Deletion of C-terminal residues of Escherichia coli ribosomal protein L10 causes the loss of binding of one L7/L12 dimer: ribosomes with one L7/L12 dimer are active

Escherichia coli ribosomal protein L10 binds the two L7/L12 dimers and thereby anchors them to the large ribosomal subunit. C-Terminal deletion variants (Delta10, Delta20, and Delta33 amino acids) of ribosomal protein L10 were constructed in order to define the binding sites for the two L7/L12 dimers and then to make and test ribosomal particles that contain only one of the two dimers. None of the deletions interfered with binding of L10 variants to ribosomal core particles. Deletion of 20 or 33 amino acids led to the inability of the proteins to bind both dimers of protein L7/L12. The L10 variant with deletion of 10 amino acids bound one L7/L12 dimer in solution and when reconstituted into ribosomes promoted the binding of only one L7/L12 dimer to the ribosome. The ribosomes that contained a single L7/L12 dimer were homogeneous by gel electrophoresis where they had a mobility between wild-type 50S subunits and cores completely lacking L7/L12. The single-dimer ribosomal particles supported elongation factor G dependent GTP hydrolysis and protein synthesis in vitro with the same activity as that of two-dimer particles. The results suggest that amino acids 145-154 in protein L10 determine the binding site ("internal-site") for one L7/L12 dimer (the one reported here), and residues 155-164 ("C-terminal-site") are involved in the interaction with the second L7/L12 dimer. Homogeneous ribosomal particles containing a single L7/L12 dimer in each of the distinct sites present an ideal system for studying the location, conformation, dynamics, and function of each of the dimers individually.     

From structure and dynamics of protein L7/L12 to molecular switching in ribosome

Based on the (1)H-(15)N NMR spectroscopy data, the three-dimensional structure and internal dynamic properties of ribosomal protein L7 from Escherichia coli were derived. The structure of L7 dimer in solution can be described as a set of three distinct domains, tumbling rather independently and linked via flexible hinge regions. The dimeric N-terminal domain (residues 1-32) consists of two antiparallel alpha-alpha-hairpins forming a symmetrical four-helical bundle, whereas the two identical C-terminal domains (residues 52-120) adopt a compact alpha/beta-fold. There is an indirect evidence of the existence of transitory helical structures at least in the first part (residues 33-43) of the hinge region. Combining structural data for the ribosomal protein L7/L12 from NMR spectroscopy and x-ray crystallography, it was suggested that its hinge region acts as a molecular switch, initiating "ratchet-like" motions of the L7/L12 stalk with respect to the ribosomal surface in response to elongation factor binding and GTP hydrolysis. This hypothesis allows an explanation of events observed during the translation cycle and provides useful insights into the role of protein L7/L12 in the functioning of the ribosome.     

Chicken liver ribosomes: Characterization of cross-reaction and inhibition of some functions by antibodies prepared against Escherichia coli ribosomal proteins L7 and L12

Antibodies prepared in rabbits against Escherichia coli ribosomal proteins L7/L12 are reported to be immunologically cross-reactive with some ribosomal proteins on the 60 S subunit of eukaryote ribosomes (Wool & Stöffler, 1974; Stöffler et al., 1974). We have confirmed these reports and extended this finding to a detailed study of the functional properties of eukaryote ribosomes which are affected by these cross-reacting antibodies. We report here the partial reactions in protein synthesis that are inhibited by the anti-L7/L12 IgG (immunoglobulin G) preparations using a chicken liver system. The following reactions were inhibited: EF-1 (elongation factor 1) dependent binding of aminoacyl-tRNA to ribosomes and GTP hydrolysis; EF-2 dependent binding of nucleotide to ribosomes and GTP hydrolysis; binding of [¹⁴C]ADP-ribosyl · EF-2 to ribosomes. This last reaction is more sensitive to the antibody inhibition than the corresponding nucleotide binding reaction. We show that the inhibitions were not simply non-specific precipitation of ribosomes by IgG, in that monovalent Fabs were also inhibitory, and peptidyl transferase activity was not inhibited. The functions inhibited with the IgG preparations in the chicken liver system are analogous to those inhibited in the homologous E. coli system. Thus the cross-reacting protein is functionally as well as immunologically conserved.     

Interaction among silkworm ribosomal proteins P1, P2 and P0 required for functional protein binding to the GTPase-associated domain of 28S rRNA

Acidic ribosomal phosphoproteins P0, P1 and P2 were isolated in soluble form from silkworm ribosomes and tested for their interactions with each other and with RNA fragments corresponding to the GTPase-associated domain of residues 1030–1127 (Escherichia coli numbering) in silkworm 28S rRNA in vitro. Mixing of P1 and P2 formed the P1–P2 heterodimer, as demonstrated by gel mobility shift and chemical crosslinking. This heterodimer, but neither P1 or P2 alone, tightly bound to P0 and formed a pentameric complex, presumably as P0(P1–P2)₂, assumed from its molecular weight derived from sedimentation analysis. Complex formation strongly stimulated binding of P0 to the GTPase-associated RNA domain. The protein complex and eL12 (E.coli L11-type), which cross-bound to the E.coli equivalent RNA domain, were tested for their function by replacing with the E.coli counterparts L10.L7/L12 complex and L11 on the rRNA domain within the 50S subunits. Both P1 and P2, together with P0 and eL12, were required to activate ribosomes in polyphenylalanine synthesis dependent on eucaryotic elongation factors as well as eEF-2-dependent GTPase activity. The results suggest that formation of the P1–P2 heterodimer is required for subsequent formation of the P0(P1–P2)₂ complex and its functional rRNA binding in silkworm ribosomes.     

Surface topography of the Bacillus stearothermophilus ribosome.

Summary The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more ¹²⁵I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of ¹²⁵I. Iodinated 70S ribosomes and subunits retained 62–78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.     

The L7Ae protein binds to two kink-turns in the Pyrococcus furiosus RNase P RNA

The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs), has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the Mg²⁺-dependent 5′ maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides proximal to the EDTA-Fe–modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae–EDTA-Fe) revealed specific RNA cleavages, localizing the binding sites of L7Ae to the RPR''s catalytic and specificity domains. These results support the presence of two kink-turns, the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which L7Ae contributes to RNase P catalysis.     

Conformational change of L7/L12 stalk in the different functional states of 50S ribosomes

Conformational change of 50S ribosomes takes place during protein synthesis. The primary change is most likely in the secondary or tertiary structure of rRNA in the L7/L12 stalk region. In order to throw further light on this conformational change, the change in fluorescence of tight couple 50S ribosomes on conversion to loose couple 50S ribosomes containing 5-(iodoacetamido ethyl)-aminonaphthalene-l-sulphonic acid-labelled L7/L12, following the treatment with elongation factor-G and 5′-guanylyl methylene diphosphate was measured. It was enhanced in agreement with the results reported earlier. Further, the quenching of fluorescence of 50S ribosomes containing 5-(iodoacetamido ethyl)-aminonaphthalene-1-sulphonic acid-labelled L7/L12 by acrylamide was studied. The quenching is more in case of loose couples. On conversion of loose couple 50S ribosomes to tight couple ones the quenching becomes less whereas the reverse happens on conversion of tight couple 70S ribosomes to loose couples. These results indicate the conformational change of L7/L12 stalk in the different functional states of 50S ribosomes.     

Gel electrophoresis of ribosomal components from seeds of Pisum sativum L

The ribosomes of dry pea seeds were analysed by polyacrylamide gel electrophoresis. Ribosomes, ribosomal subunits, rRNA and ribosomal proteins were separated by variations of this same basic technique. Pea seed ribosomes were shown to have a subunit structure, rRNA complement and ribosomal protein distribution similar to other eukaryotic ribosomes. A total of 52 ribosomal proteins were identified, 24 on the small and 28 on the large RSU. The molecular weights were mostly in the range 10–35 × 10³.     

PGJ2 and ΔPGJ2 inhibit cell growth accompanied with inhibition of phosphoinositide turnover in human astrocytoma cells

PGJ₂ and Δ¹²PGJ₂ (1 μM to 30 μm) inhibited the growth of human astrocytoma cells (1321N1) in a time-dependent manner within 48 hrs, determined by [³H]thymidine incorporation into acid-insoluble fraction or amounts of protein. The EC₅₀ values for PGJ₂ and Δ¹²PGJ₂ were approximately 8 μM and 6 μM, respectively. [³H]Thymidine incorporation to acid insoluble fraction was inhibited by these PGs within 1 hr, indicating that these PGs rapidly affect cell functions. Although it has been reported that an increase in cyclic AMP inhibits cell growth, PGJ₂ and Δ¹²PGJ₂, but not PGE₁, reduced isoproterenol (10 μM)-induced accumulation of cyclic AMP, suggesting that PGJ₂ and Δ¹²PGJ₂ may disturb adenylate cyclase system, which might be independent on cell growth. On the other hand, these PGs inhibited the incorporation of [³H]inositol into phospholipid fraction within 6 hrs. Furthermore, PGJ₂ and Δ¹²PGJ₂ inhibited carbachol- and/or histamine-induced accumulation of inositol phosphates with a similar dose-dependency to their inhibitions of cell growth. In membrane preparations, however, PGJ₂ and Δ¹²PGJ₂ failed to inhibit GTPγS (10 μM)- nor Ca²⁺ (1mM)-induced accumulation of inositol phosphate. The site of PGJ₂ or Δ¹²PGJ₂ in inhibition of inositol phosphate accumulation would not be phospholipase C nor a putative GTP binding protein involved in activation of phospholipase C. The present results indicate that PGJ₂ and Δ¹²PGJ₂ inhibit cell growth in human astrocytoma cells and the inhibition of phosphoinositide turnover by these PGs might be involved in the inhibition of cell growth.     

Overexpression of L7/L12 protein with mutations in its flexible region.

Using restriction enzymes and polymerase chain reaction, three mutated forms of L7/L12 proteins with deleted 38-46, 44-52 and 38-52 residues were obtained. These mutant proteins were isolated in a preparative scale and were shown to bind to ribosomes and to affect ribosomal function.     

Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif

The archaeal L7Ae and eukaryotic 15.5kD protein homologs are members of the L7Ae/15.5kD protein family that characteristically recognize K-turn motifs found in both archaeal and eukaryotic RNAs. In Archaea, the L7Ae protein uniquely binds the K-loop motif found in box C/D and H/ACA sRNAs, whereas the eukaryotic 15.5kD homolog is unable to recognize this variant K-turn RNA. Comparative sequence and structural analyses, coupled with amino acid replacement experiments, have demonstrated that five amino acids enable the archaeal L7Ae core protein to recognize and bind the K-loop motif. These signature residues are highly conserved in the archaeal L7Ae and eukaryotic 15.5kD homologs, but differ between the two domains of life. Interestingly, loss of K-loop binding by archaeal L7Ae does not disrupt C′/D′ RNP formation or RNA-guided nucleotide modification. L7Ae is still incorporated into the C′/D′ RNP despite its inability to bind the K-loop, thus indicating the importance of protein–protein interactions for RNP assembly and function. Finally, these five signature amino acids are distinct for each of the L7Ae/L30 family members, suggesting an evolutionary continuum of these RNA-binding proteins for recognition of the various K-turn motifs contained in their cognate RNAs.     

Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M _r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K _a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 10⁷ M^–1 and 3.1 × 10⁷ M^–1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.     

Prostaglandin D2 metabolite stimulates collagen synthesis by human osteoblasts during calcification

We investigate the effect of the prostaglandin D₂ metabolite Δ¹²−PGD₂ (9−Deoxy−Δ⁹, Δ¹²−13,14-dihydroprostaglandin D₂) on collagen synthesis in human osteoblast. Δ¹²-PGJ₂ at 10⁻⁵M enhanced collagen synthesis in the presence of 2 mM α-glycerophosphate-2Na. The stimulative effect appeared as early as 3 days after addition and continued until 22 days. The enhancement of type I collagen synthesis was confirmed by polyacrylamide gel electrophoresis. The potency was the same as 10^1t-8M 1 α, 25 dihydroxy vitamine D₃ (1,25(OH)₂D₃). Northern blot analysis showed that 10⁻⁵M Δ ¹²-PGD₂ and 10⁻⁸M 1,25(OH)₂D₃ enhanced the transcribtion of type 1 procollagen (α₁) mRNA levels in osteoblasts.     

Growth phase and growth rate dependence of ribosomal peptidyltransferase activity status in Escherichia coli

Ribosomes from a clinical isolate of E coli were purified and characterized. The structural features of these ribosomes were identical to wild-type E coli ribosomes, with the exception that rRNA in general, but especially 23S rRNA, was degraded as a result of the transition from early to late logarithmic growth phase, on different growth media. Analysis of the ribosomal protein by gel electrophoresis indicated that the L12/L7 molar ratio increases during early logarithmic phase, reaching a maximum value of about 1.6 at midlogarithmic phase, and then falling to 0.7 in late logarithmic phase. Concomitantly with L12/L7 alterations, the activity status of ribosomal peptidyltransferase was found to undergo a striking shift. Reconstitution experiments demonstrated that the two effects are closely related. Moreover, L12/L7 molar ratio as well as peptidyltransferase activity increased with increasing growth rate. In the latter case, however, the acetylation level of L12 protein per se seemed to be inadequate to modulate the peptidyltransferase activity.     

Changes in Ribosomal Activity of Escherichia coli Cells during Prolonged Culture in Sea Salts Medium

The activity of ribosomes from a clinical isolate of Escherichia coli, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction. No alterations in the extent of peptide bond formation were observed during starvation. In contrast, a 50% reduction in the k_max/K_s ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in the k_max/K_s value. {k_max is the apparent catalytic rate constant of peptidyl transferase, and K_s is the dissociation constant of the encounter complex between acetyl (Ac)[³H]Phe-tRNA-poly(U)-ribosome and puromycin.} Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation. Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period. In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period. Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level. An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period.     

The selective release of one of the two L7/L12 dimers from the Escherichia coli ribosome induced by a monoclonal antibody to the NH2-terminal region

Two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12 were prepared and characterized as reported previously (Sommer, A., Etchison, J.R., Gavino, G., Zecherle, N., Casiano, C., and Traud, R.R. (1985) J. Biol. Chem. 260, 6522-6527). Both antibodies strongly inhibited polyuridylic acid-directed polyphenylalanine synthesis, ribosome-dependent GTPase activity, and the binding of elongation factor G to the ribosome at mole ratios over ribosomes of 4:1 or less. One epitope was shown to be within residues 1-73 (Ab 1-73) and the other within 74-120 (Ab 74-120). Incubation of 50 S ribosomal subunits or 70 S ribosomes with Ab 1-73, but not with Ab 74-120, leads to a partial loss of L7/L12 from the particle with no loss of any other protein. The experiment was repeated with ribosomes reconstituted with pure radioactive L7/L12 of determined specific activity in order to quantify the L7/L12 in the antibody-treated particle. The protein-deficient core particles isolated by sucrose gradient centrifugation after incubation with Ab 1-73 were found to contain, on average, two copies of L7/L12 and one Ab 1-73. The constancy of this stoichiometry in many experiments and the demonstration of Ab 1-73 on all particles indicate the presence of a homogeneous population of ribosomes, each with only one of the two L7/L12 dimers originally present. The results show a difference in the interactions of the two dimers with the ribosome and present a means of preparing ribosomes with one dimer in a specific binding site. The accompanying paper (Olson, H.M., Sommer, A., Tewari, D. S., Traut, R.R., and Glitz, D.G. (1986) J. Biol. Chem. 261, 6924-6932) shows by immune electron microscopy the location of the two antibody-binding sites and the effect of Ab 1-73 on structure.     

Monoclonal antibodies to epitopes in both C-terminal and N-terminal domains of Escherichia coli ribosomal protein L7/L12 inhibit elongation factor binding but not peptidyl transferase activity

Two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12, one within residues 74-120 and the other within residues 1-73, shown before to inhibit the binding of EF-G, have been tested for their effects on the binding to E. coli ribosomes of EF-Tu-aminoacyl-tRNA-GTP ternary complex and on peptidyl transferase activity. Both antibodies inhibit the binding of ternary complex and EF-Tu-dependent GTPase but have no inhibitory effect on peptidyl transferase activity. The inhibition of binding of both elongation factors is indicative of overlapping binding sites for EF-G and EF-Tu. The inhibition by both antibodies implies the contribution of both domains of L7/L12 to this binding site. This implies the location of one or more of the C-terminal domains of L7/L12 on the body of the 50S subunit. The absence of any inhibition of peptidyl transferase activity shows distinct separation of this site from the factor binding site.     

Preparation and characterization of two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12

Two monoclonal antibodies with specificities for Escherichia coli 50 S ribosomal subunit protein L7/L12 were isolated. The antibodies and Fab fragments thereof were purified by affinity chromatography using solid-phase coupled L7/L12 protein as the immunoadsorbent. The two antibodies were shown to recognize different epitopes; one in the N-terminal and the other in the C-terminal domain of protein L7/L12. Both intact antibodies strongly inhibited polyuridylic acid-directed polyphenylalanine synthesis, ribosome-dependent GTPase activity, and the binding of elongation factor EF-G to the ribosome. Ratios of antibody to ribosome of 4:1 or less were effective in inhibiting these activities. Neither antibody prevented the association of ribosomal subunits to form 70 S ribosomes. The Fab fragments showed similar effects.     

Solution structure of human P1?P2 heterodimer provides insights into the role of eukaryotic stalk in recruiting the ribosome-inactivating protein trichosanthin to the ribosome

Lateral ribosomal stalk is responsible for binding and recruiting translation factors during protein synthesis. The eukaryotic stalk consists of one P0 protein with two copies of P1•P2 heterodimers to form a P0(P1•P2)₂ pentameric P-complex. Here, we have solved the structure of full-length P1•P2 by nuclear magnetic resonance spectroscopy. P1 and P2 dimerize via their helical N-terminal domains, whereas the C-terminal tails of P1•P2 are unstructured and can extend up to ∼125 Å away from the dimerization domains. ¹⁵N relaxation study reveals that the C-terminal tails are flexible, having a much faster internal mobility than the N-terminal domains. Replacement of prokaryotic L10(L7/L12)₄/L11 by eukaryotic P0(P1•P2)₂/eL12 rendered Escherichia coli ribosome, which is insensitive to trichosanthin (TCS), susceptible to depurination by TCS and the C-terminal tail was found to be responsible for this depurination. Truncation and insertion studies showed that depurination of hybrid ribosome is dependent on the length of the proline-alanine rich hinge region within the C-terminal tail. All together, we propose a model that recruitment of TCS to the sarcin-ricin loop required the flexible C-terminal tail, and the proline-alanine rich hinge region lengthens this C-terminal tail, allowing the tail to sweep around the ribosome to recruit TCS.     

Protein binding to two-dimensional hydrophobic binding-site lattices: adsorption hysteresis on immobilized butyl-residues

Long-lived metastable states involving multiple binding sites of a protein ligand with immobilized alkyl residues on a solid phase can be observed at high ionic strength between butyl agaroses (5.21 μ mol/ml packed gel) and phosphorylase b by perturbations enforcing either the on-reaction (adsorption) or the off-reaction (desorption). These apparent equilibrium states are suggested because the adsorption isotherms of phosphorylase b on butyl agaroses are not retraced by the desorption isotherms. In this first example of macromolecular adsorption hysteresis on immobilized alkyl residues, it can be shown that the irreversible entropy (Δ_iS) produced in an adsorption-desorption cycle lies between 6 (5 μ mol/ml packed gel) and 40 (21 μ mol/ml packed gel) J mol ¹ K⁻¹. For the latter gel the apparent standard entropy of adsorption (Δ_aS_i⁰′) is 160 J mol⁻¹ K⁻¹. The metastable state observed during adsorption is probably due to an energy barrier which must be overcome for the nucleation of protein binding on the matrix. Other metastable states may possibly be encountered during desorption when the adsorbed enzyme resists the breakage of hydrophobic interactions. In the transition from the adsorption branch to the desorption branch of the hysteresis loop, the apparent affinity of the enzyme-matrix interaction is enhanced. For the desorption branch, the apparent association constant of half-maximal saturation corresponds to K_d,0.5′ = 4.2 × 10⁹ ]m⁻¹ as compared to the respective constant of adsorption K_{a, 0.5}′ = 1.6 × 10⁵m⁻¹ (gel: 21μ mol/ml packed gel). Since the area of the hysteresis loops (see also Δ_iS) depends strongly on the density of butyl residues on the gel, it is concluded that the number of alkyl residues interacting with the protein molecule is crucial for the metastable states and hysteresis. It is unlikely that hysteresis is due to the pore structure of the agarose or to nearest neighbour interactions of ligand molecules. Since thermodynamic irreversibility and hysteresis may be encountered when macromolecules, such as proteins, are adsorbed to cell membranes or cell organelles: an analysis and understanding of these phenomena should be of general biological significance.