Colorimetric measurement of dermatan sulphate

The periodate-Schiff reaction has been adapted for the measurement of dermatan sulphate. The method is specific for this glycosaminoglycan, provided that glycogen and glycoproteins are removed. Measurements of dermatan sulphate present in the urine of patients affected by various mucopolysaccharidoses indicate a good agreement between the values obtained with enzymic methods and those obtained with the colorimetric method described.     

Regulation of ornithine decarboxylase synthesis. Effect of a nutritional deficiency of vitamin B6

In vitamin B₆ deficiency there is an increase in the activity of the pyridoxal phosphate dependent enzyme ornithine decarboxylase. In the rat liver: the apoenzyme and holoenzyme activity increased 1.6 and 4 fold respectively. Concomitantly, putrescine and spermidine concentrations were halved. The lack of correspondence between product concentration and enzymic activity suggests a control mechanism other than ornithine decarboxylase activity.     

In vitro transformation of dehydroepiandrosterone or its sulphate into androstenediol or its sulphate by rat brain and blood preparations

Abstract— –Enzymic transformation of [4-¹⁴C]dehydroepiandrosterone or [4-¹⁴C]dehydro-epiandrosterone sulphate to androstenediol or its sulphate occurred when incubated with a microsomal preparation of rat brain or a whole rat blood homogenate. The brain enzyme which appeared to cause this transformation had a pH optimum at 60, was NADPH₂-dependent, and had an apparent K_m of 4·6 × 10^?6m . When the subcellular fractions of rat brain were compared for transformation, microsomes had the highest specific activity, followed by the cytosol. The crude nuclear and mitochondrial fractions had no significant activity. The level of enzymic activity in the brain microsomes increased from that for rats sacrificed at 7 days of postnatal age to a maximum for rats sacrificed at 1 month of age; then the activity appeared to level off in rats older than 1 month. Microsomes obtained from the cerebellum had the highest specific activity in comparison to that obtained from the cerebral cortex, the diencephalon, and the brain stem. The incubated preparations of rat brain also converted dehydroepiandrosterone sulphate to androstenediol sulphate without hydrolysis. The enzyme in rat blood which was similar to that in the brain was also partially characterized. The blood enzyme had a pH optimum at 6–5, was nearly exclusively present in erythrocytes, was also NADPH₂-dependent, and had an apparent K_m of 2·7 × 10^?4m . The developmental pattern of the blood enzyme specific activity was similar to that of the rat brain enzyme. Upon haemolysis, most activity was recovered in the haemolysate.     

The dissociation of glucose oxidase by sodium n-dodecyl sulphate.

1. The enzymic activity of glucose oxidase was determined as a function of pH and sodium n-dodecyl sulphate (SDS) concentration. 2. Glucose oxidase is not deactivated by SDS at pH 6 even after prolonged incubation, but is deactivated at pH 4.3 and 3.65. 3. Sedimentation-rate analysis showed that glucose oxidase dissociates into its two subunits at pH 5 and below, and sedimentation-equilibrium experiments in the presence of SDS gave a subunit molecular weight of 73,500. 4. SDS binds to glucose oxidase in acid solutions; specific binding occurs ap pH 3.65, but at pH 6 only co-operative binding was observed. 5. Glucose oxidases in which some of the carboxy groups were blocked with glycine methyl ester were deactivated by SDS at pH 6.0; the rate of deactivation increased with the extent of esterification. 6. Deactivation of esterified glucose oxidases correlated with thermal analysis of the initial SDS interaction, the exothermicity of the interaction increasing with the extent of esterification. 7. The results show that carboxy groups confer resistance to deactivation by SDS on glucose oxidase by screening cationic residues and inhibiting specific interactions that facilitate dissociation into subunits.     

Studies on metabolism of vitamin A. The effect of the stage of vitamin A deficiency on sulphate activation in rat liver

1. The synthesis of ;active sulphate' in rat liver was studied at various stages of vitamin A deficiency, with the corresponding pair-fed controls. 2. The activity was significantly decreased even at the onset of the deficiency, and at the acute stage there was further loss. 3. Only at the earlier stages of the deficiency was the addition of retinol, in vitro, fully effective in restoring the lost activity; retinoic acid was partially active. No such restoration was possible at the acute deficiency stage.     

Formation and interrelationship of the activity of several dehydrogenases during rat development]

The specific and general enzymic activity of some dehydrogenases of the liver, kidneys, heart, thymus and spleen was studied on rats in early postnatal period. Some regularities of the development of enzymes under study were established: ergontic correlations of the "maturing" enzymic organ systems and the coordination of the enzymic status of organs and the lymphocytes circulating in the blood. It may be supposed that the lymphocyte is a carrier of information about formation of metabolic processes of the developing organism. The mechanisms of appearance of coordination of the enzymic activity are discussed.     

Thermogenesis of mesophilic, thermotolerant and thermophilic strains of microorganisms--producers of fungal cell wall--destroying enzymes]

Investigations were carried out to clarify the relationship between thermogenesis and production of yeast wall lyzing enzymes by the mesophilic strain of Bacillus subtilis, thermotolerant strain of Actinomyces sp. II and thermophilic strain of Actinomyces sp. 10. The enzymic lyzing activity was measured in the culture liquid filtrate of those microorganisms. The thermophilic strain of Actinomyces sp. 10 showed the highest enzymic activity. The thermogenetic curves of the cultures had several inflections. The mesophilic culture of Bacillus subtilis whose enzymic lyzing activity was the lowest displayed the highest heat release.     

Regeneration of vitamin E in human platelets

Human platelets possess active lipoxygenase and cyclooxygenase which convert arachidonic acid to (12S)-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) plus (12S)-12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and thromboxane B2 plus 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), respectively. When platelet homogenates were incubated with arachidonate, there was a rapid consumption of platelet tocopherol. Time course analysis revealed that within 0.5 min, over half of arachidonate and tocopherol were metabolized. Mass formation of 12-HPETE and 12-HETE or thromboxane B2 and HHT exceeded that of the mass of tocopherol oxidized. Preincubation with the lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) completely abolished this arachidonate-induced tocopherol oxidation whereas cyclooxygenase inhibitors (indomethacin and aspirin) further potentiated tocopherol oxidation, indicating that this oxidation is closely linked with platelet 12-lipoxygenase activity. Incubation with lipoxygenase metabolites of arachidonic acid showed that only 12-HPETE caused a rapid tocopherol oxidation which was followed by a gradual tocopherol regeneration. By using nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor which is also a strong reductant, over 60% of the arachidonate-induced oxidized tocopherol was regenerated. Tocopherol regeneration declined with increasing oxidation time induced by arachidonate, and after 30-60 min virtually no regeneration could be observed, suggesting that the precursor molecule was unstable. We postulate that the precursor molecule is the tocopheroxyl radical. In the presence of ETYA, a lipoxygenase inhibitor without antioxidant properties, either ascorbate or GSH provided significant tocopherol regeneration. Kinetic studies showed that tocopherol regeneration after the addition of ascorbate was essentially completed by 1 min. By contrast, GSH addition caused a steady increase in tocopherol which peaked after 10 min of its addition. To determine whether this rapid regeneration is chemical or enzymic, regeneration was studied in the presence of chloroform and methanol. Comparison of various reductants in this denaturing condition for enzymes showed that ascorbate and NDGA afforded significant regeneration whereas GSH was ineffective, indicating that there are distinct enzymic and non-enzymic mechanisms for tocopherol regeneration. This study provides direct evidence from mass analysis that tocopherol can be regenerated in human cell homogenates. This finding implies that maintenance of membrane tocopherol status may be an essential function of ascorbate and GSH which operate in concert to ensure maximum membrane protection against oxidative damage.     

Mitochondrial malate dehydrogenase of bovine cerebrum. Characterization and mechanisms of inhibition by silver ions.

Attempts were made to characterize mitochondrial malate dehydrogenase [L-malate: NAD+ oxidoreductase, EC 1.1.1.37] (M-MDH) purified from bovine cerebrum and to elucidate the mechanisms responsible for inhibition of the enzymic activity by Ag+. The molecular weights of the native enzyme and its subunits were 54,000-55,000 and 30,000-32,000, respectively. In general, the physiochemical and catalytic properties of bovine cerebral M-MDH was not very different from those of other corresponding mammalian enzymes. Incubation of the enzyme with Ag+ caused the loss of equivalent amounts of sulfhydryls with a parallel decrease of the enzymic activity. When the enzyme was exposed to 2-, 3.5-, and 5-fold molar excesses of Ag+, the enzymic activity showed an initial rapid fall and a subsequent slow restoration to a partially inactivated level (60-70, 45-50, and 15-20% of an untreated control, respectively), while the alpha-helical content of the enzyme fell exponentially with time. A 7-fold molar excess of Ag+ reduced both the enzymic activity and the alpha-helical content to a much greater degree and no restoration of the enzymic activity was observed. The Km values of Ag+-inactivated enzyme for NADH and oxaloacetate were the same as those of the native enzyme. The data suggest that Ag+ could inhibit enzymic activity both by reducing the structural regularity of the enzyme molecule and by attacking sulfhydryl groups necessary for the catalytic activity of bovine cerebral M-MDH.     

Histochemical studies on cell death and histolysis during regeneration. I. Distribution of acid phosphomonoesterase activity in the normal, the regenerating and the resorbing forelimb of the larval spotted salamander, Amblystoma maculatum

Histochemical methods, especially azo dye methods for detecting acid phosphomonoesterase activity were applied to normal, regenerating and denervated, amputated limbs from larval Amblystoma maculatum. Efforts were made to control inactivation of enzymic activity and diffusion of both enzyme and reaction product. “Base-line” values for enzymic activity were determined for normal limbs. Activity appeared most intense in macrophages, less intense in epidermis and cartilage matrix. Some activity was detected in Schwann cells, peri- and endoneurium and muscle fibers form normal limbs. Enzymic activity in regenerating limbs was strongest within macrophages which appeared in increased numbers especially in early stages. Wound tissue showed little increased activity. As the blastema formed, increased enzymic activity was detected in epidermis and within increased numbers of macrophages. Chondrocytes showed increased activity especially during cartilage matrix deposition. Amputated, denervated limbs showed large numbers of active macrophages beneath and within epidermis and along muscle. As regression commenced, areas of cartilage matrix breakdown showed increased enzymic activity but, in general, greatest activity was in macrophages. The various possible roles of acid phosphomonoesterase activity in the specific biological situations dealt with are considered in light of such observations.     

Immobilization of wood-rotting fungi laccases on modified cellulose and acrylic carriers

Extracellular laccases produced by three different wood-rotting fungi, Cerrena unicolor, Heterobasidion annosum and Trametes versicolor, were immobilized via covalent bonds formation on DEAE-Granocel 500, CM-Granocel 500, and acrylic carriers. Out of the tested carriers, only the DEAE-Granocel 500, which was activated by divinyl sulphone appeared to be a suitable matrix for the expression of enzymic activity. Only one laccase of all the tested enzymes produced by C. unicolor showed the best binding to the carrier and a satisfactory enzymic activity. The immobilized laccase exhibited the highest enzymic activity at pH 5.2 and it was more resistant to thermal denaturation than the native enzyme. At 90 °C, it retained 75% activity compared to the free enzyme. It was also more stable during storage at 4 °C: after 4 months the immobilized laccase retained 98% of initial activity. Immobilized C. unicolor laccase was active in 10–60% concentration of methanol, acetone, isopropanol or acetonitrile. The best enzymic activity was observed in 20% solution of acetonitrile in buffer.     

Identification and quantification of dehydroepiandrosterone sulphate in saliva

3β-Hydroxy-5-androsten-17-one (dehydroepiandrosterone) sulphate has been separated from an extract of human saliva by ion exchange gel chromatography and identified by high resolution gas chromatography-high resolution mass spectrometry of the $\text{tert}$-butyldimethylsilyl derivative of the neutral steroid obtained by enzymic hydrolysis. Quantitative analyses, employing 7,7?²H-dehydroepiandrosterone sulphate as the internal standard, have indicated concentrations in the saliva of young adult subjects to be generally in the range 0.9?5.7 nmol/l, though concentrations as high as 1O.7 nmol/l have been observed.     

Activation and stabilization of asparaginase by anti-asparaginase IgG and its Fab

Modified asparaginase, in which 4 tryptophan residues were modified with 2-hydroxy-5-nitrobenzyl bromide, had little enzymic activity and retained immunoreactivity [(1976) FEBS Lett. 65, 11-15]. Addition of IgG or its Fab towards asparaginase to the modified asparaginase gave rise to marked enhancement of the enzymic activity. Native asparaginase (4 subunits) lost the enzymic activity due to dissociation into subunits by dilution of the enzyme solution. However, in the presence of Fab, asparaginase did not lose enzymic activity on dilution, probably due to no dissociation into subunits occurring.     

Modification of an enzymic system of Ca2+ transport in sarcoplasmic reticulum membranes during lipid peroxidation. Induction and regulation systems of lipid peroxidation in skeletal and heart muscles

The enzymic and non-enzymic systems which induce and control lipid peroxidation (LPO) in muscle cells were studied. The maximal activity of enzymic NADH- and NADPH-dependent LPO was observed in sarcoplasmic reticulum (SR) membranes. It was found that an essential role in enzymic LPO induction belongs to superoxide radical anions and to hydroxyl radicals. The maximal concentration of the natural LPO inhibitor, alpha-tocopherol, was detected in SR membranes. The glutathione peroxidase and superoxide dismutase activities were determined in the cytosol fraction of myocytes. The role of compartmentation of enzymic and non-enzymic systems of LPO induction in muscle cells is discussed.     

Chemical modification of intracellular cytosine deaminase fromChromobacterium violaceum YK 391

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase fromChromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1 mM NBS, chloramine-T, ρ-CMB, ρ-HMB and iodine, and was strongly inhibited by 1 mM PMSF and pyridoxal 5′-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by ρ-CMB was also reversed by 1 mM cysteine-HCl, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase fromC. violaceum YK 391 was assumed to be a thiol enzyme.     

The role of cholesteryl 14-methylhexadecanoate in peptide elongation reactions

1. Peptide-elongation factors were purified from rat liver and human tonsils and the contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during enzyme purification. The relative contents of this compound in purified enzyme preparations was several times higher than that in the crude starting material. Elongation factors from human tonsils contained a significantly larger quantity of the cholesteryl ester than enzyme from rat liver. 2. Transfer enzymes extracted with various organic solvents showed variable decreased activities in both binding and peptidization assay. The decrease of enzymic activity was proportional to the amount of cholesteryl 14-methylhexadecanoate extracted from a given enzymic preparation. In systems containing both extracted elongation factors the polyphenylalanine synthesis was limited by the residual activity of the less active transfer factor. 3. The original enzymic activity of extracted transferases was fully recovered by the addition of pure cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. Increase of the relative contents of this cholesteryl ester during enzyme purification, decrease of the enzymic activity after the extraction and its recovery by the addition of this compound indicates that the presence of this ester in elongation factors is essential for the normal function of these enzymes.     

Polyethylene glycol-modified enzymes trap water on their surface and exert enzymic activity in organic solvents

Summary Polyethylene glycol-modified enzymes dissolved and had high enzymic activity in organic solvents. A trace amount of water was found to be necessary for the activity. It was reasoned that the amphipathic polymer covalently attached to enzymes kept water molecules around them. This was supported by findings that : (1) high enzymic activity was found in water- immiscible solvents, whereas activity was never observed in water-miscible solvents; (2) enzymic activity was inhibited by increasing the concentration of dimethyl sulfoxide in benzene; (3) activity of lipase was inhibited by a water-miscible alcohol substrate, but was steadily elevated by increasing the concentration of a water-immiscible alcohol substrate; (4) water was not absorbed from benzene solution containing a modified enzyme by molecular sieves, while it was easily absorbed in the presence of a water-miscible organic solvent, dimethyl sulfoxide.     

Antigenicity of proteinases from Streptomyces griseus (pronase)

The five pronase fractions, A(1), A(2), B, C (trypsin-like), and D (elastolytic), obtained by ion-exchange chromatography, were found to be antigenically distinct. Antibodies to pronase inhibited the enzymic activity of each of the enzyme fractions. Pronase trypsin and bovine trypsin, although resembling each other in enzymic activity and in amino acid sequence around their active sites, did not cross-react antigenically with, nor was their enzymic activity inhibited by, the respective homologous antibodies. Inactivation of pronase trypsin by complexing with soya-bean inhibitor AA, was not associated with a decrease in capacity to precipitate with its antibody. It is assumed that the antigenic sites are located far enough from the catalytic site of the enzyme to allow it to precipitate immunologically even when the catalytic site was blocked.     

Inhibitors of the plasma membrane redox system of Zea mays L. roots. The vitamin K antagonists dicumarol and warfarin.

The action of the 4-hydroxycoumarins dicumarol and warfarin, antagonists of probable vitamin K type components of the plasma membrane electron-transport system, on plasma membrane redox activity of intact maize roots was compared. Both effectors inhibited electron transfer to extracellular hexacyanoferrate III. While the effect of the strongly lipophilic dicumarol on the electron-transport system was irreversible by rinsing, the inhibition caused by the hydrophilic warfarin could be reverted completely by exchange of the incubation medium. We take these results as possible evidence for the integration of dicumarol into the plasma membrane. The action of warfarin may be confined to enzymic sites freely accessible from the aqueous apoplasmic solution.     

Potentials of enzyme tests and radioisotope hepatography in detecting early functional changes in the liver

The enzymic tests and radionuclide hepatography were used to study and compare liver function after rabbits were exposed to tetrachloromethane poisoning. The activity of serum enzymes of cholinesterase, alkaline phosphatase, aldolase and leucine aminopeptidase was determined. Hepatography was made with the use of 198Au-colloid with an activity 0.74 MBC. The enzymic tests were demonstrated to be more sensitive than radionuclide hepatography in detecting the earliest parenchymatous lesions in the liver. The data obtained correlate with the data of the pathohistological examinations, which demonstrated the presence of marked vacuole parenchymatous fatty dystrophy. The authors recommend that the enzymic tests should be used for detecting early hepatic lesions induced by tetrachloromethane.