Peter M. D. Hardwicke

A Ca2+-dependent Tryptic Cleavage Site and a Protein Kinase A Phosphorylation Site Are Present in the Ca2+ Regulatory Domain of Scallop Muscle Na+-Ca2+ Exchanger

Digestion of scallop muscle membrane fractions with trypsin led to release of soluble polypeptides derived from the large cytoplasmic domain of a Na+-Ca2+exchanger. In the presence of 1 mm Ca2+, the major product was a peptide of ∼37 kDa, with an N terminus corresponding to residue 401 of the NCX1 exchanger. In the presence of 10 mm EGTA, ∼16- and ∼19-kDa peptides were the major products. Polyclonal rabbit IgG raised against the 37-kDa peptide also bound to the 16- and 19-kDa soluble tryptic peptides and to a 105–110-kDa polypeptide in the undigested membrane preparation. The 16-kDa fragment corresponded to the N-terminal part of the 37-kDa peptide. The conformation of the precursor polypeptide chain in the region of the C terminus of the 16-kDa tryptic peptide was thus altered by the binding of Ca2+. Phosphorylation of the parent membranes with the catalytic subunit of protein kinase A and [γ-32P]ATP led to incorporation of 32P into the 16- and 37-kDa soluble fragments. A site may exist within the Ca2+ regulatory domain of a scallop muscle Na+-Ca2+ exchanger that mediates direct modulation of secondary Ca2+ regulation by cAMP.

Effect of phosphorylation on scallop sarcoplasmic reticulum

SummaryFragmented sarcoplasmic reticulum prepared from the cross-striated adductor muscle of the deep sea scallop (Placopecten magellanicus) was phosphorylated with inorganic phosphate to the E2P (ADP-insensitive) form. Negative staining of these preparations showed that the Ca-ATPase was organized into a quasi-crystalline array, which differed from the ‘dimer ribbon’ structure previously reported for the membrane under relaxing conditions (Castellani & Hardwicke,J. cell. Biol.97 (1983) 557–61; Castellaniet al., J. molec. Biol.185 (1985) 579–94). In this new form there was only a single Ca-ATPase per unit cell. Dephosphorylation of the E2P membranes and incubation with substrate or substrate analogues in the absence of Ca2+ caused the ‘dimer ribbon’ structure to appear. These results imply that rotation of at least half of the Ca-ATPase subunits in the scallop sarcoplasmic reticulum may occur about an axis perpendicular to the plane of the membrane on conversion from the E2P state to the state corresponding to that existing in the relaxed muscle.

Experimental leprosy: a model of epithelioid cell granuloma

An animal model of granulomatous hypersensitivity has been developed, which reproduces some features of the pathologies of important chronic granulomatous disorders, including tuberculosis, tuberculoid leprosy, sarcoidosis, berylliosis, Crohn’s disease, and sensitivity to zirconium. The lesions consist of focal collections of epithelioid cells surrounded by lymphocytes to form tubercles. The epithelioid cell has a secretory function and is not phagocytic. Plasmacytoid dendritic cells are precursors of epithelioid cells, which are therefore part of the innate immune system. Subplasmalemmal linear densities are also present in these cells. This autoimmune model has been induced in rabbits using a non‐myelin sensory peripheral antigen to reproduce the features of tuberculoid leprosy. The antigen is probably present only in human tissue. A granuloma antigen, which is tissue specific similar to that in peripheral nerves, could be present in sarcoidosis and Crohn’s disease. In multiple sclerosis, mononuclear cells in the brain parenchyma are not phagocytic and are therefore similar to epithelioid cells. The induction of tolerance leading to the development of a vaccine to prevent the lesions in multiple sclerosis, sarcoidosis, and Crohn’s disease is possible after purification of the granuloma antigen.

Effect of K+, and other ligands on the thiol reactivity and tryptic cleavage pattern of scallop sarcoplasmic reticulum

SummaryThe kinetics of reaction of the thiol groups of both membranous and non-ionic detergent-solubilized Ca-ATPase of scallop sarcoplasmic reticulum towards 5,5′-dithiobis(2-nitrobenzoate) were greatly modified in different ways by the presence of the following combinations of ligands: Ca2+, EGTA (no Ca2+), (ATPMg2−+EGTA) and (ATP+Ca2+). K+ was found to influence greatly the pattern of reactivity of the thiol groups of the scallop Ca-ATPase, modifying the kinetics of reaction differently according to the types of other ligand present. While all the thiol groups on the non-ionic detergent-solubilized Ca-ATPase were available for reaction in the absence of K+, whatever the combination of ligands, in the presence of K+, several groups became completely unreactive towards the reagent. In some cases the rate of inactivation of Ca-ATPase activity could be related to the rates of reaction of different kinetic classes of thiol group, according to the ligands present. Large differences were also seen in the tryptic cleavage pattern in the presence of the different ligands, and K+ led to major modifications in the products of digestion in the absence of nucleotide. An 80 kDa tryptic fragment was observed, as with lobster Ca-ATPase but unlike rabbit skeletal muscle Ca-ATPase. A complete amino-acid analysis of the scallop Ca-ATPase was carried out. Differences in the non-polar amino-acid content from rabbit skeletal muscle Ca-ATPase may relate to the different lipid composition of the two membranes.

Effect of Orthophosphate, Nucleotide Analogues, ADP, and Phosphorylation on the Cytoplasmic Domains of Ca2+-ATPase from Scallop Sarcoplasmic Reticulum

The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E1 and E2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E2 (Ca2+-free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca2+-dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E1 form of scallop Ca-ATPase was occupied by Pi, AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca2+. These results suggest that occupation of the catalytic site on E1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of Pi on the E2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E2-P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time.

Effect of the biochemical state of the Ca-ATPase protein of scallop sarcoplasmic reticulum on its interaction with trans-parinaric acid.

The polyene fluorescent probe trans-parinaric acid (tPA) was used to compare lipid-protein interactions in the scallop fragmented sarcoplasmic reticulum (FSR) between biochemical states where the Ca-ATPase molecules were arranged differently in the membrane and had different tertiary conformations. The state of the bulk lipid phase was examined over the temperature range -3 to +32 degrees C by exciting the tPA directly at 320 nm. The state of the system close to the Ca-ATPase protein was followed over the same temperature range by indirectly exciting the tPA through resonance energy transfer from the Ca-ATPase protein, with approximately one twenty-fifth the quantum yield of the directly excited probe. Raising the tPA/lipid ratio in the membrane to high levels (approx. 1:9), caused the quantum yield of indirectly excited tPA to reach a maximum, which may reflect saturation of the annular lipid phase with the probe, or contribution to the fluorescence from indirectly excited tPA bound directly to the protein. In the presence of 0.1 M KCl, a thermal perturbation was observed at approx. 7 degrees C using indirect excitation when the Ca(2+)-binding sites on the Ca-ATPase were occupied, and the subunits were disorganized. This transition was not detected in the presence of 0.1 M KCl and EGTA, when the Ca(2+)-binding sites were empty, and the Ca-ATPase subunits were organized in dimeric arrays. The transition seen with the E1(Ca2+)2 form of the membrane may involve an event at the protein/lipid interface, or a change in the environment of tPA bound to the Ca-ATPase. The temperature at which the perturbation occurs is close to that of a discontinuity in the Arrhenius plot of the Ca-ATPase enzyme activity determined in the presence of 0.1 M KCl (Kalabokis, V.N. and Hardwicke, P.M.D. (1988) J. Biol. Chem. 263, 15184-15188). No perturbation was observed in the bulk properties of the lipid component of the membrane in either the E1(Ca2+)2 or E2 states.

A novel small protein associated with a conjugated trienoic chromophore from membranes of scallop adductor muscle: phosphorylation by protein kinase A

Membranes enriched in sarcolemma from the cross-striated adductor muscle of the deep sea scallop have been found to contain a previously undescribed small protein of 6-8 kDa that can be released by treatment with organic solvent mixtures. This proteolipid co-purified with a non-amino acid chromophore containing a conjugated trienoic moiety. Although common in plants and algae, such a stable conjugated trienoic group is unusual for an animal cell. The N-terminal amino acid sequence of the protein was XEFQHGLFGXF/ADNIGLQ, which most strongly resembles sequences in the triacyl glycerol lipase precursor and the product of the human breast cancer susceptibility gene BRCA 1, but does not show similarity to previously described proteolipids. The protein was found to be one of the major substrates in its parent membrane for the catalytic subunit of protein kinase A, which may imply a regulatory function for this molecule.

A hydrophobic region on myosin light chains modulated by divalent cations

A hydrophobic region was detected on several types of myosin light chain by enhancement of the quantum yield of 1-anilino-8-naphthalenesulfonate (ANS) fluorescence. The character of this non-polar region was altered by the binding of Ca2+ or Mg2+ to the light chain, the quantum yield of the ANS being increased, and its emission maximum undergoing a blue-shift. These changes enabled the binding of divalent cations to the myosin light chains to be monitored. When Ca2+ was bound to gizzard regulatory light chain, a biphasic enhancement of light-chain-bound ANS fluorescence occurred, the first phase taking place in the micromolar range and the second in the millimolar range of free Ca2+ concentration. Enhancement of protein-bound ANS fluorescence as divalent cations were bound was also observed with other types of myosin light chain.

Plasma cells or plasmacytoid dendritic cells in sarcoidosis

To the Editor, In the discussion included in their report of Etanercept-induced cutaneous and pulmonary sarcoid-like granulomas resolving with adalimumab, the authors state that ‘‘Histopathologically, sarcoidal granulomata show aggregates of epithelioid histiocytes and giant cells surrounded by a scant infiltrate of lymphocytes and plasma cells.’’1 In descriptions of sarcoid granulomas, plasma cells are not usually mentioned, with two notable exceptions, both in neurosarcoidosis.2,3 We have suggested that these ‘plasma cells’ may in fact be plasmacytoid dendritic cells4 and that sarcoidosis may be an autoimmune disorder involving the innate immune system.5 The problem can be resolved by staining the cells with an antibody to CD123.

Treating Multiple Sclerosis and Cerebral palsy with Choroquine

neurologic outcomes. (−)-Epicatechin (EC), a flavanol enriched in cocoa and green tea, modulates redox/oxidative stress by activating the NF-E2-related factor (Nrf) 2/antioxidant response element pathway. In this study,we examinedwhether EC can reduce early brain injury in ICH models and evaluated the underlying mechanisms of action. Oral administration of EC at 3 h after ICH and then every 24 h for 3 days significantly reduced lesion volume and ameliorated neurologic deficits in both male and female mice. After EC administration, cell death and neuronal degeneration were decreased in the perihematomal area and were associated with a similar reduction in caspase-3 activity and HMGB-1 level. These changes were accompanied by attenuation of oxidative insults, increased phase II enzyme gene expression, and increased Nrf2 nuclear accumulation. Interestingly, in addition to providing neuroprotection via Nrf2 signaling, EC administration diminished heme oxygenase-1 induction and brain iron deposition via an Nrf2-independent pathway after ICH. Furthermore, EC downregulated ICH-induced activating protein-1 activation and decreased matrix metalloproteinase 9 activity, lipocalin-2 protein levels, iron-dependent cell death, and mRNA expression of ferroptosis-related genes. Collectively, our data show that EC exerts neuroprotective effects after ICH by activation of Nrf2-dependent and -independent pathways. Thus, EC may serve as a potential intervention for patients with ICH.