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The E. coli chaperonin proteins, GroEL and GroES, assist in folding newly synthesized proteins. GroES is necessary for GroEL-assisted folding under conditions where the substrate protein cannot spontaneously fold. On the basis of photolabelling of GroES with 8-azido-ATP, a role for nucleotide binding to GroES in chaperonin function was suggested [Martin, et al., Nature, 366 (1993) 279–2821]. We confirm the photolabelling of GroES with 8-azido-ATP. However, other proteins not known to contain nucleotide binding sites also become photolabeled suggesting that labeling is non-specific. Using rigorous physical methods, isothermal calorimetry and equilibrium binding, no interaction between GroES and nucleotides could be detected. We conclude that GroES has no nucleotide binding site.

In the presence of MgATP or MgADP the E. coli chaperonin proteins, GroEL and GroES, form a stable asymmetric complex with a stoichiometry of two GroEL7:one GroES7: seven MgADP. The distribution of the ligands between the two heptameric GroEL rings is crucial to our understanding of the mechanism of chaperonin-assisted folding, being either cis (i.e. [GroEL7·MgADP7·GroES7]-[GroEL7]) or trans (i.e. [GroEL7· MgADP7]-[GroEL7·GroES7]. On the basis of cross-linking experiments with 8-azido-ATP and the heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), it was suggested that GroES and MgADP are bound to the same GroEL ring which resists proteinase K digestion [Nature 366 (1993) 228–233]. However, we find that the SPDP-promoted cross linking of GroES and GroEL occurs in the absence of Mg2+, ADP or ATP, which are required for the formation of the asymmetric complex. Cross-linking is shown to occur only when the SPDP-modified GroES is co-precipitated with GroEL by trichloracetic acid. Furthermore, there are structural grounds for questioning whether SPDP can crosslink, in a physiologically relevant manner, an amino group of GroES with any of the cysteinyl groups of GroEL.

Although the fxation of CO* during photosynthesis is a dark reaction, it is generally accepted that some of the enzymes of the reductive COZ fixation cycle are indirectly regulated by light. Fructose and sedoheptulose bisphosphatase and ribulose bisphosphate carboxylase have been identified as major regulatory sites in the reductive CO2 fixation cycle [l-3]. Ribulosebisphosphate carboxylase (EC 4.1 .1.39) converted from an inactive into an active form by reaction with COZ and Mg2+, this activation being enhanced by a pH-shift from pH 7.0-8.5 [4-61. The activation is a relatively slow process with a half-time in the range of l-3 min. The activated enzyme is stable enough to be assayed for 90 s without change of activity [4] . A number of effecters such as 3-phosphoglycerate j 6-phosphogluconate, fructose-l ,6bisphosphate and NADPH have been found to increase the activation of the enzyme especially at pH values below 8.0 [7-91. Using a rapid procedure for the lysis of chloroplasts and assay of the carboxylase activity, the factors

The mechanism of glycolate synthesis during photosynthesis is a subject of current interest [l-3] . A considerable body of evidence indicates that glycolate is synthesised primarily from rlbulose-1,5bisphosphate (RuBP), a reaction catalysed by RuBP carboxylase-oxygenase [4,5] which accounts for the incorporation of one atom of molecular oxygen into the carboxyl group of glycolate both in vivo [6,7] and in vitro [8]. However, two recent reviews [2,3] have implied that glycolate is synthesised by several different pathways without quantitatively assessing the contribution of these various pathways. In this report we establish that at least 80% of the glycolate produced by intact isolated chloroplasts is synthesised by reaction(s) which bring about the incorporation of one atom of molecular oxygen into the carboxyl group of glycolate.