Nutrition and muscle potassium: differential effect in rat slow and fast muscles

The effects of malnutrition on intracellular K+ activity, (alpha K)i, and membrane potential, Em, were measured by means of double-barrelled K+-selective microlectrodes in the soleus and gastrocnemius muscles of the rat. (alpha K)i and Em were measured in vivo in normal anaesthetized animals and in rats subjected to one of two diet restrictions: a 2-day fast or a long-term hypocaloric diet. In the soleus muscle, (alpha K)i fell by similar amounts in both 2-day fasted and long-term hypocalorically fed rats, while Em depolarized significantly only in hypocalorically fed rats. In the gastrocnemius muscle, neither the 2-day fast nor the hypocaloric diet affected (alpha K)i or Em. It is suggested that the selective loss of K+ from the soleus muscle may be related to its activity pattern.     

Abscisic Acid ELISA: Organic Acid Interference

Consideration must be exercised in determination of buffers and solutions used when carrying out enzyme-linked immunosorbent assays (ELISAs). A commercial monoclonal antibody kit for abscisic acid (Idetek, Inc.) gives significant false-positives with tricarboxylic acid cycle intermediates. The organic acids or contaminants interfered with ELISA assays for ABA as indicated by deviations in the slopes of standard curves of ABA in the organic acids. The interference, in the case of α-ketoglutarate, was caused by a contaminant. Of the organic buffers tested—Tris, Tricine, and Hepes—only Hepes showed false-positive ABA. In addition, we present data indicating the presence of ABA in commercial mannitol and provide a simple procedure for removal of the ABA.     

Forskolin stimulation of pepsinogen secretion from frog esophageal mucosa is partly mediated by intrinsic cholinergic neurons

Forskolin was found to stimulate pepsinogen secretion from frog esophageal mucosa. The stimulation was dose-dependent and was accompanied with a great increase in tissue cAMP content. The response to forskolin mimicked the action of bethanechol and was not additive with bethanechol. The stimulatory effect of forskolin was inhibited by 50% in the presence of either atropine or tetrodotoxin. On the other hand, incubation in a calcium-free medium not only reduced the response to forskolin by 45% but also eliminated the influence of atropine and tetrodotoxin. These results indicate that forskolin may stimulate pepsinogen secretion from the frog esophageal mucosa via activating adenylate cyclase, and part of its effect may arise from eliciting acetylcholine release from the intrinsic neurons.     

Incidence of three cross-reactive idiotypes on human rheumatoid factor paraproteins

The basis for rheumatoid factor (RF) production in autoimmune or lymphoproliferative diseases cannot be understood without defining the molecular factors that dictate RF structure and specificity. Recently three different mAb (6B6.6, 17.109, and G6) have been developed that define cross-reactive idiotypes (CRI) on intact L or H chains of human monoclonal RF cryoglobulins. However, the true incidence of these CRI among RF and their relationship to each other have not been delineated. In the present experiments, a panel of 163 randomly selected IgM paraproteins was evaluated for the expression of the two kappa L chain CRI, 6B6.6 and 17.109, and the H chain CRI, G6. Among the paraproteins with kappa L chains, 14% expressed the 17.109 CRI, and 9% expressed the 6B6.6 CRI. Both ELISA and Western immunoblotting experiments showed that the two L chain CRI were mutually exclusive. Anti-IgG activity was documented in 22 of the IgM-kappa paraproteins, among which mAb 6B6.6 reacted with 7 (32%) and mAb 17.109 with 6 (27%). Both CRI were expressed exclusively by L chains within the kappaIII variable gene subgroup. Although 17.109 CRI+ paraproteins had kappaIIIb L chains, none of the 6B6.6 CRI+ paraproteins possessed L chains with this kappa sub-subgroup specific Ag. The G6 CRI was found predominantly among RF paraproteins and was frequently yet not exclusively associated with the 17.109 CRI+ L chains. Additional experiments were performed on a panel of normal adult human sera and documented the presence of 6B6.6 and 17.109 CRI on a small percentage (0.1 to 2.0%) of IgM from most individuals. These data indicate that 1) the mAb 6B6.6 and 17.109 identify two major and distinct CRI among IgM-RF paraproteins, 2) both CRI are associated exclusively with kappaIII L chains, 3) kappaIIIb and kappaIII non-b L chains are equally prevalent among IgM-RF, 4) the G6 H chain CRI is frequently associated with 17.109 CRI+ L chains, but not with 6B6.6 CRI+ L chains, and 5) although the ability to make 6B6.6 and 17.109 CRI+ kappa L chains is common in humans, these CRI are present in low concentrations in normal IgM.     

Dephosphorylation of the beta 2-adrenergic receptor and rhodopsin by latent phosphatase 2

Recent evidence suggests that the function of receptors coupled to guanine nucleotide regulatory proteins may be controlled by highly specific protein kinases, e.g. rhodopsin kinase and the beta-adrenergic receptor kinase. In order to investigate the nature of the phosphatases which might be involved in controlling the state of receptor phosphorylation we studied the ability of four highly purified well characterized protein phosphatases to dephosphorylate preparations of rhodopsin or beta 2-adrenergic receptor which had been highly phosphorylated by beta-adrenergic receptor kinase. These included: type 1 phosphatase, calcineurin phosphatase, type 2A phosphatase, and the high molecular weight latent phosphatase 2. Under conditions in which all the phosphatases could dephosphorylate such common substrates as [32P]phosphorylase a and [32P]myelin basic protein at similar rates only the latent phosphatase 2 was active on the phosphorylated receptors. Moreover, a latent phosphatase activity was found predominantly in a sequestered membrane fraction of frog erythrocytes. This parallels the distribution of a beta-adrenergic receptor phosphatase activity recently described in these cells (Sibley, D. R., Strasser, R. H., Benovic, J. L., Daniel, K., and Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9408-9412). These data suggest a potential role for the latent phosphatase 2 as a specific receptor phosphatase.     

Possible role of the membrane Na+/H+ antiport in ornithine decarboxylase induction by L-asparagine

Amino acids of transport systems A and N play certain important role in cell activation. For example, the presence of these amino acids is essential in the induction of ornithine decarboxylase by growth factors and hormones. At mM concentrations, each of these amino acids, particularly L-asparagine, can also induce the enzyme without being further metabolized or incorporated into proteins. We have reported that the addition of 10 mM L-asparagine to quiescent Reuber's H-35 rat hepatoma cells caused an immediate and transient increase in intracellular pH. Here we report that concomitant with the intracellular alkalinization was an increase in H+ extrusion which was amiloride-sensitive and Na+-dependent. The induction of ornithine decarboxylase by L-asparagine was also amiloride-sensitive.     

Calcium binding to tubulin

Using flow dialysis, we found two classes of calcium-binding sites on tubulin: high-affinity binding sites (1.56 +/- 0.38 per tubulin dimer) with a dissociation constant of (4.86 +/- 0.12).10(-6) M and low-affinity binding sites (5.82 +/- 0.50 per tubulin dimer) with a dissociation constant of (6.4 +/- 0.4).10(-5) M. In the presence of 6.10(-5) M MgSO4, we found 0.64 +/- 0.18 calcium-binding sites per tubulin dimer with a dissociation constant of (4.7 +/- 0.5).10(-6) M and 1.2 +/- 0.2 sites per dimer with a dissociation constant of (3.5 +/- 0.4).10(-5) M. Under controlled conditions, trypsin and chymotrypsin selectively cleaved alpha- and beta-subunits, respectively, forming major fragments of 35 kDa and 20 kDa from the alpha-subunit, and major fragments of 31 kDa and 22 kDa from the beta-subunit. The high-affinity calcium-binding sites were detected in the carboxyl-terminal region of each tubulin subunit. Computer analysis of the subunit amino-acid sequences suggested possible locations of the putative calcium-binding sites.