Pancreatic Ribosomes Research Articles

Cholecystokinin Stimulates a Specific Ribosomal S6 Kinase in Rat Pancreatic Acini

Stimulation of intact rat pancreatic acini with cholecystokinin (CCK) enhances the phosphorylation of the ribosomal protein S6 in a dose-dependent manner with half maximal stimulation at 40 pM and maximal stimulation at 1 nM CCK octapeptide. Soluble cellular extracts contained S6 kinase activity assayed using purified rat pancreatic ribosomes as substrate. Stimulation by CCK of S6 kinase was concentration dependent, being half maximal at 50 pM and maximal at 1 nM CCK. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), an activator of protein kinase C, also increased both S6 phosphorylation in intact acini and soluble S6 kinase activity. In order to determine whether S6 kinase mediated S6 phosphorylation following CCK treatment of acini, two-dimensional phosphopeptide analysis was performed for S6 proteins phosphorylated under various conditions. These data suggest that a specific soluble S6 kinase, the activation of which appears to be directly or indirectly mediated by protein kinase C, is the functional enzyme in intact acini that mediates the action of CCK to increase S6 phosphorylation and may be involved in increased protein synthesis in pancreatic acini treated with CCK.

Insulin and ribosomal protein S6 kinase in rat pancreatic acini.

Treatment of pancreatic acini from diabetic rats with insulin resulted in a dose-dependent increase in the phosphorylation of ribosomal protein S6 when analyzed by two-dimensional gel electrophoresis. To study the presence of the protein kinase mediating this phosphorylation, soluble extracts of intact acini that had been previously treated with insulin were prepared and assayed for protein kinase activity with rat pancreatic ribosomes as a substrate. Activation of S6 kinase activity, observed in a time-dependent manner, was maximal after 20-30 min and, in a dose-dependent manner, was half-maximal at 1 nM and maximal at 10 nM insulin concentration. Based on cofactor requirements, substrate specificity, and a slow activation of the enzyme, the S6 kinase was distinct from cAMP-dependent, Ca2+-calmodulin-dependent, and Ca2+-phospholipid-dependent protein kinases and protease-activated kinase II. The S6 kinase activated by insulin was highly specific for the ribosomal protein S6 when compared with various substrates, including casein, glycogen synthase, phosphorylase b, phosvitin, histone HIII-S, and histone HVIII-S. Protein S6 phosphorylation in intact acini and activation of the S6 kinase by insulin showed similar dose-response curves, consistent with the S6 kinase being responsible for the protein S6 phosphorylation in intact acini. The comparison of the dose-response curves for S6 phosphorylation and protein synthesis in acini suggests that there is a close correlation between these two insulin actions.

Dissociation of pancreatic ribosomes by [formula omitted

On incubation of canine pancreatic ribosomes at 37° with 1.07 mM p- hydroxymercuri[ 14C]benzoate , dissociation occurred. At short incubation times, labelled monoribosomes were formed. An increase in radiospecific activity occurred on dissociation, the labelling of the small subunit being greater than the large. During a 30-min incubation period, there was a further increase in the radiospecific activity of the large subunit, while that of the small subunit remained constant. The radiospecific activities of subunits formed by EDTA dissociation of labelled monoribosomes were, however, similar.

The effects of sulfhydryl-blocking reagents on canine pancreatic ribosomes

Incubation of canine pancreatic ribosomes with 1 m m-PHMB 1 1 Abbreviations: DTNB = 5,5′-dithiobis-(2-nitrobenzoic acid); DTT = dithiothreitol; IAA = iodoacetic acid; IAAm = iodoacetamide; NEM = N-ethylmaleimide; PHMB = p-hydroxymercuri-benzoate; SDS = sodium dodecyl sulfate. at 37 ° causes dissociation to subunits, followed by degradation to species sedimenting more slowly than the small subunit. During this treatment a reduction in sedimentation coefficient of the subunits is observed. Other thiol-blocking reagents were found to be less active, the order of reactivity being PHMB ⪢ DTNB ⪢ IAA > NEM > IAAm. The observation is not solely the result of ribonuclease damage caused by inactivation of a ribonuclease inhibitor, although a trace amount of ribonuclease activity which increases slightly in the presence of PHMB is associated with the ribosomes. Electrophoretic analysis of rRNA and hyperchromicity studies support this conclusion. It is suggested that when sulfhydryl groups are blocked, more slowly sedimenting species are formed which might be more sensitive to ribonuclease than were the native ribosomes.

Canine Pancreatic Ribosomes: II. The Protein Moiety

Abstract The protein moiety of purified dog pancreatic ribosomes has been examined by starch gel electrophoresis in formate buffers, pH 3, containing 6 m urea. Densitometric tracings of the complex but reproducible electrophoretograms disclosed a minimum of 21 cationic components. Identical patterns were obtained with protein prepared by alternative extraction procedures, and these were not significantly altered by preparation of the ribosomes from a single dog pancreas or by the inclusion of puromycin during isolation of the ribosomes. Treatment of the protein from canine pancreatic ribosomes with dithiothreitol in urea resulted in very minor changes in electrophoretic properties, the suggestion being that most of the half-cystine residues of the protein are already in the reduced state. Protein from microsomal and postmicrosomal canine pancreatic ribosomes presented similar but not identical patterns. Electrophoretograms of protein moieties of purified ribosomes from dog and rabbit pancreas were compared. Most components were common to both species, but a few were characteristic of one species only. The species-specific bands are reproducible and independent of the method of preparation of the protein. Of the protein present in the 80 S monoribosome of dog pancreas, three components are localized exclusively or predominately in the large subunit, and five exclusively or predominately in the small subunit. One disappears upon dissociation and the remainder appear to be common to both subunits. Of the dog-specific bands, one appears only in the small subunit, one appears only in the large subunit, and one is present in both subunits.

Canine Pancreatic Ribosomes: I. Preparation and Some Properties

Canine Pancreatic Ribosomes: I. Preparation and Some Properties

Cytochemical study on the pancreas of the guinea pig. VII. Effects of spermine on ribosomes.

Pancreatic ribosomes (guinea pig) aggregate and lose upon treatment with polyamines, particularly spermine, their bound secretory enzymes. Spermine, at 0.5 mM, for example, causes the release of about 85 per cent of the chymotrypsinogen and RNase, and from 85 to 100 per cent of the ribosomal amylase. At the same time, the particles lose about 10 per cent of their RNA, 7 to 24 per cent of their total protein, and from 75 to 100 per cent of their Mg(++). Observations with the electron microscope confirm the heavy agglutinating of the ribosomes but otherwise show little change in the structure of the particles. Using radioactive spermine it was found that, concomitant with the loss of bound enzymes and Mg(++) from the ribosomes, spermine became bound to the particle. The extent of binding ranged from 0.29 to 1.49 micromoles per 10micromoles RNA-P. The bound radioactive spermine can be removed by subsequent treatment of the ribosomes with GTP, ATP, or P-P, which treatment also removes most of the RNA of the particles, leaving behind ribosomes with a much lower RNA/protein ratio. From this evidence it was inferred that spermine, in releasing the Mg(++) of the particle, becomes salt-linked to the free phosphate hydroxyl groups of the RNA. Freshly isolated pancreatic and hepatic ribosomes contain very little spermine, about 0.1 to 0.2 micromoles polyamine/10 micromoles RNA-P. The results are discussed in terms of the linkages between the structural protein, the bound secretory enzymes, and the RNA of the ribosomes.