Statistical clumped isotope signatures.

High precision measurements of molecules containing more than one heavy isotope may provide novel constraints on element cycles in nature. These so-called clumped isotope signatures are reported relative to the random (stochastic) distribution of heavy isotopes over all available isotopocules of a molecule, which is the conventional reference. When multiple indistinguishable atoms of the same element are present in a molecule, this reference is calculated from the bulk (≈average) isotopic composition of the involved atoms. We show here that this referencing convention leads to apparent negative clumped isotope anomalies (anti-clumping) when the indistinguishable atoms originate from isotopically different populations. Such statistical clumped isotope anomalies must occur in any system where two or more indistinguishable atoms of the same element, but with different isotopic composition, combine in a molecule. The size of the anti-clumping signal is closely related to the difference of the initial isotope ratios of the indistinguishable atoms that have combined. Therefore, a measured statistical clumped isotope anomaly, relative to an expected (e.g. thermodynamical) clumped isotope composition, may allow assessment of the heterogeneity of the isotopic pools of atoms that are the substrate for formation of molecules.

Binding and reaction studies with adenine nucleotides on purified coupling factor from Rhodospirillum rubrum.

Equilibrium binding studies with ATPase isolated from Rhodospirillum rubum chromotophores have been carried out using gel filtration. Binding experiments with variable concentrations of [14C]ADP show a biphasic saturation curve. With a parameter fitting computer program the dissociation constants for two distinct binding sites are determined as 7 x 10(-6) and 9 x 10(-5) M, respectively. The enzyme-bound radioactivity is recovered as ADP (80-90%), and the rest is converted to AMP and ATP. In the free nucleotides a large amount of AMP (about 70%) is found in addition to ADP. Analogous binding experiments with [14C]ATP are monophasic. Most of the bound radioactivity can be identified as ADP showing a dissociation constant corresponding to the high affinity site. The pattern of the free nucleotides is the same as in the experiments with ADP. These results indicate three separate binding sites on the enzyme: a low and a high affinity site for ADP, and a site at which ATP hydrolysis takes place. The analysis of the nucleotides suggests for the ADP sites a phosphoryl group transfer to produce ATP and AMP. Various experiments exclude the contamination of the enzyme preparation with adenylate kinase.