pH-dependence of carnitine acetyltransferase activity

1. The pH-dependence of the kinetic constants of the carnitine acetyltransferase reaction has been investigated with the enzyme from pigeon breast muscle. 2. Michaelis constants for (-)-carnitine and acetyl-(-)-carnitine vary in a similar fashion in the pH range 6.0-9.0. A single ionizing group on the enzyme with an apparent pK7.2 is required in the basic form for binding of these substrates. 3. Binding of CoASH or acetyl-CoA raises the apparent pK of an ionizing group on the enzyme from 7.85 to 8.25. This group is probably not directly involved in forming the enzyme-substrate complex, but its microscopic environment is presumably altered. Another group in either the substrate or the free enzyme, with an apparent pK6.4, is needed in the basic form for optimum binding of CoA substrates. 4. This last group has been unequivocally identified as the 3'-phosphate of CoA, by showing that the K(m) of carnitine acetyltransferase for the substrate acetyl-3'-dephospho-CoA is independent of pH in the range 6.0-7.8. 5. V'(max.), the maximum velocity of the catalysed reaction between acetyl-CoA and (-)-carnitine, is constant between pH6.0 and 8.8. 6. The significance of these results in terms of a previously postulated reaction scheme for this enzyme is discussed.     

The pH-dependence of the peptidase activity of aminoacylase.

Aminoacylase is a potent peptidase around pH 8.5. The pH dependence of the Km values reveals that only dipeptides with uncharged N-terminal amino acids are substrates of the enzyme. The Km values reflect the hydrophobicity of the N-terminal amino acids. Calculated on the basis of unprotonated peptides they are pH independent. Hydrophobic, deprotonated amino acids are competitive inhibitors of the enzyme, tryptophan and norleucine being the strongest inhibitors. Inhibitor constants with glycylalanine as substrate have been determined for several amino acids. From the present results it may be deduced that the N-terminal amino acids of dipeptides are bound at a strongly hydrophobic site.     

A colorimetric method for measuring the esterolytic activity of elastase

This paper describes a new colorimetric method for measuring the activity of elastase. The substrate used is acetyl-l-alanyl-l-alanyl-l-alanine-methyl ester. Its concentration is determined before and after enzymatic action by the hydroxamic acid method of Hestrin. The procedure described is simpler and more rapid than the usual potentiometric method and may be adapted to serial assays. With a 1-hr test, elastase concentrations as low as 0.2 μg/ml may be assessed accurately.     

Studies on esterolytic activity of alternative complement component factor B

Alternative complement component factor B was purified from human plasma and its esterolytic activity on various α-naphthylester derivatives was examined. Of these substrates, LeuAlaArg-naphthylester was the most susceptible. The K_m value of factor B for this substrate was about 5·10⁻⁴ M. 6-Amidino2-naphthyl-4-guanidinobenzoate inhibited the esterolytic activity of factor B. After activation of factor B, the esterolytic activity did not increase and the decayed form, Bb, cleaved the substrate to the same extent as factor B.     

Estimation of serum alpha 2-macroglobulin based on the esterolytic activity of bound alpha-chymotrypsin

A colorimetric method to estimate alpha 2-macroglobulin (MG) in human serum is described which is based on the capacity of MG:alpha-chymotrypsin complex to hydrolyze N-acetyl L-tyrosine ethyl ester in the presence of excess crude preparation of a trypsin/chymotrypsin inhibitor from redwood seed. Acetyltyrosine formed was measured using Folin's reagent (7). The method was found to be as reliable as, but at least five times more sensitive than the procedures described using trypsin and soybean trypsin inhibitor. MG level is expressed in terms of micrograms of bovine alpha-chymotrypsin bound. Serum from healthy males had a lower value (150.5 +/- 31.9 micrograms/ml, n = 20) than in females (196.8 +/- 40.4, n = 20). No significant difference between the levels in fasting and postprandial conditions was observed.     

pH-dependence characteristics of Ca-ATPase activity of heavy meromyosin with modified SH-groups]

Study of pH-dependence of Ca-ATPase activity of heavy meromyosin (HMM) at low and high ionic strength showed essential differences in the modifying effect of two sulfhydryl reagents, p-CMB and silver. Silver ions in conditions studied independently on pH and KCl concentration produce an inhibition of ATP hydrolysis by myosin and HMM, the shape of the pH-dependence curve remaining similar to that of the native enzyme up to 40% of blocking free sulfhydryl groups. At the same degree of binding of sulfhydryl groups with p-CMB at 0,5 M KCl the pH-dependence curve due to activation at neutral pH changes it's shape and becomes similar to that for dissociation of two ionizable groups (at neutral and alkaline regions). In contrast to this, a low or zero concentrations of KCl no activation was observed for the enzyme with 40-50% of SH-Groups modified by p-CMB and Ca-ATPase in this case seemed to be independent of pH. The data obtained suggest that SH-Groups are not included into the active site of myosin, and the activating effect observed for some sulfhydryl reagents, is due to conformational changes and it can be the result of the penetrance of the organic part of the reagent molecule into hydrophobic region of the protein.     

Characterization of proteolytic activity of proteases

Methods for characterization of protease activity were applied to three Bacillus sp. protease preparations of varying purity. Activities differed by several orders of magnitude between the three proteases. Fractionation by HPLC and assay of peak activity against the two substrates elucidated the presence of 1–4 active fractions in each protease preparation.     

pH-dependence of kinetic parameters in the superprecipitation reaction and ATPase activity of myometrial actomyosin

The investigation of pH-dependence of superprecipitation reaction and ATPase activity of myometrium actomyosin in the interval of pH 5.5-8.0 has detected cupola-shaped curves with maximal activity of both processes by pH 6.5. On the basis of calculating the constants of ionization it was supposed that in the case of actomyosin ATPase imidazole groups of two histidins had an essential role in reaction of ATP hydrolysis and in superprecipitation process--imidazol group of histidine and carboxyl group of asparagin acid. The investigation of [ATP]- and [Mg2+]-dependence of superprecipitation reaction by pH 6.0, 6.5 and 7.0 has demonstrated different pH-sensitiveness of Michaelis constants and maximal speeds relatively Mg2+ and ATP for both processes. It was shown that pH-optimum of ATPase activity of myometrium actomyosin coincided with maximal affinity of actomyosin with ATP and Mg2+ while as for superprecipitation reaction the correlation between value of process by certain pH and affinity with ATP and Mg2+ was not detected.     

pH-dependence of hormone-binding and enzymatic activity of estrophilic hydroxysteroid dehydrogenase from the rabbit liver

The effects of pH on the ability of NADP-dependent estrophilic hydroxysteroid dehydrogenase (HSD) from the soluble fraction of rabbit liver to bind steroids and catalyze their 3 alpha, 3 beta, 17 beta- and 20 alpha-oxidoreduction were studied. The pH optima for enzymatic transformations of various steroids were found to differ significantly by more than two units. These differences do not seem to be related to the localization of the modified group in the steroid molecule. Kinetic data suggest that pH influences the catalytic efficiency, steroid affinity for the protein and, perhaps, the degree of interdependence of steroid and cofactor binding to the protein. These assumptions were confirmed by the results of direct 3H-steroid-HSD binding studies. Furthermore, the maximal levels of binding of various steroids to the protein were found to occur at pH values differing by more than 4 units. Scatchard analysis revealed the effects of hydrogen ion concentrations both on the steroids affinity for the protein and on the concentration of steroid-binding sites of HSD. The data obtained are suggestive of some "superfluity" of the protein steroid-binding site which, in turn, ensures the multifunctionality of estrophilic HSD including a possibility of an alternative orientation of steroids in their binding site.     

Effect of aminonucleoside on the activity of pancreatic proteases

Activation of typsinogen and chymotrypsinogen was studied in pancreatic extracts from normal and aminonucleoside-treated rats with hypoproteinemia. Upon incubation of the pancreatic extracts, the usual 2–3 lag phase of the sigmoidal shaped activation curve was almost abolished in aminonucleoside-treated rats. The maximal activity of the two proteases obtained after complete activation was not affected. Evidence is presented that the early onset of autocatalytic appearance of tryptic or chymotryptic activity was due to the presence of preformed trypsin-like activity in the pancreatic extracts of aminonucleoside-treated rats. A short term treatment with aminonucleoside which did not lead to proteinuria and hypoproteinemia also resulted in an increased trypsin/trypsinogen ratio in pancreas and shortening of the lag period. A direct concentration-dependent effect of the drug was demonstrated in in vitro experiments showing a release of hydrolytic enzymes from isolated pancreatic zymogen granules. The possible physiologic implication of labilization of intracellular structures by aminonucleoside is discussed.     

pH-dependence of the triose phosphate isomerase reaction

1. Some kinetic properties of aspartate transcarbamoylase (EC 2.1.3.2), that had been purified approx. 20-fold from wheat germ, were studied. 2. A plot of enzyme activity against pH showed a low maximum at pH8.4 and a second, higher, maximum at pH10.5. A plot of percentage inhibition by 0.2mm-UMP against pH was approximately parallel to the plot of activity against pH, except that between pH6.5 and 7.5 the enzyme was insensitive to 0.2mm-UMP. 3. Kinetics were studied in detail at pH10.0 and 25 degrees C. In the absence of UMP, initial-rate plots were hyperbolic when the concentration of either substrate was varied. UMP decreased both V(max.) and K(m) in plots of initial rate against l-aspartate concentration, but the plots remained hyperbolic. However, UMP converted plots of initial rate against carbamoyl phosphate concentration into a sigmoidal shape, without significantly affecting V(max.). Plots of initial rate against UMP concentration were also sigmoidal. 4. The theoretical model proposed by Monod et al. (1965) gave a partial explanation of these results. When quasi-equilibrium conditions were assumed analysis in terms of this model suggested a trimeric enzyme binding the allosteric ligands, carbamoyl phosphate and UMP, nearly exclusively to the R and T conformational states respectively, and existing predominantly in the R state when ligands were absent. However, the values of the Hill coefficients for the co-operativity of each allosteric ligand were somewhat less than those predicted by the theory. 5. Some of the implications of these results are discussed, and the enzyme is contrasted with the well-known aspartate transcarbamoylase of Escherichia coli.     

Extracellular proteases and laccases produced by Pleurotus ostreatus PoB: the effects of proteases on laccase activity

International Microbiology - Laccases are enzymes produced by plants and white rot fungi, such as Pleurotus ostreatus, with industrial applications. Fungal laccases have been widely studied, and...     

Isolation of Actinomycetes synthesizing proteases with thrombolytic activity

Proteases with the thrombolytic activity were studied in 212 strains of actinomycetes isolated from different soils of the Soviet Union. The cultures belonged to the genera Micromonospora, Nocardia and Streptomyces. Proteases were synthesized by 41% of the studied actinomycetes and some of their strains completely dissolved in vitro artificially obtained blood thrombi within 120-240 min. In the Streptomyces genus, more active strains were found in the groups Flavus, Fradia and Globisporus. The groups Olivaceus, Violaceus and Viridis had less active strains.     

pH-dependence of inhibitory action of eosin Y on ATPase activity of smooth muscle actomyosin complex of the uterus

Research of pH-dependence of inhibitory action of eosin Y (2',4',5',7'-tetrabromofluorescin) on ATPase of contractile proteins of smooth muscles of the uterus has shown that the increase of concentration of this inhibitor (from 0.1 to 10 microM) influenced the profile of pH-dependence of ATPase activity of actomyosin: in the presence of 0.1 microM eosin Y the change of optimal value of pH has been observed in more sour side in relation to the control; at the increase of concentration of eosin Y (from 0.5 to 10 microM) the strongly pronounced optimum of pH is absents in general. The ability of eosin Y to inhibit the ATPase activity of contractile complex is dependent on pH of incubation environment. The change of pH from 6.0 to 7.2 results in a 9-fold decrease of magnitude of apparent constant of inhibition Ki (from 6.5 +/- 0.8 microM to 0.74 +/- 0.07 microM). The obtained results indicate that the diminishing of concentration of H+ in an incubation environment favors the increase of affinity ATPase of actomyosin for eosin Y and prove the important role of ionization processes in the system "enzyme-substrate-inhibitor" for realization of inhibitory action of eosin Y.