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1. The alpha and beta subforms of aspartate aminotransferase were purified from pig heart. 2. The alpha subform contained 2mol of pyridoxal 5'-phosphate. The apo-(alpha subform) could be fully reactived by combination with 2mol of cofactor. 3. The protein fluorescence of the apo-(alpha subform) decreased non-linearly with increase in enzyme activity and concentration of bound cofactor. 4. It is concluded that the enzyme activity/mol of bound cofactor is largely independent of the number of cofactors bound to the dimer. 5. The beta subform had approximately half the specific enzyme activity of the alpha subform, and contained an average of one active pyridoxal 5'-phosphate molecule per molecule, which could be removed by glutamate, and another inactive cofactor which could only be removed with NaOH. 6. On recombination with pyridoxal 5'-phosphate the protein fluorescence of the apo-(beta subform) decreased linearly, showing that each dimeric enzyme molecule contained one active and one inactive bound cofactor. 7. The results are not consistent with a flip-flop mechanism for this enzyme.

1. The reaction of iodoacetate, 2-chloromercuri-4-nitrophenol and 5,5'-dithiobis-(2-nitrobenzoate) with thrombin-cleaved Factor XIII (i.e. Factor XIII(a)) was accompanied by enzyme inhibition. 2. The reaction with iodoacetate and 5,5'-dithiobis-(2-nitrobenzoate) was absolutely dependent on Ca(2+), and the rate of reaction increased with the Ca(2+) concentration up to very high, non-physiological concentrations. 3. 2-Chloromercuri-4-nitrophenol reacted with Factor XIII(a) in the absence of Ca(2+), but at a much slower rate. 4. Stopped-flow methods were used to quantify the reaction with 5,5'-dithiobis-(2-nitro-benzoate) because of the Ca(2+)-dependent dissociation of Factor XIII(a) (a'(2)b(2)) and subsequent aggregation of the a' chains into turbid precipitates. 5. The 3-carboxy-4-nitrothio-phenolate released was consistent with the reaction of 2 thiol groups/molecule of Factor XIII(a). The isolated b chains of Factor XIII did not react with either of the chromophoric reagents. This indicated that the a' chains of Factor XIII(a) were responsible for the thiol reactivity of the enzyme. 6. The Ca(2+) dependence of the enzyme inhibition by these thiol reagents was very dependent on protein concentration. This is discussed in relation to the Ca(2+)-induced dissociation of Factor XIII(a). 7. The acceptor substrate, casein, decreased the Ca(2+) concentration required for enzyme inhibition by both the mercurial and the aromatic disulphide compounds. Dansylcadaverine did not affect Ca(2+) dependence of inhibition.

1. Supernatant pig heart malate dehydrogenase is completely inhibited by reaction with diethyl pyrocarbonate at pH6.5, when 0.58+/-0.1 residue of ethoxycarbonylhistidine is formed per NADH-binding site. 2. Oxaloacetate and hydroxymalonate protect the enzyme from inhibition in the absence of coenzyme. 3. Limited ethoxycarbonylation does not alter the binding of NADH to the enzyme but prevents the enzyme-NADH complex from interacting with hydroxymalonate in a ternary complex.

1. Large quantities of human Factor XIII were prepared from ethanol precipitates of outdated human plasma. 2. Material homogeneous after chromatography on DEAE-cellulose was further resolved into two proteins, A and B, after filtration on Sepharose 6B. 3. Protein A has a molecular weight of 350000 and a subunit structure a(2)b(2) and is activated by thrombin and calcium. Protein B is inactive and probably has a subunit structure b(2). 4. Calcium causes protein A, after thrombin cleavage, to fragment to give protein B and a protein, containing only a' subunits, which is catalytically active. The latter protein slowly forms a misty precipitate which is still active and not cross-linked covalently. This confirms the suggestion of Schwartz et al. (1971) that catalytic activity is only associated with a' subunits. 5. Iodoacetate, which inhibits the enzyme, does not inhibit dissociation and aggregation of protein A. 6. The existence of two proteins and the fragmentation are possible explanations for the wide range of molecular weights given for Factor XIII in the literature.

1. Changes in the tryptophan fluorescence and the visible absorption spectrum resulting from the combination of apo-ovotransferrin with Fe3+, F,E2+, Cu2+, Zn2+, Mn2+, and Cd2+were measured. 2. As expected for a radiationless transfer of electronic excitation energy, only the ions Fe3+, Fe2+and Cu2+, which gave complexes with large extinctions between 300 and 370nm, resulted in large decreases in trytophan fluorescence. 3. The decrease in protein fluorescence was non-linear with increasing occupancy of the Fe3+ -and Cu2+ - binding sites. The decrease in fluorescence on binding of Fe3+ was biphasic and showed that the two metal-binding sites were being occupied sequentially at pH7.4-8.4. The first site reacted with Fe3+ instantaneously, the second was occupied over a minute. 5. The nonidentity of the two sites was also demonstrated by the preparation of a stable hybrid containing both Cu2+ and Zn2+.h Cu2+ and Zn2+

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.

1. A continuous fluorimetric assay for blood-clotting Factor XIII based on the incorporation of dansylcadaverine into casein was investigated. 2. Hammarsten casein was fractionated to yield the beta-casein, which was dephosphorylated and acetylated to give a substrate which itself did not bind calcium and which was not cross-linked by the activated Factor. 3. The modified beta-casein was used as substrate for a continuous fluorimetric assay and as substrate for the incorporation of radioactive glycine ethylester. 4. A linked fluorimetric assay for the zymogen is described. 5. The K(m) for calcium was redetermined and at 0.2mm was in the physiological range and much lower than the values reported by others using substrates which interact with calcium. 6. The K(m) for casein is about 14mum.

1. Bovine, porcine and chicken liver glutamate dehydrogenases were irreversibly inhibited by a tenfold excess of radioactive 4-iodoacetamidosalicylic acid at pH7.5. 2. Inhibition was accompanied by the covalent incorporation of 1.1 mol of labelled inhibitor/mol of polypeptide chain. Acid hydrolysis yielded N(epsilon)-carboxymethyl-lysine as the sole labelled amino acid. No labelled S-carboxymethylcysteine was recovered from the bovine or porcine enzymes. 3. The labelled bovine enzyme was hydrolysed with trypsin. The radioactivity was found at lysine-126 in a peptide comprising residues 119-130 of the sequence. 4. The amino acid compositions of the tryptic peptides containing labelled lysine from the porcine and chicken enzymes were similar to that of the bovine peptide.

1. The decrease in the protein fluorescence (F) of Neurospora crassa glutamate dehydrogenase is linearly related to the increase in the fraction of the coenzyme sites occupied by NADPH (alpha) at pH6.35. Under these conditions NADPH causes this enzyme to dissociate to monomers. 2. There is a non-linear relationship of F to alpha for NADH binding to give the alcohol dehydrogenase-NADH-isobutyramide complex, the l-glycerol 3-phosphate dehydrogenase-NADH complex and the bovine glutamate dehydrogenase-NADH-glutamate complex. The non-linearity is accurately represented by F=[1-alpha(1-x)](n) where n is the number of NADH-binding sites per protein molecule. 3. The co-operative binding of GTP to bovine glutamate dehydrogenase in the presence of NADH gives a linear relationship between F and alpha. 4. The prediction from the equation F=[1-alpha(1-x)](n) that initial tangents to non-linear protein-fluorescence-quenching curves will intercept the fluorescence when alpha=1 at a value of total ligand concentration less than the sum of the concentration of binding sites in the solution plus the dissociation constant of ligand is quantitatively fulfilled. 5. Non-linear protein-fluorescence titrations may be used to obtain information about the distribution of ligand among the protein molecules in solution.

1. The binding of oxamate to pig heart and pig muscle isoenzymes of lactate dehydrogenase in the presence of NADH was studied by fluorescence titration. The dissociation constant of oxamate from the heart enzyme complex is 3mum and from the muscle isoenzyme 25mum at pH5. These values quantitatively increase with pH as predicted if oxamate can bind only to the enzyme-NADH complex if a group with pK6.9 is protonated. There are four non-interacting oxamate-binding sites per tetramer. 2. o-Nitrophenylpyruvate is a poor substrate for both isoenzymes but has a reasonable affinity to the heart isoenzyme. Initially, it forms an enzyme-NADH-substrate complex, which can be detected either by protein-fluorescence quenching or by NADH-fluorescence quenching. The pH-dependence of the dissociation constant of nitrophenylpyruvate also shows that this ternary complex can only form if a group with pK6.8 is protonated. Taken with the results of chemical-modification experiments, these results allow the pK of 6.8 to be assigned to a system probably involving the imidazole side chain of histidine-195. Formation of a ternary complex from a binary one at pH8 is predicted to result in a proton being taken up from solution. 3. Isotope-effect studies with NADH and its deuterium analogue show that the rapidly formed ternary complex with o-nitrophenylpyruvate slowly isomerizes to give an active ternary complex, which then rapidly decomposes to NAD(+). The isomerization is pH-independent, and it is suggested that histidine-195 is still protonated in the activated ternary complex, which is present before hydride transfer. 4. All four subunits of the enzyme are kinetically equivalent with respect to the oxidation of bound NADH by o-nitrophenylpyruvate. 5. A partial mechanism for the enzyme is described which emphasizes the isomerizations and ionizations involved in forming the reduced ternary complex at pH6 and 8.

1. The reaction of 4-iodoacetamidosalicylate with bovine liver glutamate dehydrogenase is dependent on pH. The pH-activity curve is bell-shaped and can be described by apparent pK values of 7.8+/-0.2 and 9.1+/-0.2. 2. Enzyme in which lysine-126 has been modified by 4-iodoacetamidosalicylate has unaltered sedimentation characteristics except when measured in the presence of GTP and NADH. 3. GTP binding to the inhibited enzyme is unaltered. However, GTP can no longer promote the binding of a second molecule of NADH, since this is already bound to the inhibited enzyme without GTP. 4. The equilibrium binding of ADP, GTP, NAD-sulphite and NADH (when measured at low concentrations) was largely unchanged by modification. 5. The number of binding sites for 2-oxoglutarate to the enzyme-NADH complex were decreased by 60% in an enzyme that has been inhibited by 70%.

1. Inhibition of ox liver glutamate dehydrogenase with N-(N'-acetyl-4[(35)S]-sulphamoylphenyl)maleimide (ASPM) is more specific at pH7.3 than at pH6.9. At pH7.3 inhibition accompanies the incorporation at 1 mole of ASPM residues into about 53000g. of protein. 2. Digestion of the modified protein with chymotrypsin and trypsin yields a unique radioactive peptide. 3. Acid hydrolysis of 1 mole of this peptide yields 1 mole of N(in)-succin-2-yl-lysine. The in-amino group of a lysyl residue is thus the site of modification of the protein. 4. The sequence containing the modified lysyl residue is: [Formula: see text] where Asx respresents either aspartic acid or asparagine.

1. One mol of diethyl pyrocarbonate will react with one mol of glutamate dehydrogenase polypeptide chains to form one mol of N(1)-carbethoxyhistidine. Reaction is prevented by NADH. 2. The 1:1 complex has an increased specific activity (1.4-2.0-fold). 3. The reason for the activation is discussed. The results are not consistent with NADH dissociation from the enzyme-glutamate-NADH complex being rate-limiting in the steady state measured. 4. The effects of modification on the properties of the enzyme were investigated. The effects of GTP and NAD(+) on the enzyme activity are unaltered by activation. NADH binding is unaltered and there is no apparent change in the molecular weight. However, the activated enzyme can still be further activated by ADP. K(s) for ADP is decreased fivefold.

1. Activated human plasma factor XIII was 65% inhibited by iodo[(14)C]acetate and incorporated 0.6 mol of label into the alpha subunit, which eventually was allowed to form a precipitate. 2. All the label was recovered as S-carboxymethylcysteine in a tetra-peptide of sequence Gly-Gln-Cys-Trp.

1. Pig heart lactate dehydrogenase is inhibited by addition of one equivalent of diethyl pyrocarbonate. The inhibition is due to the acylation of a unique histidine residue which is 10-fold more reactive than free histidine. No other amino acid side chains are modified. 2. The carbethoxyhistidine residue slowly decomposes and the enzyme activity reappears. 3. The essential histidine residue is only slightly protected by the presence of NADH but is completely protected when substrate and substrate analogues bind to the enzyme-NADH complex. The protection is interpreted in terms of a model in which substrates can only bind to the enzyme in which the histidine residue is protonated and is thus not available for reaction with the acylating agent. 4. The apparent pK(a) of the histidine residue in the apoenzyme is 6.8+/-0.2. In the enzyme-NADH complex it is 6.7+/-0.2. 5. Acylated enzyme binds NADH with unchanged affinity. The enzyme is inhibited because substrates and substrate analogues cannot bind at the acylated histidine residue in the enzyme-NADH complex.

With the use of a glass electrode it is shown that one proton is taken up from the solvent when o-nitrophenylpyruvate forms a ternary complex with lactate dehydrogenase and NADH at pH8 and before the complex has rearranged to give products.

1. No discontinuities were observed during the continuous titration with NADH of the lactate dehydrogenases of ox muscle, pig heart, pig muscle, rabbit muscle, dogfish muscle or lobster tail muscle. The binding was monitored by either the enhanced fluorescence of bound NADH or the quenched fluorescence of the protein. A single macroscopic dissociation constant, independent of protein concentration, could be used to describe the binding to each enzyme, and there was no need to postulate the involvement of molecular relaxation effects. 2. The affinity for NADH decreases only threefold between pH6 and 8.5. Above pH9 the affinity decreases more rapidly with increasing pH and is consistent with a group of about pK9.5 facilitating binding. Muscle enzymes bind NADH more weakly than does the pig heart enzyme. 3. Increasing temperature and increasing concentrations of ethanol both weaken NADH binding. 4. NADH binding is weakened by increasing ionic strength. NaCl is more effective than similar ionic strengths derived from sodium phosphate or sodium pyrophosphate. 5. Commercial NAD(+) quenches the protein fluorescence of the heart and muscle isoenzymes. Highly purified NAD(+) does not, and its binding was monitored by competition for the NADH-binding sites. A single macroscopic dissociation constant is sufficient to describe NAD(+) binding at the concentrations tested. The dissociation constant is about 0.3mm and is not sensitive to changed ionic strength and to changed pH in the range pH6-8.5.

A salt-induced cooperative conformational transition of a synthetic DNA, poly(dG-dC), is reversed by addition of ethidium bromide. Binding of the dye at high salt concentrations is highly cooperative. Circular dichroism spectra of the complex and the kinetic data support a model for this cooperative binding that is formally equivalent to the "allosteric" one proposed for oligomeric proteins by Monod et al. Thus, double-helical DNA of at least one defined sequence can undergo a cooperative conformational change in solution, with simple salts and drug molecules as antagonistic effectors. Such transitions may be involved in regulatory phenomena operating directly at the level of nucleic acid structure.

1. The preparation of a derivative of pig heart lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group is described. The derivative has unchanged s(20,w) and is catalytically inactive. 2. The rate of reaction of the essential thiol group is controlled by a system with a pK>9. 3. The essential thiol group is protected by NADH against reaction with maleimide. 4. Lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group or alkylated with maleimide still binds one molecule of NADH/subunit but with a three- to four-fold diminished affinity. 5. The inhibited enzymes also bind one molecule of NAD(+)-sulphite complex/subunit but with affinity decreased 10(3)-10(4)-fold. 6. The inhibited enzymes fail to bind C(2) and C(3) molecules to give the ternary complexes enzyme-NAD(+)-pyruvate, enzyme-NADH-oxamate and enzyme-NADH-oxalate. The 1:1:1 stoicheiometry of the last-mentioned complex with the native enzyme was established by gel filtration. 7. Structures that account for these results are discussed.

1. There is a non-linear decrease in the protein fluorescence (F) of lactate dehydrogenase with the increase in the fraction (alpha) of the coenzyme-binding sites occupied with NADH. 2. By a curve-fitting procedure it is shown that the fluorescence intensity can be represented by the equation F=[1-alpha(1-x)](n) where n is the number of identical and indistinguishable coenzyme-binding sites per protein molecule and x=F(s) (1/n) (F(s) is the protein fluorescence at alpha=1). This equation implies that the relative protein fluorescence of molecules bearing j ligands form the geometric series x(j). 3. Non-linear quenching of protein fluorescence for this enzyme is probably due to radiationless transfer of energy from the protein molecule to the bound NADH and should also be observed when other potential acceptors of protein fluorescence are bound at unique sites. 4. The intercept with F(s) of an initial tangent to a curve of protein fluorescence against alpha will be at a value of alpha equal to (K(d)+[E(0)]). (1-x(n))/n.(1-x) and not at a value equal to the sum of the dissociation constant (K(d)) and the concentration of identical ligand-binding sites ([E(0)]). 5. A use of non-linear protein fluorescence quenching to investigate the state of aggregation of a protein is discussed.