A sub-population of rat liver membrane-bound ribosomes that are detached in vitro by carcinogens and centrifugation.

The chemical-carcinogen-induced detachment of ribosomes from rat liver endoplasmic reticulum was studied in vitro. Incubation of postmitochondrial supernatant with 0.2 mM-diethylnitrosamine or N-2-acetylaminofluorene removed approx. 16% of membrane-bound ribosomes, measured as differences in RNA/protein values of membrane separated from unbound ribosomes by flotation. These ribosomes are also detached by exposure to high centrifugal forces (160000g) and are among those removed by NADPH-catalysed lipid peroxidation. Extensive lipid peroxidation prohibits any measurement. The ribosomes (polyribosomes) removed are not those detached from the membrane by exposure to high KC1 concentrations (loosely bound) or high KC1 concentrations in the presence of puromycin (tightly bound). It is concluded then that centrifugally labile and carcinogen-sensitive represent a previously unreported sub-population of membrane-bound ribosomes.     

The state of phosphorylation in vivo of membrane-bound phosphoproteins in rat brain (Short Communication)

The alkali-labile P content of membrane protein prepared from rapidly frozen rat brain was measured, CuSO(4) being used to inhibit protein phosphatase activity during subcellular fractionation. The P content of the membrane fraction was significantly increased (+12%) over the control value by incubation of homogenates with ATP before fractionation. This suggests that the membrane protein in rat brain is normally only partially phosphorylated.     

The role of membrane phospholipids in the interaction of ribosomes with endoplasmic-reticulum membrane (Short Communication)

It is shown that the ionic head groups of the membrane phospholipids cannot be solely responsible for the attachment of the ribosome and that other membrane components must also be involved in the binding process.     

Peptidyl-puromycin synthesis by free and membrane-bound ribosomes.

The peptidyl transferase reaction, as measured by the formation of peptidyl-puromycin, was compared for free ribosomes and ribosomes bound to two types of membrane, the endoplasmic reticulum and the outer nuclear membrane. In most respects the reaction catalyzed by the three types of ribosome was similar, demonstrating that interaction of the 60 S ribosomal subunit with the membrane has little effect on the functioning of peptidyl transferase, a 60 S protein. However, both the rate and extent of synthesis of peptidyl puromycin were lower for ribosomes bound to the nuclear membrane than for free or microsome-bound ribosomes. This difference appears to be a direct consequence of the ribosome-membrane interaction, since ribosomes stripped from the nuclear membrane could not be distinguished from the other classes of ribosome.     

Calcium transport and phospholipid composition of microsomal fractions from denervated rat gastrocnemius (Short Communication)

Total calcium uptake, but not the initial rate, and non-calcium-stimulated (basic) adenosine triphosphatase activity of rat gastrocnemius microsomal fractions 3, 7 and 14 days after denervation increased compared with contralateral controls. Microsomal sphingomyelin also increased but phosphatidylcholine plus choline plasmalogen was unchanged in amount. These results are contrasted with those reported for vincristine myopathy.     

pH-controlled hydrogen-bonding (Short Communication)

The pH-dependence of the degree of hydrogen-bonding between a base and its conjugate acid is considered. When only a small proportion of the total base is complexed, the amount complexed is proportional to (1+coshp)(-1) where p=2.303 (pK(a)-pH), pK(a) being the dissociation constant of the conjugate acid. This represents sharp pH-dependence. As the proportion complexed increases, the curve broadens, eventually becoming flat-topped, with more than half the base complexed over the range of pH values pK(a)+/-logKC, approximately. (K is the complex association constant and C is the formal base concentration, including all forms.) There are similarities to the extent of mono-protonation of a dibasic acid.     

A rapid method for collecting and assaying gradient fractions for radioactive macromolecules (Short Communication)

A rapid method is described for collecting gradients after centrifugation, and assaying them for acid-precipitable radioactivity. An entire gradient is collected and processed on a single glass-fibre filter disc. This method allows one easily to assay six gradients for acid-precipitable radioactivity in less than 2h.     

Acceleration of gluconeogenesis from lactate by lysine (Short Communication)

l-Lysine (2mm) causes an increase (mean 60%) in the rate of gluconeogenesis from lactate in isolated liver cells. The effect is of a catalytic nature. No other amino acid has the same effect, though ornithine is slightly active. The effect is additional to the stimulatory effects of oleate and of dibutyryl cyclic AMP.     

Proteolysis of rabbit immunoglobulin M by papain (Short Communication)

1. Digestion of rabbit immunoglobulin M (IgM) by papain for 5h, in the absence of reducing agent, gives Fabmu and Fc(5)mu fragments in high yield. Shorter periods give fragment Fc(5)mu with one or more Fabmu fragments still attached. 2. Reducing agent in the absence of a denaturant cleaves rabbit IgM into half-subunits, each containing one mu chain and one light chain. Digestion by papain in the presence of such a reducing agent destroys the Fcmu domains.     

Inhibition of mitochondrial phosphate transport by p-diazobenzenesulphonate (Short Communication)

The impermeant protein-modifying reagent p-diazobenzenesulphonate inhibits phosphate transport in rat liver mitochondria. The rate of inhibition is increased by the presence of oxidizable substrate, and decreased by uncoupling agents. These results are compared with the results on inhibition of phosphate transport by N-(N-acetyl-4-sulphamoylphenyl)maleimide (Klingenberg et al., 1974).     

Specificity of anti-nucleoside antibodies (Short Communication)

The method of conjugation of a nucleoside or related compound to a carrier protein may have a significant effect on the specificity of the antibodies elicited. It is demonstrated, by means of the membrane-filtration assay, that anti-isopentenyladenosine antibodies produced by the ;periodate procedure' are much more reactive with the periodate-oxidized form of the nucleoside than with the parent compound. In addition, the simplicity and specificity of the assay used suggests its use as a sensitive radioimmunoassay for this multifunctional nucleoside.     

Crystallization of macromolecular heparin (Short Communication)

X-ray fibre-diffraction photographs were obtained from oriented films of the sodium salt of macromolecular heparin (molecular weight approx. 10⁶) prepared from rat skin. Two distinct molecular chain conformations corresponding to two different crystal lattices were observed as a function of relative humidity. The first conformation, obtained at 78% relative humidity, has a layer-line spacing of 1.73nm, which can be interpreted as an approximate twofold helix. On increasing the relative humidity to 84% a second phase with a layer-line repeat of 1.65nm is obtained with the reflexions indexing on a triclinic unit cell similar to that obtained previously (Nieduszynski & Atkins, 1973) for pig mucosal heparin.     

Inhibition of protein synthesis by ricin: experiments with rat liver mitochondria and nuclei and with ribosomes from Escherichia coli (Short Communication)

1. Ricin, a toxic protein from the seeds of Ricinus communis which inhibits poly(U)-directed polyphenylalanine synthesis by rat liver ribosomes (Montanaro et al., 1973), does not affect protein synthesis by isolated rat liver mitochondria. 2. The toxin is ineffective also on poly(U)-directed polyphenylalanine synthesis in reconstituted systems with ribosomes isolated from rat liver mitochondria or from Escherichia coli. 3. Ricin inhibits protein synthesis by isolated rat liver nuclei, but at concentrations much higher than those affecting rat liver ribosomes.     

Specificity of glycerol kinase (Short Communication)

The activity of a number of alcohols was examined as substrates or inhibitors of glycerol kinase (ATP-glycerol phosphotransferase; EC 2.7.1.30) from Candida mycoderma. On the basis of these and other results, a modified model is proposed to account for the substrate specificity of the enzyme.