Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca 2؉ -ATPase in the course of its catalytic cycle were followed in real time using rapid scan Fourier transform infrared spectroscopy. In the presence of Ca 2؉ , the cycle was induced by the photochemical release of ATP from a biologically inactive precursor (caged ATP, P 3 -1-(2-nitro)phenylethyladenosine 5-triphosphate). Absorbance changes arising from ATP binding to the ATPase were observed within the first 65 ms after initiation of ATP release. After ATP binding, up to two subsequent partial reactions of the ATPase reaction cycle were observed depending on the buffer composition (10 mM CaCl 2 ؉ 330 mM KCl or 1 mM CaCl 2 ؉ 20% Me 2 SO): (i) formation of the ADP-sensitive phosphoenzyme (k app ‫؍‬ 0.79 s ؊1 ؎ 15% at 1°C, pH 7.0, 10 mM CaCl 2 , 330 mM KCl) and (ii) phosphoenzyme conversion to the ADP-insensitive phosphoenzyme concomitant with Ca 2؉ release (k app ‫؍‬ 0.092 s ؊1 ؎ 7% at 1°C, pH 7.0, 1 mM CaCl 2 , 20% Me 2 SO). Each reaction step could well be described by a single time constant for all associated changes in the vibrational spectrum, and no intermediates other than those mentioned above were found. In particular, there is no evidence for a delay between the transition from ADP-sensitive to ADP-insensitive phosphoenzyme and Ca 2؉ release. In 2 H 2 O a kinetic isotope effect was observed: both the phosphorylation reaction and phosphoenzyme conversion were slowed down by factors of 1.5 and 3.0, respectively.The small amplitudes of the observed changes in the infrared spectrum indicate that the net change of secondary structure is very small and of the same order of magnitude for ATP binding, phosphorylation, and phosphoenzyme conversion. Therefore, our results do not support a distinction between minor and major secondary structure changes in the catalytic cycle of the ATPase, which might be expected according to the classical E 1 -E 2 model. -transport across the SR membrane to the hydrolysis of ATP. Its reaction cycle is shown in a simplified form in Fig. 1. Ca 2ϩ is bound from the cytoplasmic side of the membrane to high affinity binding sites of the ATPase (step on the left of Fig. 1), which enables the ATPase to use ATP as a substrate (1). Phosphorylation by ATP (upper step in Fig. 1) results in the occlusion of the bound Ca 2ϩ in the protein. The subsequent conversion of the phosphoenzyme from the ADP-sensitive to the ADP-insensitive form (step on the right of Fig. 1) leads to Ca 2ϩ release into the SR lumen. Hydrolytic cleavage of the phosphoenzyme completes the reaction cycle (bottom step in Fig. 1). An ATP molecule is shown bound to the ATPase throughout the cycle, which is the case at millimolar ATP concentrations (reviewed in Ref.2).The original model of the reaction cycle from deMeis and Vianna (3) was based on the assumption of two main functional states, E 1 and E 2 , of the protein. The interconversion between E 1 and E 2 is thought to be associated with a reorientation of the Ca 2ϩ -binding sites from the cytoplasmic s...

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding were recorded in H(2)O and (2)H(2)O at -7 degrees C and pH 7.0. The reaction cycle was triggered by the photochemical release of nucleotides (ATP, ADP, and AMP-PNP) from a biologically inactive precursor (caged ATP, P(3)-1-(2-nitrophenyl) adenosine 5'-triphosphate, and related caged compounds). Infrared absorbance changes due to ATP release and two steps of the Ca(2+)-ATPase reaction cycle, ATP binding and phosphorylation, were followed in real time. Under the conditions used in our experiments, the rate of ATP binding was limited by the rate of ATP release (k(app) congruent with 3 s(-1) in H(2)O and k(app) congruent with 7 s(-1) in (2)H(2)O). Bands in the amide I and II regions of the infrared spectrum show that the conformation of the Ca(2+)-ATPase changes upon nucleotide binding. The observation of bands in the amide I region can be assigned to perturbations of alpha-helical and beta-sheet structures. According to similar band profiles in the nucleotide binding spectra, ATP, AMP-PNP, and ADP induce similar conformational changes. However, subtle differences between ATP and AMP-PNP are observed; these are most likely due to the protonation state of the gamma-phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the gamma-phosphate group in the interactions between the Ca(2+)-ATPase and the nucleotide. Nucleotide binding affects Asp or Glu residues, and bands characteristic of their protonated side chains are observed at 1716 cm(-1) (H(2)O) and 1706 cm(-1) ((2)H(2)O) and seem to depend on the charge of the phosphate groups. Bands at 1516 cm(-1) (H(2)O) and 1514 cm(-1) ((2)H(2)O) are tentatively assigned to a protonated Tyr residue affected by nucleotide binding. Possible changes in Arg, Trp, and Lys absorption and in the nucleoside are discussed. The spectra are compared with those of nucleotide binding to arginine kinase, creatine kinase, and H-ras P21.

A microsystem integrating electrochemical detection for the simultaneous detection of protein markers of breast cancer is reported. The microfluidic platform was realized by high precision milling of polycarbonate sheets and features two well distinguishable sections: a detection zone incorporating the electrode arrays and the fluid storage part. The detection area is divided into separate microfluidic chambers addressing selected electrodes for the measurement of samples and calibrators. The fluidic storage part of the platform consists of five reservoirs to store the reagents and sample, which are interfaced by septa. These reservoirs have the appropriate volume to run a single assay per cartridge and are manually filled. The liquids from the reservoirs are actuated by applying a positive air pressure (i.e.via a programmable syringe pump) through the septa and are driven to the detection zone via two turning valves. The application of the realised platform in the individual and simultaneous electrochemical detection of proteic cancer markers with very low detection limits are demonstrated. The microsystem has also been validated using real patient serum samples and excellent correlation with ELISA results obtained.

Changes in the vibrational spectrum of the chaperonin GroEL in the presence of ADP and ATP have been followed as a function of time using rapid scan Fourier transform infrared spectroscopy. The interaction of nucleotides with GroEL was triggered by the photochemical release of the ligands from their corresponding biologically inactive precursors (caged nucleotides; P 3 -1-(2-nitro)phenylethyl nucleotide). Binding of either ADP or ATP induced the appearance of small differential signals in the amide I band of the protein, sensitive to protein secondary structure, suggesting a subtle and localized change in protein conformation. Moreover, conformational changes associated with ATP hydrolysis were detected that differed markedly from those observed upon nucleotide binding. Both, high-amplitude absorbance changes and difference bands attributable to modifications in the interaction between oppositely charged residues were observed during ATP hydrolysis. Once this process had occurred, the protein relaxed to an ADP-like conformation. Our results suggest that the secondary structure as well as salt bridges of GroEL are modified during ATP hydrolysis, as compared with the ATP and ADP bound protein states.