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The specific antigenicity of pentaglycine bridges in cell walls ofS. aureus was investigated. The antistaphylococcal sera (against11 Oeding strains ofS. aureus) were found to contain antibodies of two different specificities against polyglycyl peptides; one type reacted with polyglycyl peptides with a free amino terminus and the other with polyglycyl peptides with a free carboxyl terminus. These antibodies were assayed by passive hemagglutination with tanned sheep red cells sensitized with N-polyglycyl-BSA (containing polyglycyl peptides with a free amino terminus) or with C-polyglycyl-BSA (containing polyglycyl peptides with a free carboxyl terminus). Only the antibodies against N-polyglycyl peptides were absorbed byS. aureus strains and byS. epidermidis. Heterologous bacterial species failed to absorb antibodies of either specificity. Antibodies against polyglycyl peptides were used to confirm the mechanism of action of penicillin onS. aureus cell wall.

The heme-regulated translational inhibitor (HRI) has been purified 4800-fold. On electrophoresis in sodium dodecyl sulfate/polyacrylamide gel, the purified HRI showed one major polypeptide band. The purified HRI inhibits protein synthesis in lysates containing optimal levels of hemin with inhibition kinetics which parallel those observed in heme-deficiency. Data are presented which are consistent with an enzymatic function of HRI in the inhibition of protein synthesis. The HRI is an adenosine 3':5'-cyclic monophosphate independent protein kinase which phosphorylates the small subunit (38,000) but not the large subunits (52,000 and 50,000) of the initiation factor which forms a ternary complex with Met-tRNAf and GTP. This evidence supports the hypothesis that inhibition of protein synthesis by HRI involves the phosphorylation of the initiation factor. These findings are discussed in relation to various models for the regulation of protein kinase activity by heme. (see article).

Summary 80S ribosomes from skeletal muscle of diabetic rats were more susceptible than normal to dissociation by the initiation factor EIF-3; the increased sensitivity to dissociation was a property of the 60S subunit. Increased dissociation may be a reflection of the instability of diabetic 80S couples resulting from a change in the structure of the large subunit.

In the absence of added hemin, protein synthesis in rabbit reticulocyte lysates proceeds at maximal linear rates for several minutes and then ceases abruptly. Inhibition involves the action of a translational inhibitor whose formation is regulated by hemin. Addition of the isolated inhibitor to hemin-supplemented lysates produces an inhibition of protein chain initiation similar to that observed in heme-deficiency. The inhibitor has been purified over 300-fold and contains a protein kinase activity that copurifies with the inhibitory function. With calf thymus histone II as the phosphate receptor, the inhibitor-associated protein kinase requires ATP as the phosphorylating agent. Cycle AMP stimulates kinase activity 5- to 8-fold; the concentration of cycle AMP required for halfmaximal activity is 4 X 10-8 M. Preincubation of the inhibitor in the presence of cyclic AMP significantly reduces cyclic AMP-dependent phosphorylation and inhibitory activity. The corresponding protein kinase activity from hemin-supplemented lysates displays reduced cyclic AMP-dependency and little or no inhibitory activity. These findings suggest that the protein kinase activity associated with the purified translational inhibitor is involved in the mechanism of inhibition of initiation observed in hemedeficient reticulocyte lysates.

Physical, dielectric, piezoelectric and electromechanical parameters have been reported for bone and its two major constituents i.e. collagen and apatite, for their characterization as ultrasonic transducer materials. Collagen and apatite have been extracted from full bone using well known methods. These materials have been used to prepare simple disc-shaped test pieces (dimensions: diameter 10 mm and thickness 1.0 ± 0.01 mm). The variation of various electrical parameters with frequency in the region (1–108 MHz) is examined for these materials. These include impedance, phase angle, relative voltage output, quality factor ‘Q’, dielectric constant and resistivity. The data so obtained are compared with those for ceramic and quartz transducers. The observations on impedance, phase and relative voltage output for bone materials indicate that the first resonance peak falls around 56 MHz followed by second and third harmonics around 112 MHz and 168 MHz respectively. A low ‘Q’ value suggests a fairly wide band transducer, while other parameters compare favourably with ceramic and quartz materials. Bone material has also been used to obtain a transducer in the standard configuration and velocity dispersion in the frequency range 1–108 MHz is examined.

Vanadate inhibits protein chain initiation in rabbit reticulocyte lysates. The evidence that supports this conclusion is as follows: (i) the biphasic kinetics of inhibition in which protein synthesis is maintained at the control rate for 1-2 min is followed by an abrupt decline in the rate of synthesis; (ii) inhibition is associated with a marked disaggregation of polyribosomes and a concomitant increase in 80S ribosomes; and (iii) vanadate concentrations that inhibit protein chain initiation do not inhibit polypeptide chain elongation or the aminoacylation of tRNA. In partial reactions of protein chain initiation, vanadate concentrations that inhibit protein synthesis have no detectable effect on the formation of eukaryotic initiation factor eIF-2-promoted ternary complex with Met-tRNAf and GTP and on the assembly of 40S ribosomal subunit-Met-tRNAf complexes. On the addition of mRNA, the 40S ribosomal subunit-Met-tRNAf complexes also are transformed into 80S ribosome-mRNA-Met-tRNAf complexes, termed 80S initiation complexes. In vanadate-treated samples, however, these 80S initiation complexes are defective and unable to proceed beyond this step.

Discrimination between eukaryotic and prokaryotic, and formylated and non-formylated, initiator tRNAs by eukaryotic initiation factor EIF-3

Protein chain initiation in reticulocyte lysates is inhibited by (a) heme-deficiency, (b) low levels of double-stranded RNA, and (c) a purified translational inhibitor isolated from heme-deficient lysates. Previous studies have shown that the inhibitions produced by heme-deficiency and double-stranded RNA are prevented by 3': 5'-cyclic AMP, and that GTP, but not ATP, prevents the inhibition of heme-deficiency. In view of the recent finding that the inhibitor purified from heme-deficient lysates is associated with a protein kinase which appears to be involved in the mechanism of inhibition, the effects of cyclic AMP, GTP, and ATP on the three modes of inhibition were examines. In all three types of inhibition, cyclic AMP or GTP (a) prevents the onset of inhibition when added at zero time, and (b) restores protein synthesis in inhibited lysates. In contrast to these effects, ATP potentiates each inhibition, and blocks reversal of inhibition by cyclic AMP or GTP. On the basis of these and earlier findings, we propose that (a) these inhibitions involve the phosphorylation by protein kinases of the Met-tRNAf binding factor and/or a related site(s) on the 40S ribosomal subunit; and (b) cyclic AMP, GTP, and ATP exert their effects by their actions on this phosphorylation mechanism.

Fracture of the fibrous networks of normal and irradiated rat dermis have been studied using the SEM. The implications of these observations are discussed with regard to the mechanical behaviour of the tissue in the living animal. It is shown that after radiation the normal fibrous structure of collagen is replaced by a solid mat which is ill adapted to support stress, so that rupture at low stress levels is inevitable.

To define the mechanism of regulation of the protein kinase that is activated in heme deficiency and that inhibits initiation of protein synthesis, we have isolated and purified the heme-reversible form of the protein kinase from rabbit reticulocytes. The inhibitory activity is found in a single band after polyacrylamide gel electrophoresis under nondenaturing conditions. It migrates as a 95,000-dalton polypeptide in 15% sodium dodecyl sulfate/polyacrylamide gels. This purified inhibitor becomes self-phosphorylated in the presence of ATP; the phosphorylated protein and the inhibitory activity copurify. The inhibitor produces characteristic biphasic kinetics of inhibition in reticulocyte lysates and phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2); the inhibition is reversed by added eIF-2. In contrast to the heme-irreversible inhibitor, this heme-reversible inhibitor is no longer inhibitory after incubation with 20 micron hemin. Incubation with hemin also inhibits self-phosphorylation. Preincubation of the heme-reversible inhibitor in the presence of ATP potentiates the inhibition of protein synthesis in the subsequent incubation, as does treatment with N-ethylmaleimide. Phosphorylation of the heme-reversible inhibitor and inhibition of protein synthesis in the lysate due to phosphorylation of eIF-2 appear to be related. These findings suggest that hemin acts directly on the heme-reversible inhibitor.

Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA f and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA f complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg 2+ , ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA f . In the absence of Mg 2+ , however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity—hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.

Protein synthesis in rabbit reticulocytes and their lysates is regulated by heme. In heme-deficient reticulocyte lysates, protein synthesis proceeds at the initial rate for several minutes and then declines abruptly. Inhibition of protein synthesis is due to the activation of a heme-regulated translational inhibitor (HRI) which blocks the initiation of protein synthesis. Addition of the isolated HRI to hemin-supplemented lysates causes inhibition of initiation similar to that observed in heme-deficiency. HRI has been shown to be a protein kinase that specifically phosphorylates the Met-tRNA f binding factor (eIF-2). We have isolated an inhibitor (LI) of protein chain initiation from rat liver which displays properties similar to those of HRI: ( i ) the chromatographic behavior of LI on DEAE-Sephadex, DEAE-cellulose, and phosphocellulose is similar to that of HRI; ( ii ) both LI and HRI inhibit protein chain initiation in rabbit reticulocyte lysates with the same kinetics of inhibition—i.e., an initial period of synthesis for several minutes at the control rate followed by an abrupt decline in the rate of initiation; ( iii ) both inhibitions are prevented or reversed by eIF-2; ( iv ) GTP (2 mM) prevents, and ATP (2 mM) potentiates, the inhibition of protein synthesis induced by either inhibitor; ( v ) LI is associated with a protein kinase that also phosphorylates the 38,000-dalton subunit of elF-2. These findings indicate that a mechanism for the regulation of protein synthesis similar to that found in rabbit reticulocytes may be present in rat liver.

Purified eukaryotic initiation factor, EIF-3, prepared from ascites cells dissociated rat liver 80S ribosomes into subunits. Ribosomes bearing endogenous mRNA and nascent peptide were not dissociated by EIF-3. When 80S ribosomes reconstituted from subunits were used as substrate the reaction had the following characteristics: Dissociation was rapid--the reaction being completed within 2 min at 30°. The extent of dissociation was directly proportional to the amount of EIF-3; with 21 μg of EIF-3 about 70% (or 10.5 μg) of the 80S monomers were dissociated. The dissociation of 80S monomers by EIF-3 decreased with increasing concentrations of magnesium. The reaction was not catalytic: 28 moles of EIF-3 were required to dissociate 1 mole of 80S ribosomes. The characteristic of the dissociation reaction promoted by EIF-3 and by E. coli initiation factor IF-3 are remarkably similar. The dissociation reaction provides a practical assay for EIF-3 independent of complimentation of other initiating factors.

Tanned sheep erythrocytes have been considered incapable of sensitization with the precipitinogen of protein A of Staphylococcus aureus , so that the activity of this antigen in serological reactions has so far been studied by means of the agar diffusion test (ADT) only. The precipitinogen of the protein A in this study was found to become attached to the tanned erythrocytes and to sensitize them for the passive hemagglutination test (PHT). It was determined that, in contrast to nonspecific reactivity between normal human serum and the precipitinogen in the ADT, the reaction in the PHT was of specific nature. Of seven species studied, all normal human, dog, and hog sera tested were positive in the PHT. However, the hemagglutinin titers of the sera of the two animal species by far exceeded those of the human sera. The data emphasized the usefulness of the highly sensitive PHT for assaying antibodies to protein A.