Acid-induced dissociation of alpha A- and alpha B-crystallin homopolymers.

Homopolymers were constructed from the alpha A and alpha B polypeptides isolated from the lens protein alpha-crystallin. As the pH is lowered from 7.0 to 3.4, these homopolymers dissociate to smaller species with molecular masses ranging from 80 to 250 kDa for the alpha A and around 140 kDa for the alpha B dissociation products. The pKa for this dissociation was 3.8 +/- 0.2 for alpha A and 4.1 +/- 0.1 for alpha B homopolymers. Further decreases in pH, to 2.5, resulted in the presence of only denatured alpha B polypeptides, whereas the alpha A dissociation products remained intact. Fractionation of the acid dissociation products from the alpha A homopolymer at pH 2.5 yielded stable species with molecular masses of 220 +/- 30, 160 +/- 20, and 90 +/- 10 kDa. The majority of the population at acid pH consisted of the 160 kDa species. Conformational analysis of these species revealed that most of the secondary structure of the original alpha A homopolymer was retained but that the tertiary structure was perturbed. Fluorescence quenching and energy transfer measurements suggested that the molecule had undergone acid expansion, with the greatest perturbation observed in the smallest particles. The results from this work suggest that alpha A homopolymers are heterogeneous populations of aggregates of a "monomeric" molecule with a molecular mass of 160 kDa. This "monomeric" molecule may be formed from the association of two tetrameric units.     

On the structure of alpha-crystallin: construction of hybrid molecules and homopolymers

The alpha A2 and alpha B2 subunits of bovine alpha-crystallin were purified by chromatofocussing in urea and assembled into homopolymers. Light-scattering measurements indicated their molecular masses were 360 and 420 kDa. The alpha A2 and alpha B2 polypeptides were also used to construct a series of hybrid molecules with alpha A/alpha B ratios ranging from 7:1 to 1:7. Sedimentation velocity analyses, isoelectric focussing under non-deaggregating conditions, circular dichroism spectroscopy and immunochemical analysis indicated that all of the subunits had copolymerized to alpha-crystallin-like aggregates with complete regeneration of the native structure. The polymers could be distinguished on the basis of their differing affinities for the antiserum. This was directly related to the proportion of alpha A2 subunits in each polymer. It was concluded that the alpha A2 and alpha B2 subunits are structurally equivalent and occupy equivalent site in the alpha-crystallin aggregates. It was also concluded that a micellar-like quaternary structure was consistent with most previous observations on the protein.     

Specific dissociation of alpha B subunits from alpha-crystallin

Exposure of bovine alpha-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the alpha B chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only alpha A chains. The decrease in the molar mass corresponds to the mass of the alpha B chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo alpha-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for alpha-crystallin and 4.0 for a homopolymer constructed from purified alpha B2 polypeptides. An alpha A2 homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The alpha B chains were found to be completely denatured, whereas the structure of the alpha A chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the alpha B chain was responsible for the specific dissociation of this polypeptide.     

Expression and aggregation of recombinant alpha A-crystallin and its two domains.

The 20 kDa alpha A and alpha B subunits of alpha-crystallin from mammalian eye lenses form large aggregates with an average molecular weight of 800,000. To get insight into the interactions responsible for aggregate formation, we expressed in Escherichia coli the putative N- and C-terminal domains of alpha A-crystallin, as well as the intact alpha A-crystallin chain. The proteins are expressed in a stable form and in relatively high amounts (20-60% of total protein). Recombinant alpha A-crystallin and the C-terminal domain are expressed in a water-soluble form. Recombinant alpha A-crystallin forms aggregates comparable with alpha-crystallin aggregates from calf lenses, whereas the C-terminal domain forms dimers or tetramers. The N-terminal domain is expressed in an initially water-insoluble form. After solubilization, denaturation and reaggregation the N-terminal domain exists in a high molecular weight multimeric form. These observations suggest that the interactions leading to aggregation of alpha A-crystallin subunits are mainly located in the N-terminal half of the chain.     

A dynamic quaternary structure of bovine alpha-crystallin as indicated from intermolecular exchange of subunits

The structural bovine eye lens protein alpha-crystallin was dissociated in 7 M urea and its four subunits, A1, A2, B1, and B2, were separated by means of ion-exchange chromatography. Homopolymeric reaggregates of these subunits were prepared by removal of the denaturant via dialysis. It was found that subunits were exchanged upon incubation of mixtures of two homopolymers under native conditions. New hybrid species were formed within 24 h as demonstrated by isoelectric focusing. Moreover, native alpha-crystallin molecules also exchanged subunits when incubated with homopolymeric aggregates of B2 subunits. Subunit exchange between native alpha-crystallin molecules is postulated, and a "dynamic quaternary structure" is presented that allows the polydisperse protein to adapt to changes in cytoplasmic conditions upon aging of the lens tissue.     

Structural features of Ca2+/calmodulin-dependent protein kinase II revealed by electron microscopy

The molecular conformation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from the rat forebrain and cerebellum was studied by means of EM using a quick-freezing technique. Each molecule appeared to be composed of two kinds of particles, with one larger central particle and smaller peripheral particles and had shapes resembling that of a flower with 8 or 10 "petals". A favorable shadowing revealed that each peripheral particle had a thin link to the central particle. We predicted that the 8-petal molecules and 10-petal molecules were octamers and decamers of CaM kinase II subunits, respectively, each assembled with the association domains of subunits gathered in the center, and the catalytic domains in the peripheral particles. Binding of antibodies to the enzyme molecules suggested that molecules with 8 and 10 peripheral particles were homopolymers composed only of beta subunit and of alpha subunit, respectively, specifying that CaM kinase II consists of homopolymer of either alpha or beta subunits.     

Micellar subunit assembly in a three-layer model of oligomeric alpha-crystallin

Differential scanning calorimetry was performed to monitor the heat-induced changes that occur in the structural domain of lens alpha-crystallin. Circular dichroism and fluorescence also were used to resolve the controversial issue of the quaternary structure of alpha-crystallin. Based on the thermal behavior as monitored by these techniques, a model is proposed that can account for all previous data as well as the currently reported thermal data. The proposed model of native alpha-crystallin has a three-layer structure in which the inner layer (core) is a micelle containing 12 subunits arranged in cuboctahedral symmetry. The apolar region is directed inward constituting a hydrophobic core similar to a micelle and adding structural stability. A second layer of six subunits has a similar but not identical structure to the first layer, directing its apolar face toward the hydrophobic core. Thus, these two layers constitute a micelle-like structure with octahedral symmetry. The third layer adds more subunits for a total of not more than 24. Differential scanning calorimetry, circular dichroism, and fluorescence studies indicated that the inner two-layer structure of molecular mass 360 kDa is highly stable and is most likely of the alpha m form. The three-layer structure of the native protein, however, is rather unstable. At 35-45 degrees C the outer layer dissociates from the inner two layers, and at higher temperatures rapidly reassociates to a slightly modified two-layer structure with a stability similar to that of alpha m. The proposed model does not require any specific assembly of the alpha A and alpha B subunits in each layer, but the fluorescence results suggest that the native inner two layers probably contain mostly alpha A.     

Significance of alpha-crystallin heteropolymer with a 3:1 alphaA/alphaB ratio: chaperone-like activity, structure and hydrophobicity

The small heat-shock protein alpha-crystallin isolated from the eye lens exists as a large (700 kDa) heteropolymer composed of two subunits, alphaA and alphaB, of 20 kDa each. Although trace amounts of alphaA-crystallin are found in other tissues, non-lenticular distribution of alpha-crystallin is dominated by the alphaB homopolymer. In most vertebrate lens, the molar ratio of alphaA to alphaB is generally 3:1. However, the importance of this ratio in the eye lens is not known. In the present study, we have investigated the physiological significance of the 3:1 ratio by determining the secondary/tertiary structure, hydrophobicity and chaperone-like activity of alphaA- and alphaB-homopolymers and heteropolymers with different ratios of alphaA to alphaB subunits. Although, under physiologically relevant conditions, the alphaB-homopolymer (37-40 degrees C) has shown relatively higher activity, the alphaA-homopolymer or the heteropolymer with a higher alphaA proportion (3:1 ratio) has shown greater chaperone-like activity at elevated temperatures (>50 degrees C) and also upon structural perturbation. Furthermore, higher chaperone activity at elevated temperatures as well as upon structural perturbation is mainly mediated through increased hydrophobicity of alphaA. Although homopolymers and heteropolymers of alpha-crystallin did not differ in their secondary structure, changes in tertiary structure due to structural perturbations upon pre-heating are mediated predominantly by alphaA. Interestingly, the heteropolymer with higher alphaA proportion (3:1) or the alphaA-homopolymer seems to be better chaperones in protecting lens beta- and gamma-crystallins at both normal and elevated temperatures. Thus lens might have favoured a combination of these qualities to achieve optimal protection under both native and stress (perturbed) conditions for which the heteropolymer with alphaA to alphaB in the 3:1 ratio appears to be better suited.     

Identification by 1H NMR spectroscopy of flexible C-terminal extensions in bovine lens alpha-crystallin.

Two-dimensional 1H NMR spectroscopy of bovine eye lens alpha-crystallin and its isolated alpha A and alpha B subunits reveals that these aggregates have short and very flexible C-terminal extensions of eight (alpha A) and ten (alpha B) amino acids which adopt little preferred conformation in solution. Total alpha-crystallin forms a tighter aggregate than the isolated alpha A and alpha B subunit aggregates. Our results are consistent with a micelle model for alpha-crystallin quaternary structure. The presence of terminal extensions is a general feature of those crystallins, alpha and beta, which form aggregates.     

Phosphorylation of an alpha 1-like subunit of an omega-conotoxin-sensitive brain calcium channel by cAMP-dependent protein kinase and protein kinase.

Antibodies that recognize the alpha 2 delta and alpha 1 subunits of skeletal muscle L-type calcium channels have been used to investigate the subunit components and phosphorylation of omega-conotoxin (omega-CgTx)-sensitive N-type calcium channels from rabbit brain. Photolabeling of the N-type channel with a photoreactive derivative of 125I-omega-CgTx results in the identification of a single polypeptide of 240 kDa. MANC-1, a monoclonal antibody recognizing alpha 2 delta subunits of L-type calcium channels from skeletal muscle, immunoprecipitates the omega-CgTx-labeled 240-kDa polypeptide and approximately 6% of the digitonin-solubilized 125I-omega-CgTx-labeled N-type channels. MANC-1 also immunoprecipitates a phosphoprotein of 240 kDa that comigrates with 125I-omega-CgTx-labeled N-type calcium channels, but not with L-type calcium channels, in sucrose gradients. Both cAMP-dependent protein kinase and protein kinase C are effective in the phosphorylation of this polypeptide. Similar to the alpha 1 subunits of skeletal muscle L-type calcium channels, the immunoprecipitation of the 240-kDa phosphoprotein by MANC-1 is prevented by the detergent Triton X-100. Anti-CP-(1382-1400), an antipeptide antibody against a highly conserved segment of the alpha 1 subunits of calcium channels, immunoprecipitates the 240-kDa phosphopeptide in Triton X-100. The 240-kDa protein is phosphorylated to a stoichiometry of approximately 1 mol of phosphate/mol of omega-CgTx-binding N-type calcium channels by both cAMP-dependent protein kinase and protein kinase C. Our results show that the 240-kDa polypeptide is an alpha 1-like subunit of an omega-CgTx-sensitive N-type calcium channel. The N-type calcium channels containing this subunit are phosphorylated by cAMP-dependent protein kinase and protein kinase C and contain noncovalently associated alpha 1-like and alpha 2 delta-like subunits as part of their oligomeric structure.     

Interaction between the constituent A and B lens alpha-crystallin subunits leading to formation of alpha-neoprotein molecules

A and B constituent subunits associated in lens alpha-crystallin were found to interact with added B chains forming alpha-neoprotein molecules with lower A to B chain ratios than 2 A to 1 B in alpha-crystallin. Addition of 1% excess of B chains to the one in alpha-crystallin, which resulted in a ratio of 1.98 A to 1 B in the mixture, caused a change of quaternary structure in 30% of alpha-crystallin molecules within 18 h. At a ratio of 1.86 A to 1 B, all alpha-crystallin molecules were affected at this time. A maximum number of 495 B chains was found to form an association with 1 A chain, initially bound in alpha-crystallin. Such a high number may indicate that the reaction involves monomeric A chains binding aggregated macromolecules of B chains. It is in such form that B chains occur as macromolecules with an average molecular weight of 0.7 X 10(6) in aqueous solution. The alpha-neoprotein molecules selected for studies in this report had A to B chain ratios of 1.75:1, 1:1, and 0.2:1. Each behaved in immunodiffusion tests like single molecular entities. Antigenic determinants located on A as well as on B chains associated with each other in alpha-crystallin were found to be identical with determinants on the chains associated in the above alpha-neoprotein molecules. Determinants dependent on the quaternary structure of alpha-neoprotein and of alpha-crystallin molecules were completely different. Some of the quaternary determinants of various alpha-neoproteins were type specific and did not occur in molecules with different A to B chain ratios. Other quaternary determinants occurred in all alpha-neoproteins. An excess of A chains did not revert alpha-neoproteins to alpha-crystallin. However, alpha-neoprotein molecules did interact with added B chains forming neomolecules with lower A to B chain ratios.     

The phosphorylation sites of the B2 chain of bovine alpha-crystallin

The B2 chain of bovine lens alpha-crystallin is phosphorylated in a cAMP-dependent reaction. By analysis of 32P-labelled chymotryptic peptides isolated from alpha-crystallin obtained from lenses labelled in organ culture, two phosphorylated B2 chain fragments were found. Sequence analysis of the fragments gave the following results: Arg-Ala-Pro-Ser-Trp-Ile-Asp-Thr-Gly-Leu and Ser-Leu-Ser-Pro-Phe corresponding to residues 56 to 65 and 43 to 47, respectively. It is established by this work that B1 is a phosphorylated post-translational product of B2. Both the A2 and B2 chains of alpha-crystallin are phosphorylated at a similar site with the sequence Arg-(X)-Pro-Ser. This is an unusual site for cAMP-phosphorylation since the phosphorylated serine is preceded by a proline residue. It may also be of significance that the other B2 chain phosphorylation site even more radically differs from previously reported cAMP-dependent phosphorylation sites.     

Chaperone activity of alpha-crystallins modulates intermediate filament assembly.

Intermediate filaments are generally regarded as one of the most insoluble and resilient cytoskeletal structures of eukaryotic cells. In extracts from the ocular lens, we noticed an unusually high level of vimentin in a soluble, non-filamentous form. Immunoprecipitation of this soluble vimentin resulted in the co-precipitation of alpha-crystallins. The alpha-crystallins are homologous to the small heat shock proteins (sHSPs) and have recently been identified as molecular chaperones, capable of preventing the heat-induced aggregation of proteins. We find that the alpha-crystallins dramatically inhibit the in vitro assembly of GFAP and vimentin in an ATP-independent manner. This inhibition is also independent of the phosphorylation state of the alpha-crystallin polypeptides and each one of the four polypeptides, either alpha A1-, alpha A2-, alpha B1- or alpha B2-crystallin, are equally effective in this inhibition. Furthermore, we show that alpha-crystallins can increase the soluble pool of GFAP when added to preformed filaments. Electron microscopy demonstrated that alpha-crystallin particles could bind to intermediate filaments in a regular fashion, the spacing coinciding with the molecular length of GFAP. This is the first report, as far as we are aware, of a chaperone being involved in intermediate filament assembly and implicates chaperones in the remodeling of intermediate filaments during development and cell differentiation.     

Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine.

Crystallins are structural proteins responsible for establishing the remarkable optical properties of the lens. Yet many of these highly conserved proteins are also expressed in nonocular tissues, where they have alternative functions apparently unrelated to their structural role in the lens. Here we report that lens alpha-crystallins, some of which function as heat-shock proteins in other tissues, are modified with O-linked N-acetylglucosamine (O-GlcNAc). An in vitro enzymatic assay that transfers [3H]Gal to terminal GlcNAc moieties labels alpha A and alpha B crystallins in lens homogenates from man, rhesus monkey, rat, cow, and rhea (an ostrich-like bird). O-Linkage of the saccharide is demonstrated by sensitivity to base-catalyzed beta-elimination and resistance to peptide:N-glycosidase F treatment. Chromatographic analyses of the beta-elimination products and fast atom bombardment-mass spectrometry of [3H]Gal-labeled tryptic peptides confirm the saccharide structure. Isoelectric focusing of [3H]Gal-labeled bovine lens proteins reveals the presence of O-GlcNAc on all four alpha-crystallin subunits, A1, A2, B1, and B2. Electrospray mass spectrometry of bovine alpha-crystallin demonstrates the presence of a single O-GlcNAc substitution on alpha A2. Gas-phase protein sequencing and fast atom bombardment-mass spectrometry of the major radiolabeled tryptic peptide from bovine alpha-crystallin reveal that GlcNAc is attached to the alpha A subunits at serine 162. This post-translational modification may play an important role in the molecular organization of lens alpha-crystallin.     

The reaction of human alpha 2-macroglobulin with alpha-chymotrypsin. A stopped-flow kinetic investigation

The kinetics of the conformational changes of human alpha 2-macroglobulin (alpha 2M) induced by reaction with pure alpha-chymotrypsin, have been analyzed using three fluorescent probes, namely protein tryptophan groups and the dye 6-(4-toluidino)-2-naphthalenesulfonate, to monitor alterations of the alpha 2M structure, and a covalent conjugate of chymotrypsin and fluorescein isothiocyanate (Chy-FITC). The main reaction sequence exhibits a triphasic time course with any of the labels used. Each phase is first-order. The fixation of a single molecule of chymotrypsin to one protease-binding site of alpha 2M (site A) initiates the whole process and determines the access to the second site (site B). Of the three exponential phases of the reaction (20 degrees C), phase I (k1 approximately 19.6 min-1) and phase II (k2 approximately 5.3 min-1) belong to site A. Phase III is related to site B transformation. It contains two steps with different responses from tryptophan (k3 approximately 0.77 min-1) and Chy-FITC (k3 approximately 0.19 min-1) fluorescence measurements. The point to be stressed is that site A and site B, while presumably identical in the native form, are not equivalent with regard to their fluorescence and kinetic properties. However, the activation energy (E = 30.1 +/- 2.7 kJ mol-1) is the same for the three phases of the reaction. When present in sufficient excess, free chymotrypsin or native alpha 2M is able to form reversible complexes with the above-related chymotrypsin-alpha 2M adducts. Only the alpha 2M site A core seems to be involved in this parallel process. In addition the conformational state of the chymotrypsin-alpha 2M complexes is shown to depend on the pH, with a pKa of 6.4.     

Temperature-dependent chaperone activity and structural properties of human alphaA- and alphaB-crystallins

The chaperone activity and biophysical properties of recombinant human alphaA- and alphaB-crystallins were studied by light scattering and spectroscopic methods. While the chaperone function of alphaA-crystallin markedly improves with an increase in temperature, the activity of alphaB homopolymer appears to change very little upon heating. Compared with alphaB-crystallin, the alphaA-homopolymer is markedly less active at low temperatures, but becomes a more active species at high temperatures. At physiologically relevant temperatures, the alphaB homopolymer appears to be modestly (two times or less) more potent chaperone than alphaA homopolymer. In contrast to very similar thermotropic changes in the secondary structure of both homopolymers, alphaA- and alphaB-crystallins markedly differ with respect to the temperature-dependent surface hydrophobicity profiles. Upon heating, alphaA-crystallin undergoes a conformational transition resulting in the exposure of additional hydrophobic sites, whereas no such transition occurs for alphaB-crystallin. The correlation between temperature-dependent changes in the chaperone activity and hydrophobicity properties of the individual homopolymers supports the view that the chaperone activity of alpha-crystallin is dependent on the presence of surface-exposed hydrophobic patches. However, the present data also show that the surface hydrophobicity is not the sole determinant of the chaperone function of alpha-crystallin.     

Fluorescence correlation spectrometry of the interaction kinetics of tetramethylrhodamin alpha-bungarotoxin with Torpedo californica acetylcholine receptor

Fluorescence correlation spectroscopy (FCS) is suited to determine low concentrations (10(-8) M) of slowly interacting molecules with different translational diffusion coefficients on the level of single molecule counting. This new technique was applied to characterize the interaction dynamics of tetramethylrhodamin labelled alpha-bungarotoxin (B( *)) with the detergent solubilized nicotinic acetylcholine receptor (AChR) of Torpedo californica electric organ. At pseudo-first-order conditions for AChR, the complex formation with B( *) is monophasic. The association rate coefficient of the monoliganded species AChR . B is k(ass)' = 3.8 . 10(3) s(-1) at 293 K (20 degrees C). The dissociation of bound B( *) from the monomer species AChR . B( *) . B (and AChR . B(2)( *)), initiated by adding an excess of nonlabelled alpha-bungarotoxin (B), is biphasic suggesting a three state cascade for the B-sites: R(alpha) --> R(alpha)' --> R(alpha)' with the exchange dissociation constants: (k(diss)')(B) = 5.5(+/-1) . 10(-5) s(-1) and (k(diss)')(B) = 3(+/-1) . 10(-6) s(-1) at 293 K. The data are consistent with dissociative intermediate steps of ligand exchange on two different interconvertible conformations of one binding site. The dissociation of bound B( *) by excess of the neurotransmitter acetylcholine (ACh) is biphasic. At [ACh] = 0.1 M both B( *) are released from the AChR . B(2)( *) species. The mechanism involves associative ternary intermediates (AChR . B( *)A, AChR . B( *)A(2) and AChR . B(2)( *)A(2)). The equilibrium constants (K(A)) and dissociation rate constants (k(-A)) for ACh in the ternary complex state R(alpha)' and R(alpha)', respectively, are K(A)' = 1.1 . 10(-2) M and k(-A)' = 3 . 10(5) s(-1) and K(A)' = 7.5 . 10(-2) M and k(-A)' = 2 . 10(6) s(-1). It is of physiological importance that the FCS data indicate that the AChR monomer species (M(r) = 290 000), which normally at [ACh] 1 mM only binds one ACh molecule, does bind two ACh molecules at [ACh] 0.1 M.     

An unusually long non-coding region in rat lens alpha-crystallin messenger RNA.

Most of the mRNA sequence coding for the alpha A2 chain of the ocular lens protein alpha-crystallin from rat, has been determined by sequencing cloned DNA copies of this mRNA. The 892-base pair cDNA sequence encompasses all but 52 N-terminal amino acids of the alpha A2 chain. It lacks the sequence characteristic for the 22 extra amino acids inserted in the alpha A2 -like chain, named alpha AIns. A stretch of 583 nuceotides, representing more than 50% of the entire mRNA sequence, is located 3' wards of the alpha A2 coding sequence. It contains the characteristic AAUAAA signal involved in poly(A) -addition and represents an unexpectedly long non-coding region. Examination of the total cytoplasmic poly(A) RNA of rat lens by filter-hybridization and subsequent translation of the electrophoretically separated mRNA fractions shows that the alpha A2 chain is encoded by mRNA species which are distinct from the alpha AIns encoding mRNA. No evidence is obtained for an extensive size heterogeneity in the 3' untranslated regions of these two different rat lens mRNAs.     

The rotation of the alpha subunit of F1 relative to minor subunits is not involved in ATP synthesis. Evidence given by using an anti-alpha subunit monoclonal antibody

To test whether ATP synthesis could occur via a mechanism of rotational catalysis in which the alpha and beta subunits of F1 would rotate with respect to the minor subunits, we have measured the rate of ATp synthesis after binding various masses of antibodies to F1. If the rotation was an essential feature of the mechanism, the rate of ATP synthesis should be inhibited either completely or proportionately to the load carried by F1. Bivalent immunoglobulins (IgG) or monovalent Fab fragments of an anti-alpha monoclonal antibody (7B3) were bound to F1 present in electron-transport particles in a ratio of 2 Fab or 2 IgG per F1. This binding similarly inhibited the rate of ATP synthesis by a maximum of about 50%. When anti-mouse immunoglobulins were added to the F1-7B3 (IgG) complex, no significant change in the rate of inhibition was observed. In conclusion, the rate of ATP synthesis was the same when F1 was loaded with 100 kDa (2 Fab), 300 kDa (2 IgG, 7B3) or 900 kDa (2 IgG + 4 ant-mouse IgG). It is concluded that the rotation of the alpha subunits is extremely unlikely to play an essential role in the mechanism of ATP synthesis.