Ruth Miskin

Inactivation and reactivation of ribosomal subunits: Amino acyl-transfer RNA binding activity of the 30 s subunit of Escherichia coli☆

Abstract The ability of the 30 s ribosomal subunit to bind phenylalanyl-transfer RNA in the cold in response to polyuridylic acid is lost if the subunit is subjected, even transiently, to either of two treatments: (a) the removal of certain specific monovalent cations (NH+4, K+, Rb+ or Cs+), or (b) the reduction of the Mg2+ concentration below a critical concentration of about 2 m m . If the depleted cation is restored, the subunit reverts to an active form in a process that is greatly enhanced by heat. Thermally reactivated subunits retain full activity when rechilled, showing that the inactivation and reactivation processes involve changes, presumably conformational, in the subunit itself. Reactivation follows first-order kinetics with respect to the appearance of active subunits, with an Arrhenius activation energy of 26 kcal./mole between 30 °C and 40 °C. On storage at 0 °C, inactive 30 s subunits gradually lose the ability to be reactivated. Part of this loss is due to the oxidation of one or more sulphydryl groups and is prevented or reversed if a sulphydryl reducing agent is included in the storage or the reactivation medium, respectively. Active and inactive 30 s subunits have the same sedimentation coefficient and there is no direct evidence that they differ in conformation. However, two kinds of indirect evidence are in accord with the existence of conformational differences: (a) under appropriate conditions inactive 30 s subunits form dimers sedimenting at 50 s while active subunits do not, and active 30 s subunits associate more readily with 50 s subunits to form 70 s ribosomes; (b) inactive 30 s subunits undergo sulphydryl oxidation much more rapidly than do active ones. Although differing in certain details, the 30 s inactivation and reactivation processes are generally similar to those previously described for the 50 s subunit. Both subunits can exist in active and inactive forms which are easily and reversibly interconverted, suggesting that the structure of the functional ribosome is flexible and easily altered. The interconversions affect a number of ribosomal activities in parallel. It is possible that many previously described phenomena pertaining to ribosomal activity can be interpreted, at least in part, in terms of ribosomal inactivation and reactivation.

Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells.

The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities.

Inactivation and reactivation of ribosomal subunits: The peptidyl transferase activity of the 50 s subunit of Escherichia coli

The ability of the 50 s ribosomal subunit to catalyze the peptidyl transferase reaction is absolutely dependent on the continued presence of certain monovalent cations in the ribosomal medium. This ability is maintained as long as one of these cations is present and is lost if the cation is removed. The effective cations are NH4+ ≧ Rb+ > K+ > Cs+. Na+ and Li+ are ineffective. Activity is restored, with the same order of effectiveness, if an appropriate cation is added back. Although at high salt concentrations there may be a slow reactivation in the cold, heat greatly accelerates the process at all salt concentrations. Although reactivating cations are present in the peptidyl transferase assay medium, inactive ribosomes remain inactive if the assay conditions do not promote reactivation; i.e. if the assay is performed in the cold. This shows that the effect is on the ribosome and not on the assay. The ribosome can exist in two different states, active and inactive, and can be brought from either state to the other. The rate of reactivation increases markedly with rising temperature or monovalent cation concentration. Reactivation is also accelerated by the presence of aliphatic alcohols in the medium and by substrate, fMet-tRNA, provided that the substrate is actually bound to the ribosome during the heat treatment. Reactivation of ribosomes follows first-order kinetics under a wide variety of conditions. The energy of activation of the process is 40 to 50 kcal./mole below 30 °C but becomes less at higher temperatures. Inactive subunits lack no essential components and have the same sedimentation pattern and coefficient as active ones. However, it seems likely that the interconversion between the active and inactive form involves a conformational change. It is not known if the inactivation-activation process occurs in vivo; the possibility is discussed.

Plasminogen activator in the rodent brain

The cellular origin(s), the biochemical properties and the developmental pattern of the protease plasminogen activator (PA) were investigated in the rodent brain. PA activity was localized in frozen brain sections by a novel autoradiography technique. PA levels and electrophoretic mobility were determined in homogenates prepared from major regions of the developing and the mature brain, and both the localization and the specific activity of the enzyme were examined in X-irradiated brain regions. PA activity was shown to be correlated with cell bodies in neuronal-enriched regions and also with endothelial, meningeal and ependymal layers. PA levels increased in a transient manner and at different rates and time periods in the various brain regions that were analyzed. PA in neuronal, but not in epithelial cell layers was affected by X-irradiation and one of the brain PA species had a similar molecular weight of that of neuroblastoma cells. Our findings suggest that in the brain PA is produced by neurons and by epithelial cells, and that it may have additional functions to that of thrombolysis both in the developing and the mature brain.

[40] The inactivation and reactivation of Escherichia coli ribosomes

Publisher Summary When E. coli ribosomes or either of their subunits are assayed under certain conditions for any of a number of biological activities, they can be shown to exist in one of several different states: active, inactive, or partially active. The state is determined by the past treatment of the ribosome. These states are reversibly inter convertible and ribosomes can be brought from one to another by relatively mild treatments. Among the factors that influence these inter conversions in vitro are temperature, the ionic environment, and interactions between subunits or between the ribosome and certain other macromolecules that participate in protein synthesis. 70 S, 50 S, and 30 S ribosomes all undergo these inter conversions, although the characteristics of the process may vary somewhat in each case.

N-ethyl maleimide as a probe for the study of functional sites and conformations of 30 S ribosomal subunits

Escherichia coli 30 S ribosomal subunits are inactive in a number of specific functions when Mg2+ concentration is reduced to 1 mM, and activity is recovered on heating under appropriate ionic conditions. When active and inactive forms were treated with N-ethyl maleimide, both forms reacted to a similar extent, but the reagent attached mostly to different proteins. Moreover, it caused irreversible inactivation only when reacting with the inactive form of the subunit. Though the activating treatment failed to restore activity to these subunits it did expose the same sulfhydryl groups as are available in the active state for reaction with the maleimide. Different ribosomal activities were eliminated at different maleimide concentrations, permitting the assignment of specific functions to sulfhydryl groups of specific ribosomal proteins. Protein S18 appears to be involved in subunit association, binding of fMet-tRNA and of aminoacyl-tRNA to the P-site. Proteins S1, S14 and S21 are all or in part involved in the binding of aminoacyl-tRNA to the A-site and in the binding of the antibiotic dihydrostreptomycin. The reaction with N-ethyl maleimide thus provides a criterion other than biological activity for characterizing different ribosomal forms and a tool for mapping the 30 S subunit for specific functional sites.

Interconversions between inactive and active forms of ribosomal subunits

We have previously reported that the peptidyl transferase activity of the 50 S subunit of E. coli ribosomes is lost if the ribosomes are exposed to media lacking NH: and K’. Activity can be restored, but this requires both (a) the readdition of one of these ions, and (b) heat. The heat requirement is discerned only if the ribosomes are assayed at O’, where they are active only if they were previously heated in the presence of NH: or K+ [ 11. We have now studied a specific function of the 30 S subunit, the non-enzymatic binding of phenylalanyltRNA directed by poly U and assayed at 0”, and have found a similar inactivation and reactivation, with similar effects of specific monovalent cations and heat. In addition, the 30 S subunit is inactivated if the M

mRNAs encoding urokinase-type plasminogen activator and plasminogen activator inhibitor-1 are elevated in the mouse brain following kainate-mediated excitation.

+ concentration is lowered to 1 mM (as is commonly done to dissociate 70 S ribosomes), even if NH: is present. It is of particular interest, however, that ribosomes that have been inactivated toward non-enzymatic binding are active at 0’ in the enzymatic binding reactions mediated by initiation factors or transfer factor T, even if they have not been previously heated.

Localization of urokinase-type plasminogen activator mRNA in the adult mouse brain

Urokinase-type plasminogen activator (uPA) is an inducible extracellular serine protease implicated in fibrinolysis and in tissue remodeling. Recently, we have localized uPA mRNA strictly in limbic structures and the parietal cortex of the adult mouse brain. Here, we tested whether the systemic treatment of mice with kainic acid (KA), an amino acid inducing limbic seizures, could elevate in the brain mRNAs encoding uPA and its specific inhibitor, plasminogen activator inhibitor-1 (PAI-1), a major antifibrinolytic agent. Brain sections encompassing the hippocampus were tested through in situ hybridization using radiolabeled riboprobes specific for the two mRNA species. The results showed that KA greatly enhanced both mRNA species in sites of limbic structures and cortex. However, in the hypothalamus and brain blood vessels only PAI-1 mRNA was elevated. Those were also the only two locations where PAI-1 mRNA was detected in the non-treated control brain, although at a low level. For both mRNAs, KA enhancement was first evident 2-4 h after treatment, and it was most prolonged in the hippocampal area, where prominent hybridization signals persisted for three days. Here, both mRNAs were initially elevated in the hilar region of the dentate gyrus and in the molecular and oriens layers; however, PAI-1 mRNA became evident throughout the area, while uPA mRNA became especially pronounced in the CA3/CA4 subfield. In the cortex both mRNA types were induced, but only uPA mRNA was elevated in the retrosplenial cortex, and also in the subiculum. In the amygdaloid complex, uPA mRNA was restricted to the basolateral nucleus, whereas PAI-1 mRNA was seen throughout the structure, however, excluding this nucleus. These data show that seizure activity enhances the expression of uPA and PAI-1 genes in the brain; the patterns of enhancement suggest that the protease and its inhibitor may act in brain plasticity in synchrony, however, also independently of each other. Furthermore, the results suggest that by elevating PAI-1 mRNA in brain blood vessels, limbic seizures generate a risk for stroke.

Plasminogen activator in the developing rat cerebellum: Biosynthesis and localization in granular neurons

Urokinase-type plasminogen activator (uPA) is an inducible serine protease, secreted by a variety of cell types, that functions in fibrinolysis and has been implicated also in events such as cell migration and tissue remodeling and repair. To explore the role of uPA in the adult brain we have now screened the whole mouse brain for cells expressing the uPA gene through in situ hybridization using 35S-complementary RNA. uPA mRNA was visualized predominantly in three regions: (1) the subicular complex, (2) the entorhinal cortex, (3) the parietal cortex, where the signal was somewhat lower and confined to layers IV and VI. Weaker signals were seen in the basolateral nucleus of the amygdala and in the anterodorsal thalamic nucleus, and also in the hilus of the dentate gyrus where labeling was slightly over background. Cells exhibiting uPA mRNA signaling were large neurons according to morphological criteria. These results support the view of uPA being involved in neuronal functions of the adult brain, specifically in the hippocampal formation and the parietal cortex.