Subunit structure and activity of glyceraldehyde-3-phosphate dehydrogenase from spinach chloroplasts.

Glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NADP+ oxidoreductase (phosphorylating), EC 1.2.1.13) from spinach chloroplasts is a polymeric protein of approx. 600,000 daltons and sodium dodecyl sulphate gel electrophoresis shows that it consists of two subunits of molecular weight 43,000 and 37,000. Comparison of amino acid analyses and tryptic peptide maps indicates that the two subunits have a different primary structure. The native enzyme contains 0.5 mol of NADP+ and 0.5 mol of NAD+ per protomer of 80,000 daltons, no reduced pyridine nucleotides have been detected. Almost complete inactivation is obtained by reaction of two cysteinyl residues per 80,000 daltons with tetrathionate or iodo[14C2]acetic acid; since the same amount of radioactivity is incorporated in the two subunits it is likely that they are both essential for the catalytic activity. Charcoal stripping of native glyceraldehyde-phosphate dehydrogenase produces an apoprotein which still retains most of the enzymatic activity but, unlike the holoenzyme, is gradually inactivated by storage at 4 degrees C and does not react with iodoacetate under the same conditions in which the holoenzyme is completely inactivated.     

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.     

Isolation and sequence determination of a peptide located in or near the active site of bovine muscle pyruvate kinase

Bovine muscle pyruvate kinase was inactivated by treatment with trinitrobenzenesulfonic acid; approximately one trinitrophenyl group was incorporated per subunit. ADP or Mg-ADP decreased the rate of inactivation but Mg⁺⁺ alone or phosphoenolpyruvate had no effect. The inactivated protein was treated with trypsin and the trinitrophenylated peptide isolated by gel filtration. Homogeneity of the isolated peptide was shown by high voltage electrophoresis and high pressure liquid chromatography. Amino acid analysis and sequence determination revealed the presence of an acidic peptide 34 amino acids long and containing ?-trinitrophenylated lysine.     

Recombinant rat guanidinoacetate methyltransferase: structure and function of the NH2-terminal region as deduced by limited proteolysis

Recombinant rat liver guanidinoacetate methyltransferase, a monomeric protein with Mr 26,000, is inactivated upon incubation with low concentrations of trypsin. Examination of the reaction products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography followed by amino acid analysis and sequencing of isolated peptides reveals that the inactivation is due to the cleavage of the NH2-terminal segment after Arg20. The cleaved peptide is not tightly associated with the rest of the protein. The rate of inactivation is not affected by the presence of either S-adenosylmethionine (AdoMet) or guanidinoacetate, but a substantial retardation of inactivation is observed when both substrates are present. The cleavage at Arg20 is also slowed by cross-linking Cys15 and Cys90 by a disulfide bond. An equilibrium binding study shows that guanidinoacetate methyltransferase in the free form binds AdoMet but not guanidinoacetate. The trypsin-modified enzyme, despite having no catalytic activity, can weakly bind AdoMet and guanidinoacetate in the presence of AdoMet. Chymotrypsin rapidly hydrolyzes the peptide bond after Trp19, and elastase cleaves the bond after Ala24, leading in both cases to loss of activity. The results obtained in this study suggest that the portion of the methyltransferase around residues 19-24 is highly exposed to the solvent and flexible. The results also indicate that the NH2-terminal region is not directly involved in substrate binding but plays a role in catalysis.     

A functional, thioester-containing alpha 2-macroglobulin homologue isolated from the hemolymph of the American lobster (Homarus americanus)

An alpha 2-macroglobulin-like protease inhibitor was isolated from the cell-free hemolymph of the american lobster (Homarus americanus) by ion-exchange chromatography and gel filtration. Whereas the undissociated molecule has a molecular weight of 342,000 as determined by ultracentrifugation studies, under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a subunit molecular weight of 180,000. On the basis of this and other evidence, we conclude that the lobster protein is a dimer consisting of two disulfide-bonded monomers. The purified protein inhibits proteolytic enzymes but protects the esterolytic activity of trypsin toward low molecular weight substrates from inactivation by soybean trypsin inhibitor. The methylamine sensitivity of this activity suggests the presence of an internal thioester bond. This was confirmed by the covalent incorporation of [14C]methylamine, by the formation of Mr 55,000 and 125,000 autolytic cleavage fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and, more directly, by the amino acid sequence of a tryptic peptide containing the putative thioester region. Whereas the N-terminal amino acid sequence (22 residues) of the protein revealed an overall identity of only 18% when compared with the human protein, the sequence of the thioester-containing peptide was highly conserved, both with respect to human alpha 2-macroglobulin and to other proteins having a thioester bond. The protein showed the "slow to fast" conformational change typical in alpha 2-macroglobulins in nondenaturing gel electrophoresis after treatment with trypsin, but not after incubation with methylamine.     

Kinetics of RecA protein-directed inactivation of repressors of phage lambda and phage P22

Escherichia coli recA protein directs the inactivation of the repressor of Salmonella typhimurium phage P22 in vitro. As is true for repressor of the E. coli phage λ, inactivation of P22 repressor is accompanied by proteolytic cleavage of the repressor into two detectable fragments.We have investigated the kinetics of inactivation of the λ and P22 repressors in vitro. The fraction of λ repressor inactivated per unit time decreases as its concentration in the reaction is increased. However, high concentrations of λ repressor do not inhibit the inactivation of P22 repressor. Thus, it does not appear that the inactivation system is saturated by λ repressor, but rather that λ repressor is a less efficient substrate at higher concentrations.     

Inactivation mechanism of tetrameric beta-galactosidase by gamma-rays involves both fragmentation and temperature-dependent denaturation of protomers

The radiation inactivation method is widely used to estimate the molecular size of membrane-bound enzymes, receptors, and transport systems in situ. The method is based on the principle that exposure of frozen solutions or lyophilized protein preparations to increasing doses of ionizing radiations results in a first-order decay of biological activity proportional to radiation inactivation size of the protein. This parameter is believed to reflect the "functional unit" of the protein defined as the minimal assembly of structure (protomers) required for expression of a given biological activity. We tested the functional unit as a concept to interpret radiation inactivation data of proteins with Escherichia coli beta-galactosidase, where the protomers are active only when associated in a tetramer. Gamma-Irradiation of beta-galactosidase at both -78 and 38 degrees C followed by quantitation of the residual unfragmented promoter band by SDS-polyacrylamide gel electrophoresis yielded the protomer size, indicating that only one protomer is fragmented by each radiation hit. By following the enzyme activity as a function of dose it was found that only the protomer that has been directly hit and fragmented at -78 degrees C was effectively inactivated. In contrast, at 38 degrees C, it was the whole tetramer that was inactivated. beta-Galactosidase cannot have two different functional units depending on temperature. The inactivation of the whole beta-galactosidase tetramer at 38 degrees C is in fact related to protomer fragmentation but also to the production of stable denatured protomers (detected by gel-filtration HPLC and differential UV spectroscopy) due to energy transfer from fragmented protomers toward unhit protomers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction, isolation and implications of repressor-galactosidase - beta-galactosidase hybrid molecules.

Escherichia coli heterogenotes, which produce hybrid molecules between the chimaeric protein repressor-galactosidase and the enzyme beta-galactosidase, were constructed. Repressor-galactosidase in which fully active lac repressor is covalently linked to active beta-galactosidase, is an aggregate with a core structure of four beta-galactosidase parts and two peripheral lac repressor dimers. The lac repressor dimers, which are separated by tetrameric beta-galactosidase, retain all the biological activities of tetrameric lac repressor. Substitution of repressor-galactosidase subunits with beta-galactosidase subunits leads to hybrid molecules with y beta-galactosidase subunits aggregated with (4-y) repressor-galactosidase subunits (where y = 1, 2 or 3). A 2:2 hybrid, i.e. a tetrameric beta-galactosidase core with one lac repressor dimer grafted to it, binds at least 100 times less strongly to 32P-labelled lambdaplac DNA than pure lac repressor or repressor-galactosidase. The data suggest a model in which lac repressor binds with two subunits to lac operator and with the other two subunits elsewhere on the DNA, possibly on sequences like the lac operator.     

Lambda repressor inactivation: properties of purified ind- proteins in the autodigestion and RecA-mediated cleavage reactions

Under physiological conditions, lambda repressor can be inactivated in vivo or in vitro by RecA-mediated cleavage of the polypeptide chain. The repressor protein is thought to cleave itself, with RecA acting to stimulate autodigestion. ind- repressor mutants are resistant to RecA-mediated inactivation in vivo. In this paper, we report the purification of 15 ind- repressor proteins and the behaviors of these proteins in the RecA-mediated and autodigestion cleavage reactions. None of these proteins undergoes substantial RecA-dependent cleavage. However, eight mutant proteins autodigest at the same rate as wild-type repressor, six mutants do not autodigest or autodigest slower, and one mutant autodigests faster than wild-type. We discuss these results with respect to repressor structure and RecA-binding, and suggest possible roles for the RecA protein in the cleavage reaction.     

Structure-function relationship of islet-activating protein, pertussis toxin: biological activities of hybrid toxins reconstituted from native and methylated subunits

Islet-activating protein (IAP), pertussis toxin, is a hexameric protein composed of an A protomer and a B oligomer, the residual pentamer having such a subunit assembly that two different dimers, dimer 1 and dimer 2, are connected with each other by means of the smallest C subunit. Incubation of IAP with formaldehyde and pyridine-borane produced the modified toxin in which most of the free amino groups were dimethylated. The methylated and nonmethylated (native) IAP were disintegrated into their respective constituent components, which were then cross combined to reconstitute hybrid toxins with the original hexameric structure. The binding of the B oligomer to the mammalian cell surface via dimer 2 was, but the binding via dimer 1 was not, seriously impaired by methylation of amino groups in the protein. The binding of the B oligomer allowed the A protomer to enter cells and to catalyze ADP-ribosylation of a membrane Mr 41 000 protein. The diverse biological activities of IAP occurring by this mechanism were mimicked by not only methylated IAP but also all hybrid toxins, indicating that the free amino groups in the protein were not essential for the enzyme activity of the A protomer and that the A protomer was able to enter cells if the B oligomer bound to cells "monovalently" via dimer 1. An additional effect of the B oligomer binding, i.e., the direct stimulation, without the transport of the A protomer, of cells leading to mitosis in lymphocytes in vitro or increases in circulating lymphocytes in vivo, was not mimicked by hybrid toxins containing methylated dimer 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic studies of the lac repressor. XIII. Extensive amino acid replacements generated by the use of natural and synthetic nonsense suppressors

We have altered the amino acid sequence of the lac repressor one residue at a time by utilizing a collection of nonsense suppressors that permit the insertion of 13 different amino acids in response to the amber (UAG) codon, as well as an additional amino acid in response to the UGA codon. We used this collection to suppress nonsense mutations at 141 positions in the lacI gene, which encodes the 360 amino acid long lac repressor, including 53 new nonsense mutations which we constructed by oligonucleotide-directed mutagenesis. This method has generated over 1600 single amino acid substitutions in the lac repressor. We have cataloged the effects of these replacements and have interpreted the results with the objective of gaining a better understanding of lac repressor structure, and protein structure in general. The DNA binding domain of the repressor, involving the amino-terminal 59 amino acids, is extremely sensitive to substitution, with 70% of the replacements resulting in the I- phenotype. However, the remaining 301 amino acid core of the repressor is strikingly tolerant of substitutions, with only 30% of the amino acids introduced causing the I- phenotype. This analysis reveals the location of sites in the protein involved in inducer binding, tighter binding to operator and thermal stability, and permits a virtual genetic image reconstruction of the lac repressor protein.     

Induction of prophage lambda by gamma-rays, mitomycin C and tif; repressor cleavage studied by immunoprecipitation.

Summary The fate of the repressor on induction of E. coli lysogens has been studied after -irradiation, mitomycin C treatment or by temperature step-up of a tif-1 mutant. The repressor and a fragment of the repressor were selectively precipitated from labelled cell lysates by anti-repressor serum and subjected to gel electrophoresis and autoradiography. Induction of lysogens by all of these treatments leads to cleavage of the repressor. Repressor cleavage closely parallels the inactivation of its DNA binding properties and seems to be the general fate of the repressor on induction, irrespective of the nature of the inducing treatment.The effect of chloramphenicol on the cleavage has been studied in order to assess the requirement for inducible protein synthesis in this process. In contrast to results obtained by mitomycin C treatment, tif mediated cleavage of the repressor does not require the induction of new protein synthesis. Although inactivation of the DNA binding properties of the repressor after induction by -irradiation does not appear to be inhibited by chloramphenicol (West, Powell and Emmerson, 1975), cleavage of the repressor is substantially inhibited by this compound. The possible implications of these findings are discussed in relation to inducible repair functions and the mechanism of repressor inactivation.     

The bacteriophage 434 repressor dimer preferentially undergoes autoproteolysis by an intramolecular mechanism

Inactivation of the lambdoid phage repressor protein is necessary to induce lytic growth of a lambdoid prophage. Activated RecA, the mediator of the host SOS response to DNA damage, causes inactivation of the repressor by stimulating the repressor's nascent autocleavage activity. The repressor of bacteriophage lambda and its homolog, LexA, preferentially undergo RecA-stimulated autocleavage as free monomers, which requires that each monomer mediates its own (intramolecular) cleavage. The cI repressor of bacteriophage 434 preferentially undergoes autocleavage as a dimer specifically bound to DNA, opening the possibility that one 434 repressor subunit may catalyze proteolysis of its partner subunit (intermolecular cleavage) in the DNA-bound dimer. Here, we first identified and mutagenized the residues at the cleavage and active sites of 434 repressor. We utilized the mutant repressors to show that the DNA-bound 434 repressor dimer overwhelmingly prefers to use an intramolecular mechanism of autocleavage. Our data suggest that the 434 repressor cannot be forced to use an intermolecular cleavage mechanism. Based on these data, we propose a model in which the cleavage-competent conformation of the repressor is stabilized by operator binding.     

Bovine kidney alkaline phosphatase. Purification, subunit structure, and metalloenzyme properties.

Kidney alkaline phosphatase was purified to homogeneity. It is a glycoprotein of about 172,000 molecular weight. Analyses of the subunit structure by sedimentation equilibrium in 6 M guanidine hydrochloride and by gel electrophoresis in sodium dodecyl sulfate indicate that the alkaline phosphatase is a dimer comprising two very similar or identical subunits of about 87,000 molecular weight. The native enzyme contains 4.5 +/- 0.2 g atoms of zinc per mol of protein. Reconstitution experiments from the apophosphatase show that binding of 4 Zn2+ per mol of dimer is essential for full activity. The kinetic data of Zn2+ binding to the apoprotein require at least a two-step mechanism, in which one of the steps corresponds to a conformational change within the enzyme. This paper also presents data concerning amino acid composition, sugar content, enzyme stability, absorbance index, and sedimentation velocity.     

Characterization of mutations in oligomerization domain of Lac repressor protein

A series of mutant lac repressor proteins at positions 281 or 282 was isolated for detailed characterization. Although Cys281 modification by sulfhydryl reagents abrogates pH effects on inducer binding and diminishes operator binding (Daly, T. J., Olson, J. S., and Matthews, K. S. (1986) Biochemistry 25, 5468-5474), substitution at this site by alanine, serine, phenylalanine, isoleucine, or methionine did not abolish completely the pH shift nor affect operator affinity. Thus, ionization of the sulfhydryl residue does not account fully for the alterations in inducer affinity and cooperativity of binding observed with elevated pH. Substitution for Cys281 did, however, alter the kinetic parameters for inducer association with the protein. The polarity of the side chain at 281 influenced the rates of sugar binding, presumably by altering the rate of opening/closing of the binding site. Furthermore, the presence of the branched side chain of isoleucine at position 281 disrupted oligomerization of the repressor. In contrast to the tolerance for substitution at 281, the only amino acid side chain exchanges for Tyr282 which yielded tetrameric protein with near normal operator binding characteristics were phenylalanine and leucine; this result is consistent with studies of suppressed nonsense mutations at position 282 which indicated repression occurred only for the corresponding substitutions (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 221, 295-318). Despite the tetrameric character of the Y282F mutant protein, the pH dependence and cooperativity of inducer binding for this mutant protein were altered. All amino acid substitutions other than phenylalanine and leucine at this position resulted in either monomeric protein or no detectable repressor in the cell. Thus, the hydrophobic character of the side chain at position 282 is essential for tetramer formation, and the phenyl ring alone alters inducer binding parameters. The monomeric mutant proteins with substitutions for Tyr282 exhibited lower stability than their tetrameric counterparts, and the absence of dimer formation suggests alterations at this site affect both dimer and tetramer interfaces. Based on previous genetic studies and our detailed mutant characterization, the region encompassing 281 and 282, indicated by secondary structure prediction to be a turn or coil, is essential for oligomer formation and additionally exerts a strong influence on the dynamic properties of the protein, presumably mediated by interactions at the subunit interface which regulate the rate of opening and closing of the inducer binding cleft.     

lac repressor forms loops with linear DNA carrying two suitably spaced lac operators.

Tetrameric lac repressor may bind to two lac operators on one DNA fragment and induce the intervening DNA to form a loop. Electron microscopy, non-denaturing polyacrylamide gel electrophoresis, and DNase I protection experiments were used to demonstrate such DNA loops, where the distance between the centres of symmetry of the two lac operators varies between 63 and 535 bp. Formation of a DNA loop is favoured by correct phasing of the two lac operators and a low concentration of both components of the reaction. When a large excess of lac repressor over DNA is used, a 'tandem' structure is observed, in which both lac operators are occupied independently by two repressor tetramers. When the concentrations of both lac repressor and lac operator are high, a 'sandwich' structure is observed, in which two DNA molecules are connected by two lac repressor tetramers in trans.     

Sequence-specific DNA-protein interaction: the lac represser

A model is proposed for lac repressor-lac operator binding which accounts for the tetrameric subunit structure of the lac repressor and for factors involved in the strength, specificity and regulation of repressor-operator interaction. The model employs a π-helix in the amino terminal 25 residues of the lac repressor whereby three tyrosine residues of each subunit intercalate between base pairs of the lac operator. For the outer palindromic sequences of the operator, base specificity is provided by amino acids adjacent to the carboxyl sides of the tyrosine residues of two of the subunits. The inner palindromic sequences which bind the other two subunits form stems of hairpin loops in the operator. Base specificity for these two subunits is provided by amino acids adjacent to the amino sides of the tyrosine residues. In addition to 12 intercalated tyrosine residues, the model provides for a total of at least eight electrostatic interactions and ten sequence-specific hydrogen bonds.     

A novel concatenated dimer of recombinant bovine somatotropin

A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized inEscherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein.     

Mutations partially inactivating the lactose repressor of Escherichia coli

After treatment with N-methyl-N'-nitro-N-nitrosoguanidine, 133 independent mutants of a haploid strain of Escherichia coli able to use phenyl-beta-galactoside as a carbon source were obtained. The galactoside was specific in selecting for mutants with increases in their uninduced levels of beta-galactosidase. Virtually all mutants (37 in a subsample of 38) carried mutations in the lac repressor gene. There were two classes of repressor mutants. As well as the commonly identified class of mutants with completely inactivated repressors, there was a frequent class of mutants (21/37) whose repressors were partially inactivated. Most of these (15/21) repressed beta-galactosidase synthesis 4 to 50 times less than wild type, but were more numerous in the lower part of this range. Their beta-galactosidase was inducible to levels characteristic of the parent strain. The repressor activities were diverse and stably expressed under routine growth conditions. The decreased activity did not result from the formation of temperature-sensitive repressors. None of the mutants with completely inactivated repressors appeared to carry UAG or UGA chain-terminating codons. On the assumption that the partially defective repressor mutants carried missense mutations, it is argued that missense mutations in the lac repressor gene modify the repressor's affinity for the operator with high probability. An explanation is proposed for the apparent sensitivity of this repressor function to partial inactivation as the result of amino acid substitutions.