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Enzyme activators lower Km (the Michaelis constant) and/or raise Vmax (the asymptotic reaction velocity at infinite substrate concentration); conversely, inhibitors raise Km and/or lower Vmax . But what if an effector moves both Km and Vmax in the same direction? Uncompetitive inhibitors, which decrease both Km and Vmax by the same factor, are the most common example of this. A less well-known example occurs often when crowding agents are added to the buffer in order to mimic the environment commonly encountered in vivo. Crowding agents tested on different enzymes have been shown to have every conceivable effect on Km or Vmax , causing them to rise, fall, or stay the same. In this article, a mathematical analysis is presented allowing biochemists to judge whether an effector that causes Km and Vmax to both move in the same direction serves as an inhibitor, an activator, or most surprising, as both. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):446-449, 2019.

The upper bound of enzyme concentration for accurately estimating the parameters in Michaelis-Menten (MM) equation is not completely determined and still under discussion, even though many researchers have investigated the equation’s validity for a long time. In the paper, we broadly investigated the correlation between the system of ordinary differential equations for monosubstrate irreversible enzyme reaction (HMM-system) and its derivative MM equation focusing on the relationship between initial enzyme concentration [E]0 and Michaelis constant Km by numerical simulation. According to the results, the initial reaction velocity v0 is still a function of initial substrate concentration [S]0 at [E]0

A new algorithm for computation of the initial velocity of enzyme reaction at the time zero is proposed. This algorithm makes it possible to reduce the systematic error of measurements to the minimum, to estimate the reaction velocity in test samples regardless of the enzyme activity levels, and to minimize the assay time. The study is illustrated by an example of salivary alpha-amylase and standard reagent kit. The algorithm should not be applied if conjugated enzyme systems are used because there is a long initial lag phase in the kinetic curve.

Phosphotransacetylase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger and Desulfomicrobium sp. has never been well-characterized and has not been studied yet. In this paper, the specific activity of phosphotransacetylase and the kinetic properties of the enzyme in cell-free extracts of both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains were presented at the first time. The microbiological, biochemical, biophysical and statistical methods in this work were used. The optimal temperature and pH for enzyme reaction was determined. Analysis of the kinetic properties of the studied enzyme was carried out. Initial (instantaneous) reaction velocity (V0), maximum amount of the product of reaction (Pmax), the reaction time (half saturation period, τ) and maximum velocity of the phosphotransacetylase reaction (Vmax) were defined. Michaelis constants (Km) of the enzyme reaction (3.36 ± 0.35 mM for D. piger Vib-7, 5.97 ± 0.62 mM for Desulfomicrobium sp. Rod-9) were calculated. The studies of the phosphotransacetylase in the process of dissimilatory sulfate reduction and kinetic properties of this enzyme in intestinal sulfate-reducing bacteria, their production of acetate in detail can be perspective for clarification of their etiological role in the development of the humans and animals bowel diseases. These studies might help in predicting the development of diseases of the gastrointestinal tract, by providing further details on the etiology of bowel diseases which are very important for the clinical diagnosis of these disease types.

UDP-glucuronosyltransferase (UGT) 1A1-catalyzed glucuronidation is an important elimination pathway of flavonoids, and mutually inhibitory interactions may occur when two or more flavonoids are coadministered. Our recent research suggested that glucuronidation of flavonoids displayed distinct positional preferences, but whether this will lead to the mutually regioselective inhibition of UGT1A1-mediated glucuronidation of flavonoids is unknown. Therefore, we chose three monohydroxyflavone isomers, 3-hydroxyflavone (3HF), 7-hydroxyflavone (7HF), and 4'-hydroxyflavone (4'HF), and one trihydroxyflavone, 3,7,4'-trihydroxyflavone (3,7,4'THF), as the model compounds to characterize the possible mutually regioselective inhibition of glucuronidation using expressed human UGT1A1. Apparent kinetic parameters [e.g., reaction velocity (V), Michaelis-Menten constant (Km), maximum rate of metabolism (Vmax), concentration at which inhibitor achieves 50% inhibition (IC50), and the Lineweaver-Burk plots were used to evaluate the apparent kinetic mechanisms of inhibition of glucuronidation. The results showed that UGT1A1-mediated glucuronidation of three monohydroxyflavones (i.e., 3HF, 7HF, and 4'HF) and 3,7,4'THF was mutually inhibitory, and the mechanisms of inhibition appeared to be the mixed-typed inhibition. Specifically, the inhibitory effects displayed certain positional preference. Glucuronidation of 3HF was more easily inhibited by 3,7,4'THF than that of 7HF or 4'HF. Compared to 7-O-glucuronidation of 3,7,4'THF, 3-O-glucuronidation of 3,7,4'THF was more inhibited by 3HF and 4'HF, whereas glucuronidation at both 3-OH and 7-OH positions of 3,7,4'THF was more easily inhibited by 7HF than by 3HF and 4'HF. In conclusion, 3HF, 7HF, 4'HF, and 3,7,4'THF were both substrates and inhibitors of UGT1A1, and they exhibited mutually regioselective inhibition of UGT1A1-mediated glucuronidation via a mixed-type inhibitory mechanism.

Various parameters such as type and concentration of crosslinking agent, enzyme concentration, pH of the coupling medium and coupling time were optimized for the immobilization of urease on crosslinked poly(vinyl alcohol). Immobilized urease was thoroughly characterized for pH, temperature, solvents and storage stabilities and these properties were compared with the free enzyme properties. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated from Lineweaver-Burk plots for both free and immobilized enzyme systems. The energy of activation (Δ Ea) and the thermoinactivation constant (Ki) were calculated from the effect of temperature study. The urease-(PVA-F)-system shows good retention of activity even after 6–7 cycles.

A simplified methodology for assaying the caseinolysis by calpains was developed. This methodology, including the incubation of calpain with casein and direct measurement of the absorbance at 500 nm, is based on the turbidity of the reaction mixture caused by the aggregation of hydrolysates during the reaction. Unlike the typical caseinolysis assay, this novel assay does not need to separate the substrate from hydrolysates and can be continuously monitored in visible wavelength range. The activity of calpain is expressed by the maximum reaction velocity (delta A500/min) at 25 degrees C).

The efficiency of penicillin G acylase catalyzed transformations is enhanced significantly with respect to reaction velocity, and in particular, stereoselectivity by appropriate “substrate tuning”, i.e. by the introduction of a nitrogen atom into the part of the substrates which is recognized by the enzyme.

Groups of king salmon, under anesthesia with MS222, received either a single injection of 100 mg/kg body weight (β-naphthoflavone) or two injections at the same ratio (phenobarbital) three weeks prior to sacrifice. Controls received the appropriate vehicle only. Hepatic microsomes were prepared and UDP-glucuronosyltransferase (UDPGT) activities toward 4-nitrophenol, 1-naphthol and bilirubin were assayed. Activities toward 4-nitrophenol and 1-naphthol in β-naphthoflavone-treated fish were increased 1·81-and 2·15-fold respectively. Bilirubin UDPGT activity was not detectable. No induction by phenobarbital was observed. Kinetic analyses of 4-nitrophenol activity with respect to UDP-glucuronic acid (UDPGA) and 4-nitrophenol concentrations were carried out. Km and Vmax values for UDPGA were 1·7 mM and 1·1 units/mg, respectively. Between 0·05 and 4·0mM 4-nitrophenol the reaction velocity increased linearly, indicating a lack of affinity for this substrate.

Noninvasive visualization and investigation of interactions among proteins, biomolecules, and enzymes in living cells is an important goal for biologists, and fluorescence probes are powerful tools for this purpose. Because many target molecules are present in only trace amounts, high sensitivity is very important, and it is common to improve the sensitivity of fluorescence probes by focusing on high reaction velocity, K(d). (Gee, K. R.; Archer, E. A.; Lapham, L. A.; Leonard, M. E.; Zhou, Z.; Bingham, J.; Diwu, Z. Bioorg. Med. Chem. Lett. 2000, 10, 1515-1518.) So far, we have designed and synthesized various highly sensitive fluorescence probes based on the above concepts. (Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 3357-3367. Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 13447-13454.) Nevertheless, they were sometimes insufficiently sensitive to detect biomolecules in living cells, despite high chemical sensitivity in cuvette. In this report, we suggest a new approach to increase the sensitivity of fluorescence probes, focusing on their intracellular retention. Since calcein is well-retained, we investigated its structural, chemical, and optical characteristics and found that the iminodiacetic acid group (IAG) is a key structure for the intracellular retention. We next designed and synthesized novel fluorescence probes containing IAGs. They showed superior intracellular retention, making it possible to visualize low concentrations of target molecules that would be difficult to observe with conventional probes and permitting long-term observation in living cells. Improvement of intracellular retention of fluorescence probes holds great promise as a strategy for developing a wide range of highly sensitive probes for studies on various biological phenomena.

It is commonly known that consumption of foods and beverages rich in polyphenols is associated with a lower incidence of cardiovascular disease. The purpose of this study was to assess whether the application of red wine polyphenols influences the kinetic properties of renal Na(+),K(+)-ATPase in rats in which hypertension has been experimentally induced by the nitric oxide synthase inhibitor L-NAME. Treatment with polyphenols during the recovery from hypertension to normotension resulted in the complete revival of the functional properties of the Na(+),K(+)-ATPase, as indicated by the total restoration of K(m), K(Na) (concentration of Na(+) necessary to achieve half-maximal reaction velocity) and V(max) for enzyme activation by ATP and/or Na(+) to pre-hypertension values. Two positive effects of polyphenols during the recovery period are indicated: a restoration of the affinity of the ATP and Na(+) binding sites to control values and a probable increase in the number of Na(+),K(+)-ATPase molecules to a level comparable to that in control conditions, as suggested by the complete renewal of V(max).

The heparin-enhanced antithrombin III/thrombin reaction was studied under experimental conditions where the dependence of the reaction velocity on the concentrations of thrombin and antithrombin III could be determined. The results have shown that the reaction is saturable with respect to both thrombin (KT = 3.6 x 10(-8) M) and antithrombin III (KAT = 1.0 x 10(-7) M) when the heparin concentration is low relative to the initial protein concentrations. The apparent first order rate constant for the rate-limiting step in the reaction was approximately 800 min-1. The reaction was subject to inhibition by antithrombin III/thrombin, the product of the reaction. Inhibition appeared to be noncompetitive with respect to antithrombin II with KP, the apparent heparin product dissociation constant, approximately equal to KT. When the heparin-enhanced antithrombin III/thrombin reaction was studied under conditions where the heparin concentration was high relative to the initial protein concentrations the overall reaction was second order. The initial reaction velocity, under any set of experimental conditions, could be described by the general rate equation for a random order bireactant, enzyme-catalyzed reaction, which is mathematically identical with the "template" model for the mechanism of action of heparin (Griffith, M. J. (1982) J. Biol. Chem. 257, 7360-7365).

The chitin synthetase of Phycomyces blakesleeanus mycelium is a particulate enzyme sedimenting mostly at 1000xg. The activity in crude extracts or cellular fractions can be increased more than tenfold by mild trypsin treatment. Plotting the reaction velocity versus UDP-N-acetylglucosamine concentration yields a sigmoidal curve. N-acetylglucosamine, which greatly stimulates the enzyme, changes the kinetics to an almost normal hyperbolic relationship.

The Michaelis constants and maximum reaction velocity values for butyrylcholine, acetoacetylcholine, β-hydroxybutyrylcholine and crotonylcholine were determined using purified horse serum cholinesterase. The methods used for the synthesis of the above compounds are also described.

1. 1. An automated method was developed for the estimation of enzymatic activities based on the slope measurement of linear reaction curves. The procedure is applicable to most spectrophotometric assays, even when enzymatic reaction velocity is measured by means of a subsequent chemical reaction. 2. 2. The method was developed in the particular case of soluble starch hydrolysis by rat pancreatic amylase, estimated by the reduction of 3,5-dinitrosalicylic acid. Good kinetic graphs were obtained at a rate of 12 assays per hour for 1.8 min recording time.

1 The steady state kinetics of the Mg2+-dependent ATPase of beef brain microsomes are assayed by means of a rapid photometric method. Omitting K+ from the reaction medium, the Na+, K+ -Mg2+-dependent ATPase is not active. 2 The Mg2+-dependent ATPase is also active with Ca2+ and inactive in the absence of Mg2+ or Ca2+ when 40 mM Na+ is present. Hence the name “Mg2+-(Ca2+)-ATPase” is proposed. 3 The complexes MgATP2- and CaATP2− are evidently the true substrates of the ATPase. MgATP2- is split with a 2.2-fold higher velocity than CaATP2−. 4 The Mg2+-(Ca2+)-ATPase is inhibited competitively by an excess of free Mg2+- or Ca2+-ions and probably uncompetitively by an excess of free ATP4−. 5 The reaction rate of the Mg2+-(Ca2+)-ATPase as a function of the concentrations of substrate, Mg2+- resp. Ca2+-ions and ATP4− can be expressed as [MeATP2−] has to be calculated from the ATP4− and the metal ion concentrations and the dissociation constants of MgATP2− and CaATP2−. 6 MgATP2− acting as substrate yields the Michaelis-constant Km= 3.6 ± 0.4 × 10−5 M and the dissociation constants KMg= 8.3 ± 0.8 × 10−4 M and KATP= 2.0 ± 0.6 × 10−4 M; CaATP2− yields Km= 1.3 ± 0.15 × 10−5 M, KCa= 8.0 ± 0.9 × 10−4 M and KATP= 4.5 ± 1.2 × 10−5 M. On account of the magnitude of the reaction velocity constants k1, k−1 and k2, the Michaelis-constant seems to approximate k2/k1 for both substrates.

Purified soluble tyrosinase isolated from the melanosomes of Harding-Passey mouse melanoma was incubated with L-dopa. The reaction velocity of the tyrosinase thus incubated decreased significantly. There was an inverse linear relationship between the concentration of dopa in the preincubation mixture and the reaction velocity of the tyrosinase which remained. The experimental results obtained were similar to what had been observed in melanosomes. 64 Cu-exchanged tyrosinase or 11 C-amino-acid-labeled tyrosinase were incubated with L-dopa; the reaction mixture was centrifuged after varied incubation times, and the radioactivity of the obtained supernatant was determined. The time courses of the disappearance of labeled tyrosinase from the reaction systems indicated that soluble tyrosinase combined with the products of the dopa-tyrosinase reaction and precipitated out with them. Thus tyrosinase was excluded from the reaction systems. The experimental results obtained suggest that the decrease of tyrosinase activity is due to the binding of tyrosinase with reaction products, probably at the active site, and to the disappearance of the tyrosinase molecule from the reaction system.

The purification and properties of a Na+- and K+-activated ATPase from ox brain is described. The enzyme preparation is characterized by a high purity and a low content (< 1%) of Mg++-stimulated ATPase.

A method for the determination of enzyme kinetic constants Vm, Km, and Ki in a histochemical system has been devised. As a substitute for the reciprocal of the reaction velocity, the times necessary to reach a fixed amount of end product (the initial visible color) in a tissue site at various substrate concentrations are plotted, according to the method of Lineweaver and Burk, against the reciprocal of the substrate concentrations. The technique as applied to trypsin-like esterase and amidase activities in human mast cells indicates that a single enzyme or closely related enzymes in this site are responsible for the hydrolysis of both the amide and ester substrates and that typical trypsin substrates act as competitive inhibitors of their hydrolysis. Parallel biochemical studies were performed to evaluate the effect of certain aspects of the experimental histochemical method on a purified homospecific enzyme. The relative kinetic constants derived by the histochemical method afford a further means of characterizing enzymic activity in a histochemical system.

Summary. 1 Esperiments carried out with low thrombin concentrations show that sera of different species under similar conditions inactivate different quantities of thrombin. 2 The course of the thrombin inactivation by sera of 12 mammalian species, during the initial phase, is calculnted. 3 Sera of horse, cattle, rabbit, sheep and goat absorb thrombin, and the fermentative inactivation has A constant velocity during the initial phase. Another group of sera (rat, mice, dog, guinea-pig, pig, hedpehog) shows no absorption, the inactivation is more rapid. and the reaction velocity is not constant.

1. 1. The variation of the Michaelis constant (Km and the maximum reaction velocity (V) with pH for the reaction of hemoglobin with human, canine and porcine pepsins was studied. 2. 2. At pH 2 the Km's were found to be 6 · 3 × 10−4 M, 10 × 10−4 and 1 · 1 × 10−4 M, respectively, for human, canine and porcine pepsins. 3. 3. The similarity between human and canine pepsins permits valid comparisons of these enzyme activities in molar terms, and porcine pepsin appears to be a reasonable gravimetric standard for such studies.

Heparin cofactor material of M ~15,000 has been separated from Fraction I. Isolated systems containing heparin, cofactor, and thrombin exhibit a progressive, limited thrombin inactivation. Complications arise since reaction velocity, specific capacity of cofactor (units T/mg. C) and extent of reversibility (using protamine) are functions of cofactor concentration.

The rate of oxidative inactivation may be approximately described by {ie411-01}, which for constantE andT gives {ie411-02}. It was found that the effect of a certain amount of residual chlorine varied with the amount of non-viral organic or inorganic matter, but that these variations could be explained through the dependence of the reaction velocity on the oxidation potential.

This paper gives a program in BASIC for calculating the kinetic parameters Km and Vmax for an enzyme reaction from a set of paired values of reaction velocity at given substrate concentrations. An initial estimate of the two parameters is made using a weighted linear regression, these values are then used in an iterative process to fit the data to the Michaelis-Menten equation and give final values of Km and Vmax with their associated standard errors.

Chromogenic substrates allow those interested in blood clotting and fibrinolytic enzymes to follow the activity of these enzymes continuously with the aid of a spectrophotometer. This is a major new tool in the study of these enzymes. Like any new tool, it takes some time to become acquainted with its possibilities and limitations so that we can use it optimally. This communication will stress some of the more obvious pitfalls and snags that stand in the way of an advantageous use of these substances. I will discuss the following main points

The reaction pathway catalysed by pyruvate carboxylase was re-examined by using two independent experimental approaches not previously applied to this enzyme. To avoid the variable stoicheiometry associated with oxaloacetate formation, the reaction rate was measured by following release of Pi. Initial velocities, when plotted as a function of varying concentrations of either MgATP2- or HCO3-, at fixed concentrations of pyruvate, gave in double-reciprocal-form families of straight intersecting lines. Further, when the reaction velocity was determined as a function of varying MgATP2- concentrations by using pyruvate, 3-fluoropyruvate and 2-oxobutyrate as alternative carboxyl-acceptor substrates, the slopes of the double-reciprocal plots were significantly different. Both results support a sequential reaction pathway.

A rise in the extracellular concentration of glucose from zero to 5.6 and 16.7 mM caused a graded increase in the glucose-1,6-bisphosphate content of rat pancreatic islets. Glucose-1,6-bisphosphate activated phosphofructokinase in islet homogenates, when the reaction velocity was measured at low concentrations of fructose-6-phosphate. It is postulated that glucose-1, 6-bisphosphate participates, together with fructose-2,6-bisphosphate, in the regulation of glycolysis in intact islet cells.

The possibilities of computer and some mathematical and statistical considerations may renew the interest and reevaluate the advantages of the double reciprocal plot proposed by Lineweaver and Burk. From the experimental values of substrate concentration and reaction velocity, the best-fitting line can be statistically obtained using nonparametric criteria. Computer programs in FORTRAN and BASIC permit easy and elegant resolutions.

In order to investigate the extent of the relationship between the three copper-containing glycoproteins, laccases I, II and III (Mr70000, 80000 and 390000 respectively) of Podospora anserina, the following experiments were carried out on laccases II and III: (a) determination of amino acid composition; (b) determination of N-terminal and C-terminal amino acid; (c) determination of sugar composition; (d) dissociation studies on native and denatured laccases and also after removal of copper from the enzymes; (e) digestion of the carbohydrate moieties with the aid of glycosylhydrolases. A comparison between the results of these experiments and data previously obtained with laccase I allows the following conclusions to be drawn. 1. Laccases II and III are not identical. 2. Neither of these low molecular weight laccases are as complete molecules subunits of the oligomeric laccase I. 3. The possibility of partial identity of amino acid sequences of laccases I and III can not be excluded. 4. Laccase II possibly consists of subunits of Mr37000 whereas laccase III does not. 5. Digestion of 50% of the carbohydrate content leads to complete loss of serological specificity (serological reaction and cross reaction). This finding is discussed with regard to the possible role of the carbohydrate moiety as antigenic determinants and thus as the reason for the immunological relationship. As a consequence, at least three independent structural genes for laccases must be assumed.

The variation of the rate of oxidation of histamine derivatives, diamines, a monoamine and aminoalkylonium compounds by pig kidney diamine oxidase with alteration of pH has been studied. Two types of behaviour of reaction velocity as a function of pH are shown; some substrates having a straightforward optimum pH value around pH 7·4 and others showing an uneven curve instead of a smooth maximum around this pH value. This could be related to inhibition by high substrate concentration as shown by the variation of substrate inhibition with pH. Implications of these findings in the interpretation of the mechanism of binding and oxidation of histamine derivatives by diamine oxidase are discussed. In particular, the side chain αβ dehydrogenation mechanism for the oxidation of histamine is criticised since histamine derivatives lacking β hydrogens were found to be substrates of DAO.

Heparin is a substance extracted from animal tissues, formed by acidic carbohydrate long chains polymers (amino sugar containing sulfated mucopolysaccharides). It is used in therapy for its powerful anticoagulant activity (1). In fact the mucopolysaccharide achieves a dramatical increase of the reaction velocity between proteinases of the clotting system and at least two plasma proteinase inhibitors: antithrombin III (2,3) and heparin cofactor II more recently described (4,5).

When reaction velocity measurements of enzyme reactions are carried out with single beam, single monochromator spectrophotometers, stray light in the spectrophotometer can produce systematic errors in the apparent velocities when highly absorbing solutions (optical density >2.0) are used. These errors can give rise to spurious “inhibition” patterns of the steady state kinetics. Because of a suspected error of this kind, this laboratory has recently reinvestigated the kinetics of glucose 6-phosphate dehydrogenase from Escherichia coli and found that the reported noncompetitive inhibition of the enzyme by DPNH is explained more readily by an unnoticed effect of stray light on the apparent reaction velocity than by a true enzyme inhibition. Methods for estimating and correcting such errors in spectrophotometers are presented in detail.

The effects of volatile anaesthetics on bacterial luciferase were studied in vitro . It was shown that the concentration of anaesthetic required to inhibit the reaction velocity by 50% was similar to that required to reduce light output by 50% in vivo and this concentration was also in the clinical range for each agent. A kinetic response suggestive of competitive inhibition is occuring at the aldehyde binding site on the luciferase and it is postulated that this is related to the very hydrophobic nature of this site.

Resident mouse peritoneal macrophages contain a phospholipase C of high activity that is specific for phosphatidylinositol. The activity has a neutral pH optimum, is Ca2+-dependent and has a maximum reaction velocity of 525nmol/h per mg of protein. Certain phenothiazines are potent inhibitors of this activity.

The inhibition of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) by L-hydrazinosuccinate has been studied. The velocity of the enzyme reaction decreased with time when the reaction was initiated by the addition of enzyme to a mixture of the assay components and L-hydrazinosuccinate, while it increased slowly from a low level when a preincubated mixture of the enzyme and the inhibitor was added to the reaction mixture to initiate the reaction. Nearly 50% decrease in the initial reaction velocity was produced by a prolonged preincubation of the enzyme with the inhibitor, both at low concentrations of about 2 nM. These findings indicate that the inhibition is of the slow- and tight-binding type. The time-course of the reaction of the enzyme and the inhibitor, examined by the change in activity, was not in accord with single-step mechanisms, but rather appeared to follow biphasic kinetics. The inhibition could be fully reversed only in the presence of L-cysteine sulfinate or large excess of L-aspartate to convert the regenerated enzyme to its pyridoxamine form. The time-course of the reversal followed pseudo-first-order kinetics. Quantitative analysis of the experimental data has shown that the results are consistent with a mechanism of enzyme-inhibitor interaction which involves a reaction of two consecutive, reversible steps. The overall inhibition constant for L-hydrazinosuccinate was calculated to be approx. 0.2 nM.

Bilirubin was found to be a potent inhibitor of NAD+-linked isocitrate dehydrogenase (threo-ds-isocitrate:NAD+ oxidoreductase (decarboxylating), EC 1.1.1.41). It inhibited the enzyme by decreasing the affinity of enzyme for isocitrate, while the maximum reaction velocity was not affected. Bilirubin (less than 10−6 M) showed a detectable inhibition. On the other hand, neither the mitochondrial nor the cytoplasmic NADP+ enzymes (threo-ds-isocitrate:NADP+ oxidoreductase ((decarboxylating), EC 1.1.1.42) were affected by bilirubin.

Summary The first part of this lecture is devoted to some aspects of the study concerning the relationship between chemical structure and biological activity of lysozymes: role of the N -terminal sequences and of the tryptophan (modification by ozonization), histidine and cystine (heat stability) residues. In the second part are summarized some recent results obtained in the studies of goose and duck egg-white lysozymes and of human lysozymes (normal and from patients suffering from leukemia); the N -terminal sequences (53 amino acids) of two duck egg-white lysozymes are reported. Some observations on the mechanism of action of the lysozymes can be found in the conclusion (substrates, reaction velocity, specificity, transglycosylation reactions, behaviour in the presence of inhibitors).

A method has been presented for assay of PLP utilizing partially purified and highly resolved tyrosine decarboxylase. A rapid and highly sensitive measurement of the reaction velocity, and thus concentration, is afforded by capture of 14CO2 in a KOH-containing well, which can be counted directly in a scintillation vial. The conditions and specificity of this assay have been defined.

Semi-purified preparations of histidine decarboxylase from hamster placenta and from fetal rat tissues have been studied. The properties of the histamine-forming enzyme from hamster placenta are very similar to those of fetal rat histidine decarboxylase. Both enzymes can be extracted and purified by the same technique and their kinetic properties appear identical. With saturating concentrations of histidine the apparent maximal reaction velocity is around pH 5.5, but it could be established that the pH optimum of the reaction is inversely related to the substrate concentration. The apparent K m decreases with increasing pH. Enzymes with similar properties have been demonstrated in some other tissues of the rat and mouse; the apparent substrate specificity seems to justify the use of the name histidine decarboxylase to denote this enzyme, or group of enzymes.