The activation mechanism of Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) is investigated by steady-state and stopped-flow fluorescence spectroscopies. Lys(75)-labeled TA-cal [Török, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820] is used to measure binding events, and double-labeled AEDANS,DDP-T34C/T110/C-calmodulin [Drum et al. (2000) J. Biol. Chem. 275, 36334-36340] (DA-cal) is used to detect changes in calmodulin conformation. Fluorescence quenching of DA-cal attributed to resonance energy transfer is related to the compactness of the calmodulin molecule. Interprobe distances are estimated by lifetime measurements of Ca(2+)/DA-cal in complexes with unphosphorylated nucleotide-free, nucleotide-bound, and Thr(286)-phospho-alphaCaMKII as well as with alphaCaMKII-derived calmodulin-binding peptides in the presence of Ca(2+). These measurements show that calmodulin can assume at least two spectrally distinct conformations when bound to alphaCaMKII with estimated interprobe distances of 40 and 22-26 A. Incubation with ATP facilitates the assumption of the most compact conformation. Nonhydrolyzable ATP analogues partially replicate the effects of ATP, suggesting that while the binding of ATP induces a conformational change, Thr(286)-autophosphorylation is probably required for the transition of calmodulin into its most compact conformer. The rate constant for the association of Ca(2+)/TA-cal with alphaCaMKII is estimated as 2 x 10(7) M(-1) s(-1) and is not substantially affected by the presence of ATP. The rate of net calmodulin compaction measured by Ca(2+)/DA-cal is markedly slower, occurring with a rate constant of 2.5 x 10(6) M(-1) s(-1), suggesting that unproductive complexes may play a role in the activation mechanism.

Gold drugs are still amongst the most efficacious for the treatment of rheumatoid arthritis. Their mechanism of action, as well as the molecular basis of their side-effects, remain poorly understood. Current theories are reviewed, including recent potential breakthroughs. The interaction of gold(III) with peptides and proteins and its immunochemical implications are discussed.Ninety elements occur naturally on earth of which 9 are radioactive. Eighty-one elements are therefore potentially available to support life, of which 61 are metals. It is believed that about 25 are essential for human life but our knowledge of the biochemistry of several of these (e.g.V; Ni, Sn) is poor. In general, therefore, there is enormous scope for the use of inorganic compounds in medicine, and a need for research in this area. Metals currently used in medicine include gold in antiarthritic agents, platinum in anticancer drugs, lithium for manic depression, bismuth in anti-ulcer drugs, and silver and mercury in antimicrobial agents (1). The effectiveness of ruthenium complexes as anti-metastatic agents with potential use in cancer therapy is currently receiving much attention (2). However, the potential of inorganic metal compounds as drugs has yet to be fully explored. With the exception of platinum, which has attracted the most research efforts because of its importance in the therapy of some types of cancer, and which is now generally thought to be effective by interaction with DNA, the mechanism of action of other inorganic drugs is mostly unknown.One of the problems of inorganic drugs is their side-effects. For gold drugs used in the therapy of * Hypersensitivity is an adaptive immune response occurring in an exaggerated or inappropriate form causing tissue damage. It is a characteristic of the individual and is manifested on second contact with the particular antigen (in this case gold drugs). Delayed-type hypersensitivity takes more than 12 hours to develop and is mediated primarily by T cell and macrophages.

Proton NMR studies show that [AuCl 4 ] Ϫ reacts slowly with glycylglycyl--histidine (Gly-Gly--His) (t ₂  ₁ = 9.3 h at 310 K and pH* 2) in D 2 O at pH* (meter reading) values as low as 1.5 to form the stable complex [Au III (Gly-Gly--His-H Ϫ2 )]ClؒH 2 O 1 via one intermediate. Complex 1 is shown by X-ray crystallography to be square-planar with gold bound to the terminal NH 2 [Au᎐N 2.049(10) Å], two deprotonated amide nitrogens [Au᎐N Ϫ 1.941(9), 2.006(10) Å] and HisδN [Au᎐N 2.038(9) Å] giving one six-membered and two five-membered chelate rings. At pH* 7 the reaction of [AuCl 4 ] Ϫ with Gly-Gly--His follows a different course, apparently involving the formation of Au III cross-linked polymers. The anion [PdCl 4 ] 2Ϫ reacts rapidly with Gly-Gly--His also at pH* 2, and forms a similar square-planar complex [Pd II (Gly-Gly--His-H Ϫ2 )]ؒ1.5H 2 O 2 involving the terminal NH 2 [Pd᎐N 2.058(7) Å], two deprotonated amide nitrogens [Pd᎐N Ϫ 1.943(7), 1.983(6) Å] and HisδN [Pd᎐N 2.016(6) Å]. By potentiometry, pK a values of 2.58 (CO 2 H), 8.63 (HisεNH, 'pyrrole nitrogen') and 11.50 (co-ordinated NH 2 ) for 1 and 11.30 (HisεNH) for 2 were determined and confirmed by 1 H NMR spectroscopy.

Thr(286) autophosphorylation is important for the role of alphaCaMKII in learning and memory. Phospho-Thr(286)-alphaCaMKII has been described to have two types of activity: Ca(2+)-independent partial activity and Ca(2+)/calmodulin-activated full activity. We investigated the mechanism of switching between the two activities in order to relate them to the physiological functioning of alphaCaMKII. Using a fluorometric coupled enzyme assay and smooth muscle myosin light chain (MLC) as substrate, we found that (1) Ca(2+)-independent activity of phospho-Thr(286)-alphaCaMKII represents 5.0 (+/-3.7)% of the activity measured in the presence of optimal concentrations of Ca(2+) and calmodulin and (2) Ca(2+) in the presence of calmodulin activates the enzyme with a K(m) of 137 (+/-56) nM and a Hill coefficient n = 1.8 (+/-0.3). In contrast, unphosphorylated alphaCaMKII has a K(m) for Ca(2+) in the presence of calmodulin of 425 (+/-119) nM and a Hill coefficient n = 5.4 (+/-0.4). Thus, the activity of phospho-Thr(286)-alphaCaMKII is essentially Ca(2+)/calmodulin dependent with MLC as substrate. In physiological terms, our data suggest that alphaCaMKII is only activated in stimulated neurones whereas Ca(2+)/calmodulin activation of phospho-Thr(286)-alphaCaMKII can occur in resting cells (approximately 100 nM [Ca(2+)]). Stopped-flow experiments using Ca(2+)/TA-cal [Ca(2+)/2-chloro-(epsilon-amino-Lys(75))-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin] showed that at 100 nM [Ca(2+)] partially Ca(2+)-saturated Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII complexes existed. These are likely to account for the activity of the phospho-Thr(286)-alphaCaMKII enzyme at resting [Ca(2+)]. Ca(2+) dissociation measurements by a fluorescent Ca(2+) chelator revealed that the limiting Ca(2+) dissociation rate constants were 1.5 s(-1) from the Ca(2+)/cal.alphaCaMKII and 0.023 s(-1) from the Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII complex, accounting for the differences in the Ca(2+) sensitivities of the Ca(2+)/cal.alphaCaMKII and Ca(2+)/cal.phospho-Thr(286)-alphaCaMKII enzymes.

The mechanisms by which metals induce activation of T cells and thus produce allergic and/ or autoimmune reactions are still obscure, and the same is true for the mechanisms that underly T cell cross-reactivity to different heavy metal ions. In the present study, we investigated induction by metals of T cell reactions to cryptic peptides of bovine RNase A. Murine CD4+ T cell hybridomas specific for cryptic RNase peptides presented from Au(III)-treated RNase were used as detection probes. We showed that in vitro treatment of RNase with Pd(II), Pd(IV), Ni(IV), and partially Pt(IV), but not Au(I), Ni(II), or Pt(II), induced presentation of the same cryptic peptides as those presented from Au(III)-treated RNase. That the former heavy metal ions, but not the latter, were able to alter the antigenicity of RNase was reflected by their ability to induce conformational changes of RNase, as detected by circular dichroism spectroscopy. Furthermore, upon immunization against RNase pretreated with these metals, CD4+ T cell hybridomas specific for unidentified cryptic peptides were obtained. In conclusion, "metal-specific" T cell reactions may be directed against cryptic peptides, and metal cross-reactivity in allergic individuals might be due to metal-induced presentation of overlapping, but not identical, panels of cryptic peptides.

The solution behaviour of the square-planar gold (Ill)

No abstract

Intramuscular chrysotherapy is a well-established treatment for rheumatoid arthritis. Its therapeutic use has been limited by the high incidence of dermatological side-effects. The pathogenic mechanisms of these are unknown, but could include allergic reactions to gold or to nickel contaminating the gold. In order to investigate these mechanisms further, 15 patients, who developed cutaneous eruptions after chrysotherapy, were assessed using skin biopsy and lymphocyte transformation stimulated by gold and nickel salts in vitro. Chrysotherapy induced two main cutaneous eruptions: lichenoid reactions and non-specific dermatitis. Peripheral blood mononuclear cells from patients with lichenoid reaction proliferated to gold salts in vitro, while those who developed non-specific dermatitis responded mainly to nickel. Nickel was a significant contaminant of the gold preparation (sodium aurothiomalate, Myocrisin, Rhone-Poulenc Ltd), amounting to a total of 650 ng after 6 months treatment. We suggest that a significant percentage of skin reactions during chrysotherapy are due to nickel contamination of the gold preparation.