Molecular forms of purified human erythrocyte membrane acetylcholinesterase investigated by crosslinking with diimidates.

Several molecular forms of human erythrocyte membrane acetylcholinesterase have been studied after crosslinking with bifunctional diimidates. The crosslinked products were analysed by centrifugation on linear sucrose density gradients containing Triton X-100. Molecular weights of covalently linked oligomers were estimated by sodium dodecylsulfate gel electrophoresis. It was shown that acetylcholinesterase crosslinked in absence of Triton X-100 consists of molecular forms built up by dimeric protomers. These dimers were identical with the enzymatically active species sedimenting with 6.5S in linear sucrose density gradients.     

Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by alpha-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.     

Studies on the oligomeric state of isolated cytochrome oxidase using cross-linking reagents

(1) Sucrose gradient centrifugation of cytochrome oxidase in the presence of Triton X-100 gave one slowly sedimenting green band. After cross-linking with dithiobis(succinimidylpropionate) (DSP), two green bands were observed, one sedimenting like the control and the other one more rapidly. Only the slowly sedimenting band was observed if the cross-linker was cleaved by dithiothreitol before centrifugation. (2) The rapidly sedimenting band in the Triton-containing sucrose gradient is probably the internally cross-linked dimer of cytochrome oxidase; the one sedimenting slowly is the monomeric enzyme. (3) Cross-linking with DSP after monomerization yields a small fraction of internally cross-linked dimers in addition to the internally cross-linked monomers. Under similar conditions, but using the shorter cross-linker disuccinimidyl tartarate (DST), no dimers are detected. (4) Both DSP and DST cross-link the dimeric enzyme so that it could no longer be monomerized by centrifugation in Triton, unless the cross-link is cleaved. (5) Polypeptide analysis using two-dimensional gel electrophoresis of cross-linked dimers and monomers suggest that subunit VIb is involved in intermonomeric cross-linking of dimeric enzyme by DSP.     

Partial Purification and Some Properties of the Staphylococcus aureus Cytoplasmic Nitrate Reductase

The cytoplasmic nitrate reductase in heme mutant H-14 of Staphylococcus aureus was partially purified by steps which included ammonium sulfate fractionation and chromatography on Bio-Gel A 1.5m and ion-exchange columns. The active fractions from the ion-exchange columns showed two forms of the enzyme upon electrophoresis in nondenaturing gels of polyacrylamide; these corresponded to proteins of R(f) 0.16 and 0.28. Each form contained a predominant polypeptide of molecular weight 140,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The R(f) 0.16 form contained another major polypeptide of molecular weight 57,000, but the R(f) 0.28 form contained several other polypeptides. The sedimentation properties of the enzyme were examined after partial purification on Bio-Gel A 1.5m. In sucrose gradients containing Triton X-100 the enzyme sedimented as a homogeneous peak with an estimated molecular weight of 225,000; without detergent a heterogeneous profile was observed of molecular weight greater than 250,000. Treatment of the enzyme with trypsin increased the specific activity, and the enzyme sedimented as a homogeneous peak in sucrose gradients without Triton X-100, with an estimated molecular weight of 202,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that trypsin treatment converted the polypeptide of molecular weight 140,000 to a polypeptide of molecular weight 112,000. We conclude that the cytoplasmic nitrate reductase of S. aureus has a large subunit of molecular weight 140,000, which can be modified by trypsin to a polypeptide of molecular weight 112,000 without loss of catalytic activity.     

Limited proteolysis of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The hydrogenase from Paracoccus denitrificans, which is an intrinsic membrane protein, has been solubilised from membranes by Triton X-100. The partial specific volume of the solubilised protein has been determined using sucrose density gradient centrifugation in H2O and 2H2O. The values of the specific volumes of hydrogenase, measured in the presence or absence of Triton X-100, are 0.73 and 0.74 ml . g-1, respectively, indicating that hydrogenase binds much less than one micelle of Triton X-100. The sedimentation coefficient of hydrogenase is increased from 10.4 S to 15.9 S on removal of detergent. The Stokes' radius of hydrogenase, determined by gel filtration on Sepharose 6B, is 5.5 nm in the presence of Triton X-100 compared to 6.7 nm in the absence of detergent. The apparent molecular weight therefore increases from 242,500 to 466,000 on removal of detergent. In the presence of urea and sodium dodecylsulphate, the hydrogenase has an apparent molecular weight of 63,000. The enzyme therefore behaves as a non-covalently linked tetramer in the presence of Triton X-100. Removal of Triton X-100 results in association of tetramers to form octamers.     

Herpes simplex virus 1 reiterated S component sequences (c1) situated between the a sequence and alpha 4 gene are not essential for virus replication.

The MC29 virus-coded protein p110gag-myc was found exclusively in the nucleus of transformed Japanese quail (Q8) cells, and time course experiments indicated that the protein had a half-life of about 30 min. When extracts of either Q8 or chicken embryo cells infected with MC29 virus were prepared with nondenaturing detergents and then sedimented in sucrose gradients, p110 was found in the fractions expected to contain monomers (5.9S), dimers (9.3S), or mixtures of the two. The same extracts treated with denaturing detergent (0.2% sodium dodecyl sulfate) exhibited p110 only in fractions expected for the monomeric protein, but beta-mercaptoethanol had no effect on the original distribution. Gradients prepared with 0.5 or 1.0 M NaCl failed to dissociate the faster-sedimenting form. No other protein or polyribonucleotide which could increase the sedimentation rate of p110 was found, and neither RNase nor DNase altered the sedimentation pattern of p110 in nondenatured extracts. A reassociation of monomeric p110 into dimers discernible by gel electrophoresis was demonstrated.     

Molecular form of human lymphocyte membrane-bound acetylcholinesterase

The membrane-bound acetylcholinesterase (AchE) from human peripheral blood lymphocyte gives only one symmetrical peak on sucrose density gradient centrifugation in the presence of Triton X-100 detergent, with the calculated sedimentation coefficient of 6.5 S. However, this dimeric form of AchE was converted to a monomeric 3.8 S form when treated with 2-mercaptoethanol and iodoacetic acid. The results are consistent with studies which have shown by sodium dodecyl sulfate gel electrophoresis that the enzyme is built up of two identical monomers inter-linked by disulfide bond(s). Under reducing conditions, revealed a single species of 70,000 molecular weight, whereas under non-reducing conditions, another species of 140,000 molecular weight of the AchE was found. Polyacrylamide gel electrophoresis indicated a single band with AchE activity in the presence of Triton X-100. In contrast, in the absence of the same detergent multiple band pattern could be observed. These results suggest that membrane-bound AchE enzyme is present in homogenous dimeric form on human lymphocyte membrane.     

Use of resistant mutants to study the interaction of triton X-100 with Staphylococcus aureus.

Staphylococcus aureus mutants resistant to the nonionic detergent Triton X-100, isolated from the wild-type strain H and the autolysin-deficient strain RUS3, could grow and divide in broth containing 5% (vol/vol) Triton X-100, while growth of the parental strains was markedly inhibited above the critical micellar concentration (0.02%) of the detergent. Growth-inhibitory concentrations of Triton X-100 killed wild-type cells without demonstrable cellular lysis. Triton X-100 stimulated autolysin activity of S. aureus cells under nongrowing conditions, and this lytic response was markedly reduced in energy-poisoned cells. In contrast, the detergent had no effect on the activity of autolysins in cell-free systems, and growth in the presence of Triton X-100 did not alter either the cellular autolysin activity or the susceptibility of cell walls to exogenous lytic enzymes. Treatment with either Triton X-100 or penicillin G in the growth medium stimulated release of predominantly acylated intracellular lipoteichoic acid and sensitized staphylococci to Triton X-100-induced autolysis. There was no significant difference in the cell wall and membrane compositions or Triton X-100 binding between the parental strains and the resistant mutants. The resistant mutant TXR1, derived from S. aureus H, had a higher level of L-alpha-glycerophosphate dehydrogenase activity, and its oxygen uptake was more resistant to inhibition by a submicellar concentration (0.008%) of Triton X-100. Growth in the presence of subinhibitory concentrations of Triton X-100 rendered S. aureus H cells phenotypically resistant to the detergent and greatly stimulated the level of oxygen uptake. Membranes isolated from such cells exhibited enhanced activity of the respiratory enzymes succinic dehydrogenase and L-alpha-glycerophosphate dehydrogenase.     

Detergent-solubilized bovine cytochrome c oxidase: dimerization depends on the amphiphilic environment

The extent to which bovine cytochrome c oxidase (COX) dimerizes in nondenaturing detergent environments was assessed by sedimentation velocity and equilibrium. In contrast to generally accepted opinion, the COX dimer is difficult to maintain and is the major oligomeric form only when COX is solubilized with a low concentration of dodecylmaltoside, i.e., approximately 1 mg/mg protein. The dimer form is intrinsically unstable and dissociates into monomers with increased detergent concentration, i.e., >5 mg/mg protein. The structure of the solubilizing detergent, however, greatly alters detergent effectiveness by inducing either monomerization or aggregation. Triton X-100 is most effective at solubilizing COX, but it destabilizes COX dimers, even at low concentration. Undecylmaltoside, decylmaltoside, and octaethyleneglycolmonododecyl ether (C(12)E(8)) are less effective at solubilizing COX. Each prevents COX aggregation at high detergent concentration, but also destabilizes the COX dimer. Other detergents, e.g., Tween 20, sodium cholate, sodium deoxycholate, CHAPS, or CHAPSO, are completely ineffective COX solubilizers and do not prevent aggregation even at 10-40 mg/mL. The transition from dimers to monomers depends on many factors other than detergent structure and concentration, e.g., protein concentration, phospholipid content and pH. We conclude that the intrinsic dimeric structure of COX can be maintained only after solubilization with low concentrations of dodecylmaltoside at near neutral pH, and even then precautions must be taken to prevent its dissociation into monomers.     

Organization and structure of two mixed micellar phases of the sphingomyelin/Triton X-305 system

Properties of mixed dispersions of sphingomyelin and the nonionic detergent, Triton X-305, were investigated by analytical ultracentrifugation and by autocorrelation spectroscopy of scattered laser light. These properties were compared with those of the sphingomyelin/Triton X-100 mixed micellar system reported previously [S. Yedgar, Y. Barenholz, and V. G. Cooper (1974) Biochim. Biophys. Acta 363, 98-111]. The substitution of the 30-unit ethylene oxide chain of Triton X-305 for the 10-unit chain of the Triton X-100 resulted in the appearance of two micellar phases at all detergent/lipid mixture ratios studied, whereas only a single mixed micellar phase was observed using Triton X-100. Despite this difference, the properties of the mixed lipid/detergent micelles obtained using Triton X-100 have been verified in the following respects: The detergent aggregation numbers in the mixed micelles are quite constant over a wide range of detergent molar fractions, being about 70 and 400 for the lighter and heavier mixed micellar phases, respectively. The detergent aggregation numbers are larger in the mixed micelle than in the pure detergent micelle. Very large sphingomyelin aggregation numbers can be accommodated within the mixed micelles, apparently by the critical intervention of the detergent molecules to produce a stable micellar structure.     

Proportional equilibration of K, Na ions, and sucrose molecules in pig lenses incubated in the presence of the non-ionic detergent Triton X-100

The release of sodium and potassium and the uptake of sucrose molecules was studied in pig lenses incubated in isosmotic sucrose solution in either the presence or absence of 1% Triton X-100 (a non-ionic detergent). This Triton X-100 treatment has been shown to cause severe disruptions of cell membrane integrity. If sodium and potassium were free in the lens fibers as in a dilute aqueous solution, they would be expected to diffuse three to four times faster than sucrose. However, measurements of sodium and potassium release and sucrose uptake in the Triton X-100 treated lenses show a 1:1 equilibration. When pig lenses were incubated in the same solution without detergent, the sucrose uptake was significantly less than the potassium and sodium release. It is postulated that a slow, detergent mediated collapse of protein-water-ion interactions within the lens is the rate-limiting step of the observed equilibration of monovalent cations and sucrose molecules.     

Phosphatidylglycerophosphate synthease and phosphatidylserine synthase activites in Clostridium perfringens

Cytidine 5'-diphospho (CDP)-1,2-diacyl-sn-glycerol (CDPdiacylglycerol):sn-glycerol-3-phosphate phosphatidyltransferase (EC 2.7.8.5, phosphatidylglycero-P synthase) and CDPdiacylglycerol:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthase) activities were identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of phosphatidylglycero-P synthase and phosphatidylserine synthase with the cell envelope fraction of cell-free extracts was demonstrated by sucrose density gradient centrifugation, by both activities sedimenting with the 100,000 x g pellet and solubilization of both activities from the 100,000 x g pellet with Triton X-100. The pH optimum for both enzyme activities was 8.0 with tris(hydroxy-methyl)aminomethane-hydrochloride buffer. Phosphatidylglycero-P synthase activity was dependent on magnesium ions (100 mM). Phosphatidylserine synthase activity was dependent on manganese (0.1 mM) or magnesium ions (50 mM). Both enzyme activities were dependent on the addition of the nonionic detergent Triton X-100. Maximum phosphatidylglycero-P synthase and phosphatidylserine synthase activities were obtained when the molar ratio of Triton X-100 to CDP-diacylglycerol was 50:1 and 12:1, respectively. The Km for sn-glycero-3-P in the phosphatidylglycero-P synthase reaction was 0.1 mM. The Km for L-serine in the phosphatidylserine synthase reaction was 0.15 mM. Both enzyme activities were 100% stable for at least 20 min at 60 degrees C.     

Correlation between active form and dimeric structure of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart

The active form of purified mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated by crosslinking with dimethylsuberimidate and SDS-PAGE, with or without pretreatment with the inactivating detergent Triton X-100. In the absence of detergent, crosslinked isomers of the dimeric form of 208–235 kDa were obtained. Addition of detergent led to the simultaneous loss of the dimers and the bulk of the activity. Removal of the detergent led to a partial restoration of both activity and the dimeric forms. The results suggest that the active form is a dimer, and that the detergent-dependent conversion to the largely inactive monomer is reversible. It is proposed that the mechanism of inactivation of transhydrogenase by Triton X-100 involves a disruption of essential hydrophobic interactions between the membrane-spanning regions of the monomers.     

Amphiphilic,glycophosphatidylinositol-specific phospholipase C (PI-PLC)-insensitive monomers and dimers of acetylcholinesterase

1. In a recent study, we distinguished two classes of amphiphilic AChE3 dimers in Torpedo tissues: class I corresponds to glycolipid-anchored dimers and class II molecules are characterized by their lack of sensitivity to PI-PLC and PI-PLD, relatively small shift in sedimentation with detergent, and absence of aggregation without detergent. 2. In the present report, we analyze the amphiphlic or nonamphiphilic properties of globular AChE forms in T28 murine neural cells, rabbit muscle, and chicken muscle. The molecular forms were identified by sucrose gradient sedimentation in the presence and absence of detergent and analyzed by nondenaturing charge-shift electrophoresis. Some amphiphilic forms showed an abnormal electrophoretic migration in the absence of detergent, because of the retention of detergent micelles. 3. We show that the amphiphilic monomers (G1a) from these tissues, as well as the amphiphilic dimers (G2a) from chicken muscle, resemble the class II dimers of Torpedo AChE. We cannot exclude that these molecules possess a glycolipidic anchor but suggest that their hydrophobic domain may be of a different nature. We discuss their relationship with other cholinesterase molecular forms.     

Ciliary membrane tubulin and associated proteins: a complex stable to Triton X-114 dissociation

When either membranes from scallop gill cilia or reconstituted membranes from the same source are solubilized with Triton X-114 and the detergent is condensed by warming, no significant fraction of any major membrane protein partitions into the micellar detergent. Rather, most of the membrane lipids condense with the detergent phase, forming mixed micelles from which nearly pure lipid vesicles may be produced by adsorption of detergent with polystyrene beads. One minor membrane protein, with a molecular weight of about 20 000, is associated consistently with these vesicles. The aqueous phase contains a fairly homogeneous protein-Triton X-114 micelle sedimenting at 2.6 S in the analytical ultracentrifuge. Sucrose gradient velocity analysis in a detergent-free gradient indicates moderate size polydispersity but constant polypeptide composition throughout the sedimenting protein zone. Sucrose gradient equilibrium analysis (also in a detergent-free gradient) results in a protein-detergent complex banding at a density of 1.245 g/cm3. Sedimentation of the protein-detergent complex in the ultracentrifuge, followed by fixation and normal processing for electron microscopy, reveals a fine, reticular material consisting of 5-10-nm granules. These data are consistent with previous evidence that membrane tubulin and most other membrane proteins exist together as a discrete lipid-protein complex in molluscan gill ciliary membranes.     

Molecular weight of detergent-solubilized prostaglandin-F2 alpha receptor from bovine corpora lutea

A prostaglandin F2 alpha receptor localized in plasma membranes of bovine corpus luteum cells was solubilized by treatment with Triton X-100. Sepharose chromatographies of ([3H]prostaglandin F2 alpha)-receptor complex gave a Stokes' radius of 630 nm. In the absence of detergent, aggregated forms of the receptor appeared. Sedimentation experiments of solubilized receptor in sucrose/H2O and sucrose/2H2O density gradients gave the following values: sedimentation coefficient (S20, w) 4.6 S; partial specific volume (VB) 0.78 cm3/g and frictional ratio (f/fo) 1.6. Based on the sedimentation coefficient and the Stokes' radius and assuming that the receptor is a non-glycosylated protein the molar mass of the receptor-(Triton X-100) complex was 144000 g/mol. The VB value indicated that ca. 26% of the weight represented bound detergent and that the molecular weight of the prostaglandin F2 alpha receptor is approximately 107000.     

Heterogeneity of Rat Brain Acetylcholinesterase: A Study by Gel Filtration and Gradient Centrifugation

Abstract: According to their solubilization properties, two classes of acetyl-cholinesterases (AChE) can be detected in the adult rat brain: a "soluble" species (easily solubilized without detergent), and a membrane-bound species (solubilized only in the presence of detergent). The latter was found to be homogeneous by gel filtration (Stokes radius 8.05 ± 0.35 nm) and sucrose gradient centrifugation (9.75 ± 0.2 S) in the presence of Triton X-100. The "soluble" AChE gives three stable species in the presence of the same detergent with Stokes radii and sedimentation constants of 10.9 ± 0.5 nm and 16 ± 2 S; 6.75 ± 0.30 nm and 10.7 ± 0.4 S; 5.37 ± 0.35 nm and 4.37 ± 0.1 S. Co-chromatography and co-sedimentation or the reduction and alkylation of disulfide bridges show that all the soluble species are different from the membrane-bound AChE. The possibility that soluble and membrane-bound AChE are completely different molecules is discussed.     

Quantitative Development and Molecular Forms of Acetyl-and Butyrylcholinesterase During Morphogenesis and Synaptogenesis of Chick Brain and Retina

The embryonic development of total specific activities as well as of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and of butyrylcholinesterase (BChE, EC 3.1.1.8) have been studied in the chick brain. A comparison of the development in different brain parts shows that cholinesterases first develop in diencephalon, then in tectum and telencephalon; cholinesterase development in retina is delayed by about 2-3 days; and the development in rhombencephalon [not studied until embryonic day 6 (E6)] and cerebellum is last. Both enzymes show complex and independent developmental patterns. During the early period (E3-E7) first BChE expresses high specific activities that decline rapidly, but in contrast AChE increases more or less constantly with a short temporal delay. Thereafter the developmental courses approach a late phase (E14-E20), during which AChE reaches very high specific activities and BChE follows at much lower but about parallel levels. By extraction of tissues from brain and retina in high salt plus 1% Triton X-100, we find that both cholinesterases are present in two major molecular forms, AChE sedimenting at 5.9S and 11.6S (corresponding to G2 and G4 globular forms) and BChE at 2.9S and 10.3S (G1 and G4, globular). During development there is a continuous increase of G4 over G2 AChE, the G4 form reaching 80% in brain but only 30% in retina. The proportion of G1 BChE in brain remains almost constant at 55%, but in retina there is a drastic shift from 65% G1 before E5 to 70% G4 form at E7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strandedness and Complementarity of DNA from Long-Term RNA-Dependent DNA Polymerase Reactions of Soehner-Dmochowski Murine Sarcoma Virus

The DNA product of the endogenously instructed RNA-dependent DNA polymerase reaction of murine sarcoma virus continued to be synthesized for as long as 64 h in the presence of 0.008% Triton X-100. Higher detergent concentrations and actinomycin D inhibited DNA product synthesis. The DNA product from long-term polymerase reactions consisted of small DNA fragments as shown by sedimentation in alkaline sucrose gradients. The enzymatic DNA product was separated into a slow sedimenting fraction and a fast sedimenting fraction by rate-zonal centrifugation. Fast sedimenting DNA was the predominant fraction made in viral polymerase reactions containing 262 mM NaCl. By using a combination of S-1 nuclease and pancreatic RNase A, the amount of single-stranded DNA, double-stranded DNA, and DNA-RNA hybrid present in the slow-sedimenting and fast-sedimenting fractions was determined. Under standard polymerase conditions of 70 mM NaCl, single-stranded DNA was the major form of DNA found in both fractions. In contrast, the prevalent form of DNA made in the presence of 262 mM NaCl was DNA-RNA hybrid. Hybridization studies in which either S-1 nuclease or pancreatic RNase A was used to measure hybrid formation demonstrated not only that the DNA product was complementary in base sequence to the RNA genome, but also that at least 79 to 84% of the RNA genome was transcribed into complementary DNA.     

Dynamic nature of the quaternary structure of the vesicular stomatitis virus envelope glycoprotein

The envelope glycoprotein (G protein) of vesicular stomatitis virus probably exists in the viral envelope as a trimer of identical subunits. Depending on the conditions of solubilization, G protein may dissociate into monomers. G protein solubilized with the detergent octyl glucoside was shown to exist as oligomeric forms by sedimentation velocity analysis and chemical cross-linking. G protein was modified with either fluorescein isothiocyanate or rhodamine isothiocyanate. Resonance energy transfer between fluorescein and rhodamine labels was observed upon mixing the two labeled G proteins in octyl glucoside. This result provided further evidence that G protein in octyl glucoside is oligomeric and indicated that the subunits are capable of exchange to form mixed oligomers. Resonance energy transfer was independent of G protein concentration in the range examined (10-80 nM) and was not observed when labeled G proteins were mixed with fluorescein or rhodamine that was not conjugated to protein. Resonance energy transfer decreased upon incorporation of G protein into Triton X-100, consistent with sedimentation velocity data that G protein in Triton X-100 is primarily monomeric. Kinetic analysis showed that the subunit exchange reaction had a half-time of about 3 min at 27 degrees C that was independent of G protein concentration. These data indicate that the exchange occurs through dissociation of G protein trimers into monomers and dimers followed by reassociation into timers. Thus, in octyl glucoside, G protein must exist as an equilibrium between monomers and oligomers. This implies that monomers are capable of self-assembly into trimers.