Jahangir A. Khawaja

Magnesium : An update on physiological, clinical and analytical aspects

There is an increased interest in the role of magnesium ions in clinical medicine, nutrition and physiology. The characteristics of the binding of magnesium and calcium ions to various components, macromolecules and biological membranes are described. Magnesium affects many cellular functions, including transport of potassium and calcium ions, and modulates signal transduction, energy metabolism and cell proliferation. The mechanism of cellular uptake and efflux of magnesium, its intracellular transport, intestinal absorption, renal excretion and the effect of hormones on these are reviewed. Magnesium deficiency is not uncommon among the general population: its intake has decreased over the years especially in the western world. The magnesium supplementation or intravenous infusion may be beneficial in various diseased states. Of special interest is the magnesium status in alcoholism, eclampsia, hypertension, atherosclerosis, cardiac diseases, diabetes, and asthma. The development of instrumentation for the assay of ionized magnesium is reviewed, as are the analytical procedures for total magnesium in blood and free magnesium in the cytosol. The improved procedures for the assay of different magnesium states are useful in understanding the role of magnesium in health and disease.

Interaction of ribosomes and ribosomal subparticles with endoplasmic reticulum membranes in vitro: Effect of spermine and magnesium

Abstract The interaction of rat liver ribosomes and their subparticles with endoplasmic reticulum membranes has been studied in vitro under various ionic conditions. About 50 % of free ribosomes and 25 % of detergent-treated (free+bound) ribosomes were found to be associated with isolated total endoplasmic reticulum membranes stripped of ribosomes if incubated in buffers containing 5 mM Mg2+ or 0.5 mM spermine at 0° for 60 min. If Mg2+ and spermine were omitted from the incubation medium, the binding was considerably reduced. K+ was found to inhibit attachment. Only negligible attachment was observed if the ribosomes were incubated with rough membranes already studded with ribosomes. Free ribosomes, after their attachment to total endoplasmic reticulum, became relatively more resistant to attack by ribonuclease. Both ribosomal subparticles were bound to total endoplasmic reticulum in the presence of spermine or Mg2+, although the binding was relatively greater in the case of the large subparticles in the presence of spermine. It is suggested that Mg2+ and spermine may play a role in the ribosome-membrane attachment.

Effect of spermine and magnesium on the attachment of free ribosomes to endoplasmic reticulum membranes in vitro

Abstract A portion of the free ribosomes became attached to the isolated total endoplasmic reticulum membranes when incubated at 0°C for 60 min in buffers containing either 0.3–0.5 mM spermine or 5 mM Mg 2+ . This attachment was negligible if both spermine and Mg 2+ were omitted from the incubation medium. When smooth endoplasmic reticulum was tested alone, free ribosomes did not associate appreciably, whether or not spermine or Mg 2+ was present. These results suggest that spermine and Mg 2+ may function through forming ribosome-cation-membrane bridges. There is also an indication of the presence of ribosome binding sites on the endoplasmic reticulum membranes.

Synthesis and the puromycin-mediated release of nascent polypeptide chains by reconstructed rough endoplasmic reticulum from rat liver and brain cortex

The mechanism by which the cell separates the synthesis of secreted proteins from the synthesis of retained proteins is not yet fully understood. Available data suggest that secretory proteins are preferentially, if not exclusively, synthesized on the membrane-bound ribosomes but the converse is not necessarily so [ 1,2]. The endoplasmic reticulum membranes may play an important role in the selection and the translation of the template present on the attached ribosomes [3]. In recent years a number of attempts have been made to reconstruct rough endoplasmic reticulum by reacting ribosome-free membranes and ribosomes under various conditions of incubation [4-61. Recent evidence suggests that the reconstructed liver RER exhibits structural, and in some respects, functional properties analogous to those of authentic RER [4,7,8]. There are, however, conflicting reports on the ability of the reconstructed RER to discharge the nascent peptide chains vectorially through the ER membranes. Thus Burke and Redman [9] failed to obtain any evidence for the vectorial discharge of nascent polypeptides across the membrane in the reconstructed RER. On the other hand, Shires et al. [lo] have claimed that their preparation of reconstructed RER was active in the vectorial release of the newly synthesized chains. The present investigation was undertaken to resolve this controversy by recon-

Production of a lesion on liver rough endoplasmic reticulum after prolonged ethanol ingestion and its reversal through abstinence

Abstract Prolonged ethanol ingestion by adult rats resulted in an inhibition of amino acid incorporation in vitro by liver membrane-bound ribosomes. The same treatment, however, produced a stimulation in the activity of free ribosomes. The addition of endoplasmic reticulum membranes, extracted from the microsomal fraction of control or ethanol-treated rats, had an inhibitory effect on the protein synthetic activities of free ribosomes. However, the membrane preparations from the livers of ethanol-treated animals had relatively greater inhibitory activity if compared with the similar preparation from the control livers. The observed effect of ethanol intake on liver protein synthesis in vitro could be almost completely reversed if the animals abstained from ethanol for a period of 4 weeks.

Isolation and characterization of membrane-bound ribosomes from nerve cell bodies of immature rat brain-cortex

Abstract Free and membrane-bound ribosomes were isolated from neuronal perikarya of the immature rat brain-cortex. The two topographic forms of ribosomes were essentially free of contaminating organelles as shown by RNA, protein and marker enzyme analysis. Membrane-bound ribosomes amount to about a quarter of the total ribosomal population in neuronal perikarya. Both forms of ribosomes efficiently carried out cell-free protein synthesis but the membrane-bound fraction was more active than the free ribosomes.

Preferential effect of oestradiol 17βinvivo on the protein synthetic activity of uterine membrane-bound ribosomes

Membrane-bound ribosomes were isolated and quantified in uterine tissue of ovariectomized rats. Oestrogen stimulation of {14C}phenylalanine incorporation on free and membrane-bound ribosomes was 70 % and 134 % respectively. The addition of poly U to the assay mixture reduced the hormonal stimulation of incorporation to about 23 % in both classes of ribosomes. Membrane-bound ribosomes were relatively less responsive to increase in {14C}phenylalanine incorporation due to the addition of poly U. Hormone treatment resulted in an increased proportion of membrane-bound ribosomes. The results suggest that membrane-bound ribosomes are more responsive to hormonal stimulus than are free ribosomes in rat uterus.

Gradual release of spermine and RNA from rat liver microsomes treated with EDTA

It has been previously shown that chelating agents such as EDTA [l-3] and pyrophosphate [4] can release between 50-60% RNA from hepatic microsomes. Sabatini et al. [3] have shown that the addition of increasing amounts of EDTA dissociates microsomes in a gradual fashion: the small subunits being the first to be released from the microsomal membranes. The polyamines, spermine and spermidine, are associated with the isolated ribosomes of both bacterial and animal origin [5-91 . Previous work from this laboratory has shown that polyamines such as spermine may be involved in the attachment of ribosomes to membranes of the endoplasmic reticulum [9, IO]. In our preliminary experiments reported earlier [ 111, no polyamines could be detected after treatment of microsomes with 15 mM EDTA, a condition which should release most of the small subunits from the microsomes. As this finding was apparently at variance with our more recent observations [9, lo] , it was decided to re-investigate the release of the polyamine spermine from the microsomes, under conditions that would gradually remove ribosomal subunits from the microsomal membranes. The present results show that when ribosomal subunits are gradually removed from the microsomes by treatment with EDTA, there is a concomitant release of endogenous spermine as well as of “C-spermine administered in uivo to the rat. The release of spermine closely followed that of RNA from the microsomes, both reaching a limiting value of about 60% at 50 mM EDTA.

Vectorial Discharge and Localization of Nascent Polypeptides in Rough Endoplasmic Reticulum of Developing Neuronal Perikarya of Rat Brain Cortex

Abstract: Rough endoplasmic reticulum (RER) prepared from bulk‐isolated neuronal perikarya of rat brain cortex of different postnatal ages was found to be active in vectorial discharge of nascent proteins through the membrane; this activity increased with the increasing age of animals and reached maximal values in adults. RER isolated from whole cortical tissue (containing all cell types) exhibited vectorial release only up to 18 days of age; the preparation from adult animals was essentially devoid of secretory activity. Controlled proteolysis of various preparations suggested that in neuronal RER of 8‐day‐old rats the proportion of nascent proteins operationally retained in the intravesicular space was about twice that retained by cortical preparations. For the purpose of comparison, these parameters were studied also in liver RER.

Effect of ethanol ingestion on the nucleocytoplasmic transport of hepatic RNA.

Influence of prolonged ethanol ingestion on the nucleocytoplasmic transport of RNA has been examined in a cell-free system. The nucleocytoplasmic transport of RNA was energy- and temperature-dependent. Ethanol treatment of rats for 10 weeks led to a significant increase in the release of isotopically labelled nuclear RNA from the nucleus, suggesting a partial loss of nuclear restrictive control. Results from crossover experiments led to the conclusion that the observed effect of ethanol ingestion was mediated through factors present in the cytosol as well as in the nuclear fraction.