Ryanodine-affinity chromatography purifies 106 kD Ca release channels from skeletal and cardiac sarcoplasmic reticulum

A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the 'physiological' Ca2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca2+ release channels which are a minor component (0.1-0.3%) of SR proteins.     

1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain

The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0–7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ∼80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required.     

In vitro regeneration of Vigna unguiculata (L.) Walp. cv. Blackeye cowpea via shoot organogenesis

An in vitro regeneration system was developed in cowpea [Vigna unguiculata (L.) Walp.] Blackeye. Among several explants studied, shoot initiation response was observed from shoot apices of 3–5-day-old seedlings. The optimal medium for maximum shoot initiation comprised MS salts, B₅ vitamins, 8.88 μM N ⁶-benzylaminopurine, 1 gl^-1 casein hydrolysate, 342 μM L-glutamine, 3% sucrose, 0.3% phytagel, adjusted to pH 5.8. A shift in pH from 5.8 to 7.0 had no effect on shoot initiation and on number of shoots per explant. The highest shoot initiation frequency (77%) was obtained using this preferred medium, reaching a maximum of eight shoots per explant. For shoot elongation, 14 μM gibberellic acid was supplemented in the shoot initiation medium. Presence of indolebutyric acid in the rooting medium had no effect on root induction. The regenerated plants were fertile and developed normally.     

Special stem cells for bone

Mesenchymal stem cells (MSCs) are multipotential in vitro, but their endogenous properties are poorly defined. In this issue of Cell Stem Cell, Park et al. (2012) report that an MSC-like, osteolineage-directed Mx1+ population generates new osteoblasts at sites of bone damage, suggesting its potential for skeletal repair and regeneration.     

Emerging functional roles of cathepsin E

Cathepsin E is an intracellular aspartic protease of the endolysosomal pathway. It has been implicated in several physiological and pathological processes however, its exact functional role is yet to be elucidated. The present review gives an account of the major physiological functions that are associated to cathepsin E by various research groups and highlights the conditions developed in cathepsin E deficiency or the conditions where overexpression of cathepsin E is observed.     

'Calcium-activated' intracellular calcium elevation: a novel mechanism of osteoclast regulation

The osteoclast is unique in its capacity to resorb bone. An unbalanced increase in this activity causes osteoporosis, a crippling bone disease that poses a major public health problem. Despite this, our understanding of osteoclast regulation is very limited. Calcitonin is the only known physiological inhibitor of osteoclast function. We demonstrate here for the first time that the concentration of calcium ions at the resorptive site directly regulates osteoclast function by modulating the intracellular free calcium concentration. This represents an important feedback mechanism of osteoclast control.     

Selective antagonism of calcitonin-induced osteoclastic quiescence (Q effect) by human calcitonin gene-related peptide-(Val8Phe37)

Exposure of isolated rat osteoclasts to calcitonin (CT) leads to an abrupt cessation of cell motility (Q effect) followed by cell retraction (R effect). We have previously shown that these effects are mediated by two G proteins that appear to activate separate post-receptor pathways. The present study demonstrates that the Q but not the R effect of CT (0.006 microM) is abolished in the presence of human calcitonin gene-related peptide (CGRP)-(Val8Phe37) (0.5 microM), a fragment analogue of human CGRP. This selective antagonism suggests that the Q effect could result from an action of CT upon a site that is distinct from that producing the R effect. The former site ('amylin site') also appears to interact with related peptides, amylin and CGRP, whilst the latter site ('CT site') specifically interacts with CT.