTOAFFINITY-LABEL DERIVATIVES OF tRNA Position of the Affinity-labeling Group Attachment of an affinity-labeling group to aminoacyl-tRNA requires special precautions to insure single-site specificity. In most studies, advantage has been taken of the existence of the single, highly reactive α -amino group on the aminoacyl moiety of tRNA. Thus, affinity reagents have usually been bound via an amide bond with the...

(−)‐2‐Hydroxy‐N‐cyclopropylmethylmorphinan: Radioimmunoassay and pharmacokinetic profile

The pharmacokinetic profile of (−)‐2‐hydroxy‐N‐cyclopropylmethylmorphinan (HCMM), a narcotic antagonist and analgesic, has been evaulated in man following administration of 25 to 50 mg of the drug orally and 10 to 15 mg intramuscularly. A specific radioimmunoassay procedure was developed for the determination of HCMM in plasma and urine. The drug had a mean “apparent” elimination half‐life in plasma of about 11 hr following both routes of administration. A mean of 47% of the oral dose was excreted in the urine as unconjugated and conjugated HCMM and only 5% of the dose was excreted as intact HCMM. In one subject studied, the plasma levels of conjugated HCMM were as much as 5‐fold higher than the levels of un conjugated drug. Although there was considerable intersubject variability following both routes of administration, the overall pharmacokinetic parameters suggest that oral and intramuscular doses are bioequivalent.