Antioxidant and Neuroprotective Activities of Mogami-benibana (Safflower,Carthamus tinctorius Linne)

Free radical scavenging activity of the extracts of petals (bud, early stage, full blooming and ending stage), leaf, stem, root and seeds of Mogami-benibana (safflower, Carthamus tinctorius Linne), the contents of the major active components of carthamin and polyphenols, and neuroprotective effect of the petal extracts and carthamin in the brain of mice and rats were examined. Water extracts of Mogami-benibana petals scavenged superoxide, hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and singlet oxygen. The scavenging activities of the extract of safflower petals with various colors showed the order of orange, yellow and white from high to low. This order is consistent with the contents of carthamin, which is a pigment of orange color and is found highest in orange petals and lowest in white petals. There was also a relationship between DPPH radical scavenging activity and carthamin content in the petal extracts of safflower. The neuroprotective effects were examined in cellular and animal models. Mogami-benibana petal extract inhibited glutamate-induced C6 glia cell death, significantly decreased the formation of malondialdehyde in mouse cerebrum, and inhibited the increase in thiobarbituric acid reactive substances and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the cerebral cortex of rats subjected to an injection of FeCl₃ solution into the sensory motor cortex. Carthamin showed similar effects in inhibiting 8-OHdG by the petal extract in rats. These results suggest that the petal extract of Mogami-benibana has free radical scavenging activity and neuroprotective effect and carthamin is one of the major active components. Special Issue Article in Honor of Dr. Akitane Mori.     

Biosynthesis of carthamin in florets and cultured cells ofCarthamus tinctorius

Phenylalanine labelled by¹⁴C was administered to the cultured cells and the intact flowers ofCarthamus tinctorius, and the biosynthetic activity of carthamin in these two materials was compared. The cultured cells took up positively the fed substrate, but they could not incorporate the label into carthamin, while incorporation of the radioactivity from phenylalanine into the red pigment occurred in the intact flowers. The activities of polyphenol-oxidizing enzymes were screened in the cell cultures and the intact tissues from the herbal plant. Polyphenol-oxidizing enzymes were operative normally in the mother explant, whereas their activity patterns changed altogether in the cultured cells, where kurenamin, a new reddish pigment, is produced actively. The data are discussed in terms of the phenotypic changes in the polyphenol metabolism of the cultured cells propagated under restricted culture conditions.     

Purification and characterization of precarthamin decarboxylase from the yellow petals of Carthamus tinctorius L

Carthamin, a red quinochalcone pigment in safflower (Carthamus tinctorius L.), is enzymatically converted from a yellow precursor, precarthamin. The enzyme, which catalyzes the oxidative decarboxylation of precarthamin to carthamin, was purified to apparent homogeneity from yellow petals of safflower and named precarthamin decarboxylase. The molecular mass of the denatured enzyme was estimated as 33 kDa by SDS-PAGE. The molecular mass of the native enzyme was determined by gel filtration chromatography to be 24 kDa; thus, the native enzyme is a monomer. The optimum pH of the enzyme was 5.0. The enzyme activity was inhibited by Mn2+, Fe2+, and Cu2+ and sharply decreased at temperatures higher than 50 degrees C for 10 min. The activation energy and the Arrhenius frequency factor of the enzyme reaction were 19.7 kcal mol(-1) and 9.94 x 10(11) s(-1), respectively. The saturation curve of precarthamin showed that the enzyme follows Michaelis-Menten kinetics. The Km and Vmax of the enzyme were calculated as 164 microM and 29.2 nmol/ min, respectively. The turnover number (kcat) of the enzyme was calculated as 1.42 x 10(2) s(-1). The enzyme activity was severely inhibited by reducing agents such as glutathione and DTT at pH 5.0, suggesting that a disulfide bond may play an important role in enzyme function.     

Influence of UV-light on carthamin accumulation and floret elongation in a cultivar of saffron thistle (Carthamus tinctorius L.)

Pre-matured florets of Benibana, a cultivar of saffron thistle (Carthamus tinctorius L.) was irradiated with UV-B (280–320 nm) or UV-C (254 nm) light for 48 h at 23±1 °C and the influence of UV-light on carthamin accumulation and floret elongation was investigated. UV-C light enhances carthamin accumulation most prominently, showing a specific value of 52.3 nmol carthamin·dm⁻³·h⁻¹·25μm⁻² (13.9 times of control), while it restricts floret elongation by a light-suppression manner (net elongation: 0.058 mm·h⁻¹, one ninth of control). UV-B light is also promotive for the red colour appearance (25.0 nmol carthamin·dm⁻³·h⁻¹·25 μm⁻², 6.7 times of control) with suppressing floret elongation (net elongation: 0.17 mm·h⁻¹, one third of control). Heterogeneous productivity of carthamin was seen in floret tissues after continuous treating under UV-C light. Carthamin accumulation, heterogeneous carthamin productivity and decrease of floret elongation restraint under UV-lights are discussed.     

Distribution of post-proline cleaving enzyme in human brain and the peripheral tissues

Summary We have studied the distribution of post-propline cleaving enzyme activity in the various tissues in humans using 7-(succinyl-Gly-Pro)-4-methylcoumarinamide as substrate. The post-propline cleaving enzyme activity was high in muscle, testes, kidney and submandibular gland, but was low in the heart, mesenterium and aorta. In the brain, relatively high post-propline cleaving enzyme activity was observed in the cerebral cortex, but other brain regions showed a very low enzyme activity.On Sephadex G-100 column chromatography, enzyme activity in human kidney showed a major peak and a minor peak. The major peak coincided with the enzyme in human cerebral cortex, but was different from human serum enzyme. Diisopropylfluorophosphate, a serine protease inhibitor, strongly inhibited the enzyme activity of each active fraction. The enzyme in the cerebral cortex and kidney was inhibited by heavy metals and thiol blocking agents. However, inhibition of enzyme activity in the serum was not observed with such inhibitors. Therefore, we suppose that post-proline cleaving enzyme activity in the brain is similar, if not identical, to that in the kidney.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Adenylate cyclase and guanylate cyclase.

Adenylate cyclase from the guinea-pig pancreas was activated in a dose-dependent manner by both secretin and cholecystokinin-pancreozymin, but in contrast with results in other species the hormones were approximately equipotent. All other hormones and transmitter substances tested were without any effect on adenylate cyclase activity. Guanylate cyclase activity was shown to have both particulate and supernatant components in the guinea-pig pancreas. The particulate enzyme, but not the supernatant enzyme, was markedly activated by Triton X-100, and most of the induced activity was released into the supernatant. The supernatant enzyme was specifically Mn2+-dependent, but, even though Mn2+ was maximally effective at a concentration of 3 mM, activity could be raised further by increasing Ca2+ concentration. The particulate enzyme, by contrast, was relatively Mn2+-independent. Activity of the particulate guanylate cyclase was enhanced by phosphatidylserine. The supernatant enzyme displayed classical Michaelis-Menten kinetics, but the particulate enzyme deviated markedly from such kinetics. Under none of the conditions used was any significant activation of guanylate cyclase observed with any of the secretogen hormones or transmitter substances.     

Modulation of Tryptophan Hydroxylase Activity by Phospholipids: Stimulation Followed by Inactivation

The activity of tryptophan hydroxylase from the rat brainstem was stimulated rapidly three- to fourfold by the addition of phosphatidylinositol or phosphatidylserine. However, the activity of the enzyme once stimulated was decreased gradually by subsequent incubation with the phospholipid at 37 degrees C, reaching a level below the original activity after 1 h of incubation. The presence of ferrous ion almost perfectly protected the enzyme against this phospholipid inactivation. The activity of the enzyme inactivated by incubation with the phospholipid was not only restored, but also increased further by incubation at 37 degrees C with ferrous ion and dithiothreitol. Gel filtration analysis revealed that the enzyme stimulated by phosphatidylinositol was eluted in a void volume together with the phospholipid vesicles, but the enzyme inactivated by incubation with phosphatidylinositol was eluted at a later region apart from the vesicles. These results, taken together, suggest the possible involvement of cellular membranes in the regulation of tryptophan hydroxylase in the central nervous system.     

Increase in NADH-glutamate dehydrogenase activity by mercury in excised bean leaf segments

Application of Hg to excised bean leaf segments increased the glutamate dehydrogenase (NADH-GDH) activity substantially. However, specific activity of the enzyme decreased at lower concentration of Hg, and increased to lesser extent at higher concentration of Hg. Mercury supply increased the glutamate synthase (NADH-GOGAT) activity also. Mercury supply increased the NADH-GDH activity in the presence of NH4NO3, but to a lesser extent than in the absence of NH4NO3. The specific activity of the enzyme decreased considerably at lower concentration of Hg, but increased significantly at higher concentration of Hg. An increase in NADH-GOGAT activity was observed in the presence of NH4NO3, but specific activity of the enzyme decreased marginally. Increase in GDH activity due to Hg remained unaffected by the supply of sucrose, but was reduced by glutamine and glutathione and enhanced by Al. The glutamate dehydrogenase (+Hg enzyme) from mercury treated leaf segments had higher value of S0.5 for NADH than the enzyme (-Hg enzyme) from material not treated with mercury indicating that Hg binding to enzyme prevented NADH binding to the enzyme possibly at thiol groups. However, + Hg enzyme has more reactivity, as apparent Vmax value was higher for it. It has been suggested that Hg activates the NADH-GDH enzyme in the bean leaf segments by binding to thiol groups of protein and pronounced increase in activity by Hg suggests a possible role of enzyme under Hg-stress.     

Purification and partial characterization of iodothyronine 5'-deiodinase from rat liver microsomes

We have isolated and purified iodothyronine 5'-deiodinase from rat liver microsomes to homogeneity as judged by PAGE and analytical HPLC. The enzyme progressively lost activity after solubilization, and specific activity enhancement was a modest 22-fold, but the final preparation still had substantial activity and was used for molecular characterization. The enzyme had an Mr of 56,000 with a single band in SDS-PAGE, suggesting absence of subunit structure. The high Km, and the GSH-responsive low Km, activities were co-purified, but the low Km enzyme lost GSH-responsiveness upon pretreatment with dithiothreitol (DTT) and urea. The enzyme was strongly inhibited by the iron chelator, alpha,alpha'-dipyridyl and showed a broad absorbance band at 410 nm. Spectral analysis with diethylpyrocarbonate (DEPC) revealed 5 histidine residues/mol enzyme, while enzyme activity was inhibited by DEPC in a pseudo-first order process with modification of 1 histidine residue/mol.     

Phosphatidylinositol-specific phospholipase C of murine lymphocytes

Phosphatidylinositol-specific phospholipase C (PI-phospholipase C) was found primarily in the cytosolic fraction of murine splenic lymphocytes. However, small but significant amounts of the activity of the enzyme were detected in the microsome and plasma membrane fractions. Both the cytosolic and membrane-bound phospholipases C specifically hydrolyzed inositol phospholipids, phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. PI-Phospholipase C activity was detected in the cytosolic and microsome fractions from both T-cell-enriched and B-cell-enriched spleen cells. The membrane-bound enzyme was distinguishable from the cytosolic enzyme in the following properties. The cytosolic PI-phospholipase C showed optimal activity at pH 6.0 while the membrane-bound enzyme had two pH optima between pH 5.0 and 7.0. The activity of the cytosolic enzyme was first detected at 1 microM Ca2+, and maximum activity was observed at 100 microM Ca2+, while the membrane-bound PI-phospholipase C required higher Ca2+ concentrations, of millimolar order. The membrane-bound enzyme could hardly be extracted with 1 M NaCl but was extracted with 0.4% cholate.A portion of the membrane-bound PI-phospholipase C activity in the cholate extract was absorbed by concanavalin A-Sepharose and specifically eluted with an alpha-methylmannoside solution. The cytosolic enzyme, which was water soluble, did not bind to concanavalin A-Sepharose. Trypsinization of lymphocytes before subcellular fractionation caused a significant decrease in the PI-phospholipase C activity in the microsome fraction but almost no loss at all of the cytosolic enzyme activity.     

Purification and properties of a lytic enzyme from the cell wall of Chlorella ellipsoidea C-87

Cell wall lytic activity was detected in the culture medium and cell wall of 1AM Chlorella ellipsoidea C-87. The enzymes of both fractions had their highest activity at pH 5. The lytic activity bound to the cell wall consisted of a polysaccharide releasing enzyme, an exo-type enzyme releasing disaccharide, and glucosidase; but only the polysaccharide releasing enzyme was solubilized by lithium chloride. A polysaccharide releasing enzyme with a molecular weight around 40 kDa was isolated from the culture medium. Hemicellulose is degraded by the polysaccharide releasing enzyme, and the rigid wall by the exo-type enzyme.     

Purification and characterization of lipoprotein lipase from pig myocardium.

1. Lipoprotein lipase was purified from pig myocardium by a two-step purification procedure involving (a) the formation of an enzyme-substrate complex and (b) affinity chromatography on Sepharose which contained covalently linked heparin. The purified enzyme gave in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis one main band with an apparent molecular weight of 73 000. The enzyme, which was purified 70 000-fold, had a specific activity of 860 mumol of unesterified fatty acid liberated/h per mg of protein. 2. The purified enzyme hydrolysed [14C]triolein emulsions in the absence of added cofactors but its activity was increased fivefold by adding normal human serum. Of the low-density lipoprotein apoproteins only apolipoprotein CII could be substituted for serum in activating the enzyme. This lipase had maximum activity at 0.05-0.15 M-NaCl. Heparin increased the activity of the purified enzyme twofold at low concentrations, but high concentrations inhibited. The triglyceride lipase of pig myocardium thus resembles lipoprotein lipase purified from adipose tissue and from plasma, but is clearly different from pig hepatic triglyceride lipase.     

Combined action of Escherichia coli glycogen synthase and branching enzyme in the so-called "unprimed" polyglucoside synthesis.

Mutants of Escherichia coli which are unable to synthesize glycogen were used to study the so-called “unprimed” synthesis of glycogen. The glycogen synthase has been partially purified from these mutants. During the purification, attempts were made to separate the activity which requires the addition of an exogenous primer (primed activity) from the activity which does not require a primer but is highly dependent on the presence of some salts such as citrate and EDTA (unprimed activity). No separation between these two activities could be achieved but the results obtained by chromatography on DEAE-Sephadex indicate that there is a single form of glycogen synthase which is responsible for both unprimed and primed activity. The evidence that a single protein was necessary to catalyze these two reactions was given by the findings that mutants defective in glycogen synthase activity were unable to catalyze glucosyl transfer without added primer. At low concentration, the glycogen synthase purified from a branching enzyme negative mutant catalyzed the unprimed reaction at a slow rate even in presence of salts. A protein activator of this reaction was found in mutants lacking glycogen synthase but not in mutants lacking branching enzyme. The hypothesis that this activator is the branching enzyme itself was supported by the observation that it co-purified with the branching enzyme from a E. coli strain defective in glycogen synthase activity. EDTA or Triton X-100 increased the stimulation of the unprimed synthesis by the branching enzyme. The apparent affinity of the glycogen synthase for glycogen was increased twofold in the presence of EDTA but the branching enzyme further increased the effect of EDTA. The combined action of the glycogen synthase and the branching enzyme on the endogenous glucan associated with the synthase may account for the unprimed activity observed in vitro.     

Purification and characterization of a thermostable, haloalkaliphilic extracellular serine protease from the extreme halophilic archaeon Halogeometricum borinquense strain TSS101

A novel haloalkaliphilic, thermostable serine protease was purified from the extreme halophilic archaeon, Halogeometricum borinquense strain TSS101. The protease was isolated from a stationary phase culture, purified 116-fold with 18% yield and characterized biochemically. The molecular mass of the purified enzyme was estimated to be 86 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10.0 in 20% NaCl. The enzyme had high activity over the pH range from 6.0 to 10.0. Enzymatic activity was strongly inhibited by 1 mM phenyl methylsulfonyl fluoride, but activity was increased 59% by 0.1% cetyltrimethylammonium bromide. The enzyme exhibited relatively high thermal stability, retaining 80% of its activity after 1 h at 90 degrees C. Thermostability increased in the presence of Ca2+. The stability of the enzyme was maintained in 10% sucrose and in the absence of NaCl.     

Characterization of an iron- and manganese-containing superoxide dismutase from Methylobacillus sp. strain SK1 DSM 8269

A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.     

Glycogen debranching enzyme in bovine brain

Glycogen debranching enzyme was partially purified from bovine brain using a substrate for measuring the amylo-1,6-glucosidase activity. Bovine cerebrum was homogenized, followed by cell-fractionation of the resulting homogenate. The enzyme activity was found mainly in the cytosolic fraction. The enzyme was purified 5,000-fold by ammonium sulfate precipitation, anion-exchange chromatography, gel-filtration, anion-exchange HPLC, and gel-permeation HPLC. The enzyme preparation had no alpha-glucosidase or alpha-amylase activities and degraded phosphorylase limit dextrin of glycogen with phosphorylase. The molecular weight of the enzyme was 190,000 and the optimal pH was 6.0. The brain enzyme differed from glycogen debranching enzyme of liver or muscle in its mode of action on dextrins with an alpha-1,6-glucosyl branch, indicating an amino acid sequence different from those of the latter two enzymes. It is likely that the enzyme is involved in the breakdown of brain glycogen in concert with phosphorylase as in the cases of liver and muscle, but that this proceeds in a somewhat different manner. The enzyme activity decreased in the presence of ATP, suggesting that the degradation of brain glycogen is controlled by the modification of the debranching enzyme activity as well as the phosphorylase.     

Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-monooxygenase

7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase was purified from liver microsomes of phenobarbital-treated rabbits. The purification was carried out by solubilization of microsomes by cholate, fractionation with polyethylene glycol, affinity chromatography on cholate-Sepharose 4B column, hydroxylapatite column chromatography, chromatography on DEAE-Sepharose CL-6B column, and a second hydroxylapatite column chromatography. The purified preparation gave a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 9.0 nmol of cytochrome P-450/mg of protein, which corresponded to 5.3-fold purification from microsomes on the basis of specific heme content. The specific activity of the enzyme expressed as enzyme activity per mg of enzyme protein was increased 315-fold from microsomes. The molecular weight of the enzyme was estimated to be 56,000 from calibrated polyacrylamide gel electrophoresis. The enzyme-pH curve gave a peak at pH 7.0. The Michaelis constant for 7 alpha-hydroxy-4-cholesten-3-one was 27 microM. Absorption spectra of the oxidized form of the enzyme showed a Soret band at 418 nm. 7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase activity was reconstituted from the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine, and NADPH. The purified enzyme was free from steroid 25-hydroxylase activity and that of 26- or 27-hydroxylase but revealed some activity for benzphetamine N-demethylation. The enzyme activity was not inhibited by metapyrone, aminoglutethimide, and KCN, but was seriously inhibited by nonionic detergents such as Emulgen 913. The enzyme was labile under low buffer concentrations but was stabilized at least for 4 weeks under higher buffer concentration such as 300 mM phosphate buffer.     

Induction of a novel long-chain acyl-CoA hydrolase in rat liver by administration of peroxisome proliferators

The activity of long-chain acyl-CoA hydrolase in rat liver was increased by the administration of peroxisome proliferators, such as ethyl p-chlorophenoxyisobutyrate, di(2-ethylhexyl)phthalate or acetylsalicylic acid. The induced activity was mainly confined in the soluble fluid after the subcellular fractionation. The enzyme was purified nearly to homogeneity from livers of rats treated with di(2-ethylhexyl)phthalate. The specific activity of the final preparation was 247 mumol palmitoyl-CoA hydrolyzed min-1 mg protein-1. The molecular weight of the native enzyme was estimated to be 150 000 by gel filtration and that of the subunits was 41 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The activity of the enzyme was not increased but inhibited by bovine serum albumin or Triton X-100. The molecular and catalytic properties of the enzyme suggest that the induced enzyme was different from mitochondrial and microsomal long-chain acyl-CoA hydrolyses in liver.     

Purification and Properties of a Glycerol Ester Hydrolase (Lipase) from Propionibacterium shermanii

An intracellular glycerol ester hydrolase (lipase) from Propionibacterium shermanii was recovered from cell-free extracts and purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography on diethylaminoethylcellulose. Maximum enzyme activity was observed at pH 7.2 and 47 C when an emulsion of tributyrin was used as substrate. The enzyme was stable between pH 5.5 and 8. Heating the enzyme solution at 45 C for 10 min resulted in a 75% decrease in activity. Maximum rate of hydrolysis of triglycerides was observed on tripropionin, followed in order by tributyrin, tricaproin, and tricaprylin. The lipase was strongly inhibited by mercury and arsenicals, but specific sulfhydryl reagents had little or no inhibiting effect on the enzyme activity. The enzyme also showed some esterase activity, but the hydrolysis of substrates in solution was small as compared to the hydrolysis of substrates in emulsion.     

Milk Clotting Enzyme from Microorganisms

The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.

The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.

Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.

The enzyme activity was inhibited by Hg⁺⁺ or Fe⁺⁺⁺.

Trypsinogenkinase activity was not contained in this enzyme.

The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.

The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.

Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin.