Nucleotide dependent monomer/dimer equilibrium of OpuAA, the nucleotide-binding protein of the osmotically regulated ABC transporter OpuA from Bacillus subtilis

The OpuA system of Bacillus subtilis is a member of the substrate-binding-protein-dependent ABC transporter superfamily and serves for the uptake of the compatible solute glycine betaine under hyperosmotic growth conditions. Here, we have characterized the nucleotide-binding protein (OpuAA) of the B.subtilis OpuA transporter in vitro. OpuAA was overexpressed heterologously in Escherichia coli as a hexahistidine tag fusion protein and purified to homogeneity by affinity and size exclusion chromatography (SEC). Dynamic monomer/dimer equilibrium was observed for OpuAA, and the K(D) value was determined to be 6 microM. Under high ionic strength assay conditions, the monomer/dimer interconversion was diminished, which enabled separation of both species by SEC and separate analysis of both monomeric and dimeric OpuAA. In the presence of 1 M NaCl, monomeric OpuAA showed a basal ATPase activity (K(M)=0.45 mM; k(2)=2.3 min(-1)), whereas dimeric OpuAA showed little ATPase activity under this condition. The addition of nucleotides influenced the monomer/dimer ratio of OpuAA, demonstrating different oligomeric states during its catalytic cycle. The monomer was the preferred species under post-hydrolysis conditions (e.g. ADP/Mg(2+)), whereas the dimer dominated the nucleotide-free and ATP-bound states. The affinity and stoichiometry of monomeric or dimeric OpuAA/ATP complexes were determined by means of the fluorescent ATP-analog TNP-ATP. One molecule of TNP-ATP was bound in the monomeric state and two TNP-ATP molecules were detected in the dimeric state of OpuAA. Binding of TNP-ADP/Mg(2+) to dimeric OpuAA induced a conformational change that led to the decay of the dimer. On the basis of our data, we propose a model that couples changes in the oligomeric state of OpuAA with ATP hydrolysis.     

Phosphofructokinase from lycopersicon esculentum fruits-II. Changes in the regulatory properties with dissociation

The regulatory properties of PFK. from the tomato are discussed in relation to the dissociation of the oligomeric form of the enzyme. Both the oligomeric and monomeric forms of PFK were inhibited by citrate, malate, PEP, 2-phosphoglycerate, phosphoglycolate and ammonium sulphate. PEP was the most potent inhibitor of PFK activity with 9 and 10 μn PEP causing 50%, inhibition of the oligomeric and monomeric forms of PFK respectively. The inhibition by all these metabolites of the oligomeric form of PFK was sigmoidal while their inhibition of the monomeric form was hyperbolic. The magnitude of inhibition by these metabolites is affected by the levels of Mg²⁺. The oligomeric form of the enzyme is more resistant to citrate inhibition than the monomeric form. In the presence of citrate or ammonium sulphate, the kinetics of the oligomeric form of PFK with F6P yielded positive cooperativity while in their absence, the kinetics revealed negative cooperative interactions. Phosphoenolpyruvate had no effect on the nature of the kinetics with F6P. ADP is stimulatory to the oligomeric form while it is slightly inhibitory to the monomeric form. The significance of these properties and their relation with the regulation of PFK activity in vivo are discussed.     

Spatial extent of charge repulsion regulates assembly pathways for lysozyme amyloid fibrils

Formation of large protein fibrils with a characteristic cross β-sheet architecture is the key indicator for a wide variety of systemic and neurodegenerative amyloid diseases. Recent experiments have strongly implicated oligomeric intermediates, transiently formed during fibril assembly, as critical contributors to cellular toxicity in amyloid diseases. At the same time, amyloid fibril assembly can proceed along different assembly pathways that might or might not involve such oligomeric intermediates. Elucidating the mechanisms that determine whether fibril formation proceeds along non-oligomeric or oligomeric pathways, therefore, is important not just for understanding amyloid fibril assembly at the molecular level but also for developing new targets for intervening with fibril formation. We have investigated fibril formation by hen egg white lysozyme, an enzyme for which human variants underlie non-neuropathic amyloidosis. Using a combination of static and dynamic light scattering, atomic force microscopy and circular dichroism, we find that amyloidogenic lysozyme monomers switch between three different assembly pathways: from monomeric to oligomeric fibril assembly and, eventually, disordered precipitation as the ionic strength of the solution increases. Fibril assembly only occurred under conditions of net repulsion among the amyloidogenic monomers while net attraction caused precipitation. The transition from monomeric to oligomeric fibril assembly, in turn, occurred as salt-mediated charge screening reduced repulsion among individual charged residues on the same monomer. We suggest a model of amyloid fibril formation in which repulsive charge interactions are a prerequisite for ordered fibril assembly. Furthermore, the spatial extent of non-specific charge screening selects between monomeric and oligomeric assembly pathways by affecting which subset of denatured states can form suitable intermolecular bonds and by altering the energetic and entropic requirements for the initial intermediates emerging along the monomeric vs. oligomeric assembly path.     

Phosphofructokinase and ripening in lycopersicon esculentum fruits

The relative activity and the kinetic properties of phosphofructokinase (PFK) during the various stages of tomato ripening were investigated. There were no significant changes in the extractable activity of the enzyme during ripening but there was an apparent change in the molecular form of the enzyme as the fruits entered the climacteric and senescence stages. While the enzyme extracted from preclimacteric fruit existed solely in an oligomeric form, that extracted from fruits in the later stages of ripening was present as a mixture of the monomeric and oligomeric forms. Changes in the regulatory properties of the enzyme extracted at the various stages of ripening were explicable in terms of the dissociation of the oligomeric form of the enzyme to smaller units. PFK from the preclimacteric fruit is more resistant to inhibition by citrate and salts than the enzyme from the post climacteric fruit. On the other hand, the preclimacteric enzyme is stimulated by Pi and ADP while the post climacteric enzyme is not. The significance of these effects in relation to the physiology of tomato ripening and senescence is discussed.     

A Monomeric Variant of Insulin Degrading Enzyme (IDE) Loses Its Regulatory Properties

Background

Insulin degrading enzyme (IDE) is a key enzyme in the metabolism of both insulin and amyloid beta peptides. IDE is unique in that it is subject to allosteric activation which is hypothesized to occur through an oligomeric structuture.

Methodology/Principal Findings

IDE is known to exist as an equilibrium mixture of monomers, dimers, and higher oligomers, with the dimer being the predominant form. Based on the crystal structure of IDE we deleted the putative dimer interface in the C-terminal region, which resulted in a monomeric variant. Monomeric IDE retained enzymatic activity, however instead of the allosteric behavior seen with wild type enzyme it displayed Michaelis-Menten kinetic behavior. With the substrate Abz-GGFLRKHGQ-EDDnp, monomeric IDE retained ∼25% of the wild type activity. In contrast with the larger peptide substrates β-endorphin and amyloid β peptide 1–40, monomeric IDE retained only 1 to 0.25% of wild type activity. Unlike wild type IDE neither bradykinin nor dynorphin B-9 activated the monomeric variant of the enzyme. Similarly, monomeric IDE was not activated by polyphosphates under conditions in which the activity of wild type enzyme was increased more than 50 fold.

Conclusions/Significance

These findings serve to establish the dimer interface in IDE and demonstrate the requirement for an oligomeric form of the enzyme for its regulatory properties. The data support a mechanism where the binding of activators to oligomeric IDE induces a conformational change that cannot occur in the monomeric variant. Since a conformational change from a closed to a more open structure is likely the rate-determining step in the IDE reaction, the subunit induced conformational change likely shifts the structure of the oligomeric enzyme to a more open conformation.     

Characterization of monomeric L1 metallo-beta -lactamase and the role of the N-terminal extension in negative cooperativity and antibiotic hydrolysis

The L1 metallo-beta-lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in beta-lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 beta-lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form but demonstrated greatly reduced rates of beta-lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higher K(m) values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K(m) values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for beta-lactam hydrolysis, it is clearly important to its activity and substrate specificity.     

Structure and dynamics of UBA5-UFM1 complex formation showing new insights in the UBA5 activation mechanism

Ubiquitin fold modifier 1 (UFM1) is an ubiquitin-like protein (Ubl) involved especially in endoplasmic stress response. Activation occurs via a three-step mechanism like other Ubls. Data obtained reveal that UFM1 regulates the oligomeric state of ubiquitin activating enzyme 5 (UBA5) to initiate the activation step. Mixtures of homodimers and heterotrimers are observed in solution at the equilibrium state, demonstrating that the UBA5-UFM1 complex undergoes several concentration dependent oligomeric translational states to form a final functional complex. The oligomerization state of unbound UBA5 is also concentration dependent and shifts from the monomeric to the dimeric state. Data describing different oligomeric states are complemented with binding studies that reveal a negative cooperativity for the complex formation and thereby provide more detailed insights into the complex formation mechanism.     

A monomer form of the glutathione S-transferase Y7F mutant from Schistosoma japonicum at acidic pH

Dissociation and unfolding of homodimeric glutathione S-transferase Y7F mutant from Schistosoma japonicum (SjGST-Y7F) were investigated at equilibrium using urea as denaturant. The conserved residue Tyr7 plays a central role in the catalytic mechanism and the mutation Tyr-Phe yields an inactive enzyme that is able to bind the substrate GSH with a higher binding constant than the wild type enzyme. Mutant SjGST-Y7F is a dimer at pH 6 or higher and a stable monomer at pH 5 that binds GSH (K value of 1.2x10(5)+/-6.4x10(3)M(-1) at pH 6.5 and 6.3x10(4)+/-1.25x10(3)M(-1) at pH 5). The stability of the SjGST-Y7F mutant was studied by urea induced unfolding techniques (DeltaG(W)=13.86+/-0.63kcalmol(-1) at pH 6.5 and DeltaG(W)=11.22+/-0.25kcalmol(-1) at pH 5) and the monomeric form characterized by means of size exclusion chromatography, fluorescence, and electrophoretic techniques.     

Effect of dimer dissociation on activity and thermostability of the alpha-glucuronidase from Geobacillus stearothermophilus: dissecting the different oligomeric forms of family 67 glycoside hydrolases

The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. alpha-Glucuronidases are family 67 glycosidases that cleave the alpha-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of alpha-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the alpha-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial alpha-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in alpha-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35 degrees C, compared to 65 degrees C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9 degrees C, was almost identical to that of the wild-type, 73.4 degrees C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed.     

Structure and function of threonine synthase from yeast.

Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 A resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open alpha/beta architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the beta-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.     

Fluorescence studies on calmodulin binding to erythrocyte Ca2(+)-ATPase in different oligomerization states

The fluorescent spinach calmodulin derivative 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin (MIANS-CaM) was used to investigate calmodulin interaction with the purified, detergent-solubilized erythrocyte Ca2(+)-ATPase. Previous studies have shown that the Ca2(+)-ATPase exists in equilibria between monomeric and oligomeric forms. We report here that MIANS-CaM binds to both enzyme forms in a Ca2(+)-dependent manner, with a approximately 50% fluorescence enhancement. These findings confirm our previous observation that enzyme oligomers retain their ability to bind calmodulin, even though they are fully activated in the absence of calmodulin. The Ca2+ dependence of MIANS-CaM binding to monomeric Ca2(+)-ATPase is of higher affinity (K 1/2 = 0.09 microM Ca2+) and less cooperative (nH = 1.1) than the Ca2+ dependence of enzyme activation by MIANS-CaM (K 1/2 = 0.26 microM Ca2+, nH = 2.8). These Ca2+ dependences and the order of events, in which calmodulin binding precedes enzyme activation, demonstrate that calmodulin indeed could be a physiological activator of the monomeric enzyme. The calcium dependence of calmodulin binding to oligomeric Ca2(+)-ATPase occurs at even lower levels of Ca2+ (K 1/2 = 0.04 microM Ca2+), in a highly cooperative fashion (nH = 2.3), and essentially in parallel with enzyme activation (K 1/2 = 0.05 microM Ca2+, nH = 2.9). The observed differences between monomers and oligomers suggest that the oligomerized Ca2(+)-ATPase is in a conformation necessary for efficient, cooperative calcium binding at nanomolar Ca2+, which the monomeric enzyme acquires only upon interaction with calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of chaperonin GroEL by a monomer of ovine prion protein and its oligomeric forms

The possibility of inhibition of chaperonin functional activity by amyloid proteins was studied. It was found that the ovine prion protein PrP as well as its oligomeric and fibrillar forms are capable of binding with the chaperonin GroEL. Besides, GroEL was shown to promote amyloid aggregation of the monomeric and oligomeric PrP as well as PrP fibrils. The monomeric PrP was shown to inhibit the GroEL-assisted reactivation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The oligomers of PrP decelerate the GroEL-assisted reactivation of GAPDH, and PrP fibrils did not affect this process. The chaperonin GroEL is capable of interacting with GAPDH and different PrP forms simultaneously. A possible role of the inhibition of chaperonins by amyloid proteins in the misfolding of the enzymes involved in cell metabolism and in progression of neurodegenerative diseases of amyloid nature is discussed.     

Conversion of the noncooperative Bacillus subtilis aspartate transcarbamoylase into a cooperative enzyme by a single amino acid substitution.

Allosteric enzymes are part of a unique class of enzymes which regulate metabolic pathways. On the molecular level, allosteric regulation is the result of interactions between discrete binding sites on the enzyme. In order to accommodate these multiple binding sites, allosteric enzymes have evolved with oligomeric quaternary structures. However, only a few oligomeric enzymes are known to have regulatory interactions between binding sites. Is regulatory activity an inherent property of oligomeric enzymes? The trimeric Bacillus subtilis aspartate transcarbamoylase catalyzes the first committed step of the pyrimidine biosynthetic pathway and is not known to be a regulatory enzyme. When an alanine residue is substituted for the active-site residue Arg-99 by site-specific mutagenesis, the regulatory activity of homotropic substrate cooperativity (Hill coefficient of 1.5) is observed in the resulting mutant enzyme. These results suggest that homotropic regulation may have evolved by a relatively small number of mutations to an oligomeric enzyme.     

Homomeric and heteromeric interactions of the extracellular domains of death receptors and death decoy receptors

Death receptors (DRs) can induce apoptosis by oligomerization with TRAIL, whereas death decoy receptors (DcRs) cannot, due to their lack of functional intracellular death domains. However, it is not known whether DRs and DcRs can interact with one another to form oligomeric complexes prior to TRAIL binding. To address this issue, the extracellular domains (ECDs) of DR4 (sDR4), DR5 (sDR5), DcR1 (sDcR1), and DcR2 (sDcR2) were expressed in a soluble, monomeric form, and their binding interactions were quantified by surface plasmon resonance. The purified sDRs and sDcRs exhibited native-like secondary structure and bound to TRAIL with binding affinities in the nanomolar range (K(D)= approximately 10-62 nM), suggesting that they were properly folded and functional. The soluble receptors interacted homophilically and heterophilically with similar micromolar range affinities (K(D)= approximately 1-9 microM), with the exception that sDR5 did not interact with the sDcRs. Our results suggest that most DRs and DcRs can laterally interact through their ECDs to form homomeric and/or heteromeric complexes in the absence of TRAIL binding.     

Phytase activity in tobacco (Nicotiana tabacum) root exudates is exhibited by a purple acid phosphatase

Phytases are enzymes that catalyze liberation of inorganic phosphates from phytate, the major organic phosphorus in soil. Tobacco (Nicotiana tabacum) responds to phosphorus starvation with an increase in extracellular phytase activity. By a three-step purification scheme, a phosphatase with phytase activity was purified 486-fold from tobacco root exudates to a specific activity of 6,028 nkat mg(-1) and an overall yield of 3%. SDS-PAGE revealed a single polypeptide of 64 kDa, thus indicating apparent homogeneity of the final enzyme preparation. Gel filtration chromatography suggested that the enzyme was a ca. 56 kDa monomeric protein. De novo sequencing by tandem mass spectrometry resulted in a tryptic peptide sequence that shares high homology with several plant purple acid phosphatases. The identity of the enzyme was further confirmed by molybdate-inhibition assay and cDNA cloning. The purified enzyme exhibited pH and temperature optima at 5.0-5.5 and 45 degrees C, respectively, and were found to have high affinities for both p-nitrophenyl phosphate (pNPP; K(m)=13.9 microM) and phytate (K(m)=14.7 microM), but a higher kcat for pNPP (2,056 s(-1)) than phytate (908 s(-1)). Although a broad specificity of the enzyme was observed for a range of physiological substrates in soil, maximum activity was achieved using mononucleotides as substrates. We conclude that the phytase activity in tobacco root exudates is exhibited by a purple acid phosphatase and its catalytic properties are pertinent to its role in mobilizing organic P in soil.     

Uronic Acid products release from enzymically active cell wall from tomato fruit and its dependency on enzyme quantity and distribution

Isolated cell wall from tomato (Lycopersicon esculentum Mill. cv Rutgers) fruit released polymeric (degree of polymerization [DP] > 8), oligomeric, and monomeric uronic acids in a reaction mediated by bound polygalacturonase (PG) (EC 3.2.1.15). Wall autolytic capacity increased with ripening, reflecting increased levels of bound PG; however, characteristic oligomeric and monomeric products were recovered from all wall isolates exhibiting net pectin release. The capacity of wall from fruit at early ripening (breaker, turning) to generate oligomeric and monomeric uronic acids was attributed to the nonuniform ripening pattern of the tomato fruit and, consequently, a locally dense distribution of enzyme in wall originating from those fruit portions at more temporally advanced stages of ripening. Artificial autolytically active wall, prepared by permitting solubilized PG to bind to enzymically inactive wall from maturegreen fruit, released products which were similar in size characteristics to those recovered from active wall isolates. Extraction of wall-bound PG using high concentrations of NaCl (1.2 molar) did not attenuate subsequent autolytic activity but greatly suppressed the production of oligomeric and monomeric products. An examination of water-soluble uronic acids recovered from ripe pericarp tissue disclosed the presence of polymeric and monomeric uronic acids but only trace quantities of oligomers. The significance in autolytic reactions of enzyme quantity and distribution and their possible relevance to in vivo pectin degradation will be discussed.     

Structure and evolution of a novel dimeric enzyme from a clinically important bacterial pathogen

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine biosynthetic pathway. The tetrameric structure of DHDPS is thought to be essential for enzymatic activity, as isolated dimeric mutants of Escherichia coli DHDPS possess less than 2.5% that of the activity of the wild-type tetramer. It has recently been proposed that the dimeric form lacks activity due to increased dynamics. Tetramerization, by buttressing two dimers together, reduces dynamics in the dimeric unit and explains why all active bacterial DHDPS enzymes to date have been shown to be homo-tetrameric. However, in this study we demonstrate for the first time that DHDPS from methicillin-resistant Staphylococcus aureus (MRSA) exists in a monomer-dimer equilibrium in solution. Fluorescence-detected analytical ultracentrifugation was employed to show that the dimerization dissociation constant of MRSA-DHDPS is 33 nm in the absence of substrates and 29 nm in the presence of (S)-aspartate semialdehyde (ASA), but is 20-fold tighter in the presence of the substrate pyruvate (1.6 nm). The MRSA-DHDPS dimer exhibits a ping-pong kinetic mechanism (k(cat)=70+/-2 s(-1), K(m)(Pyruvate)=0.11+/-0.01 mm, and K(m)(ASA)=0.22+/-0.02 mm) and shows ASA substrate inhibition with a K(si)(ASA) of 2.7+/-0.9 mm. We also demonstrate that unlike the E. coli tetramer, the MRSA-DHDPS dimer is insensitive to lysine inhibition. The near atomic resolution (1.45 A) crystal structure confirms the dimeric quaternary structure and reveals that the dimerization interface of the MRSA enzyme is more extensive in buried surface area and noncovalent contacts than the equivalent interface in tetrameric DHDPS enzymes from other bacterial species. These data provide a detailed mechanistic insight into DHDPS catalysis and the evolution of quaternary structure of this important bacterial enzyme.     

Methionyl-tRna synthetase from wheat germ. Effect of an endogenous protease and correlations between structural characteristics and catalytic properties

Methionyl-tRNA synthetase (MetRS) has been described as a free monomeric or oligomeric enzyme; or included in a multienzyme complex. Moreover, on limited tryptic digestion, it can generate shorter forms. So, when purified from wheat-germ lysate, the possible presence of proteases able to hydrolyse this enzyme was investigated. When extraction was performed with sulfhydryl-blocking reagents, an active monomeric MetRS of Mr 105,000 was purified. This enzyme form was identical to the structure exhibiting methionyl-tRNA synthetase activity in multienzyme complexes. Without this inhibitor, MetRS was purified as an active dimeric form of Mr 165,000 with identical subunits of Mr 82,000. A protease inhibited by sulfhydryl-blocking reagents and included in a complex of Mr 2.10(6) was isolated from this wheat-germ lysate. This protease was able to hydrolyse different proteins (albumin, casein), but was without activity for a trypsin substrate, such as N-alpha-benzoyl-DL-arginine p-nitroanilide. When added to a solution of Mr-105,000 MetRS, it yielded an inactive peptide of Mr 20,000, containing numerous charged amino acids and a protein of Mr 82,000, able to give an active dimeric enzyme of Mr 165,000. Amino acid analysis of this last form, indicated an identical structure with the active dimeric MetRS of Mr 165,000, purified in the absence of protease inhibitors. Moreover, the affinity for methionine was the same for the monomeric enzyme of Mr 105,000 and the dimeric form of Mr 165,000, probably because proteolysis did not affect the catalytic domain. When enzymic activity of the proteolyzed form (Mr 2 x 82,000) was studied versus enzyme concentration, a decrease in specific activity, at low concentrations, was seen. This phenomenon was analysed on the basis of the existence of an equilibrium between an active dimer and two inactive monomers. With the active monomeric form of Mr 105,000, no change in specific activity with decreasing enzyme concentration occurred.     

Circular mitochondrial DNA molecules from petite mutants of Saccharomyces cerevisiae: resolution by polyacrylamide gel electrophoresis

Mitochondrial DNA (mtDNA) from petite strain K45 ofSaccharomyces cerevisiae contains about 7% circular DNA molecules which comprise a simple oligomeric series based on a monomeric size of 1.7 kilobase pairs. Electrophoresis of K45 mtDNA on a polyacrylamide-agarose slab gel fractionates the mtDNA into a major band (containing linear DNA) and several faster running minor bands each containing particular size class of circular DNA molecules. From study of mtDNA from K45 and two other simple petites it was found that the mobility of circles is inversely proportional to the logarithm of the circle size. Polyacrylamide gel electrophoresis thus permits the separation of circular mtDNA from the linear mtDNA of simple petites, and physically resolves circles of different size from one another.     

The Role of Acyl Lipids in Reconstitution of Lipid-Depleted Light-Harvesting Complex II from Cold-Hardened and Nonhardened Rye

The role of acyl lipids in the in vitro stabilization of the oligomeric form of light-harvesting complex II of winter rye (Secale cereale L. cv Muskateer) grown at 5 or 20°C was investigated. Purified light-harvesting complex II was enzymically delipidated to various extents by treatment with the following lipolytic enzymes: phospholipase C, phospholipase A₂, and galactolipase. Complete removal of phosphatidylcholine had no effect on the stability of the oligomeric form, whereas the removal of phosphatidylcholine plus phosphatidylglycerol caused a decrease in the ratio of oligomeric:monomeric forms from 1.86 ± 0.17 to 0.85 ± 0.17 and 3.51 ± 0.82 to 0.81 ± 0.29 for purified cold-hardened and nonhardened light-harvesting complex II, respectively, with no change in free pigment content. Incubation of delipidated cold-hardened or nonhardened light-harvesting complex with purified thylakoid phosphatidylglycerol containing trans-Δ³-hexadecenoic acid resulted in 48% reconstitution of the oligomeric form on a total chlorophyll basis with an oligomer:monomer of about 1.90. Incubation in the presence of di- 16:0 or di- 18:1 phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglyceride, or digalactosyldiacylglyceride caused no oligomerization, but rather a further destabilization of the monomeric form. These lipid-dependent structural changes were correlated with significant changes in the 77K fluorescence emission spectra for purified light-harvesting complex II. We conclude that the stabilization of the supramolecular organization of light-harvesting complex II from rye is specifically dependent upon molecular species of phosphatidylglycerol containing trans-Δ³-hexadecenoic acid.