Difference in physical and chemical properties between muscle-type and spleen-type isoenzymes of pyruvate kinase in rat.
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Amino Acids↗
Biomedical subjects
T Horio
Publications and source records attributed to T Horio.
Specific affinity of glycerol dehydrogenase from Geotrichum candidium for 10-carboxydecyl-sepharose: its application to chromatography for purification of the enzyme.
Glycerol dehydrogenase [EC 1.1.1.6] (pI 5.9) from Geotrichum candidium is effectively adsorbed in the presence of 20 mM acute buffer (pH 6.0) on either octyl-Sepharose or 10-carboxydecyl-Sepharose, among ten different kinds of n-alkyl-Sepharose derivatives tested, some of which have an amino or a carboxyl group as a terminal residue. The enzyme adsorbed on 10-carboxydecyl-Sepharose is 95% eluted with 0.26 M NaCl. n-Propanol (10% and 15%, but not 5%), and various nucleotides such as NAD+, NADH, NADP+, NADPH, AMP, ADP, and ATP (1 mM) are also effective for elution. The elution with nucleotides is facilitated by 5% n-propanol. On the other hand, the enzyme adsorbed on octyl-Sepharose is not eluted under the conditions described above. These results suggest that the adsorption-elution of the enzyme on 10-carboxydecyl-Sepharose is due to a combination of hydrophobic interaction and electrostatic repulsion between a specific locus of the enzyme surface and the 10-carboxydecyl residue. Experimental conditions are described under which the enzyme can be purified 266-fold with a yield of 79% by a single chromatography of the cell extract on a 10-carboxydecyl-Sepharose column.
Adsorption↗
Affinities of various nucleases to DNA-Sepharose under non-digestive conditions: survey for productive affinity chromatography.
1. It has been reported that DNase I can be highly purified from pancreas extract by affinity chromatography on a dDNA-Sepharose column under non-digestive conditions. In the present study, the adsorption-elution of other nucleases on the column under non-digestive conditions was studied. 2. All the seven kinds of nucleases tested were adsorbed when applied on a dDNA-Sepharose column under conditions which did not allow the enzymes to hydrolyze the DNA. The non-digestive conditions were as follows. i) For DNase II (pI=10.2), pH 3.0 in the presence of 50 mM sodium sulfate (inhibitor), ii) for micrococcal nuclease (pI=9.6), pH 4.0 in the absence of Ca2+ (activator), iii) for restriction endonucleases Eco RI (pI=5+1), Hind III (pI=5+1), and Bam HI (pI=5+1), pH 4.0 in the presence of 20% glycerol and 0.1% Neopeptone (stabilizers), and iv) for nucleases S1 (pI=5+1) and nuclease P1 (pI=4.5), pH 7.0. At the respective pH's, the enzymes other than nucleases S1 and P1 were cationic so as to exhibit electrostatic attraction to the anionic dDNA-Sepharose. Although S1 and P1 were anionic, they still adsorbed to the column. 3. All the adsorbed nucleases described above were eluted by a concentration gradient of KCl without changing pH. The ionic strengths required for elution were 0.19 for DNase II, 0.53 for micrococcal nuclease, 0.73 for Eco RI, 0.72 for Hind III, 0.37 for Bam HI, 0.17 for P1, and 0.13 for S1. The fact that the ionic strength required for the elution of DNase I (pI=5.0) was 0.39 at pH 4.0 indicates that the former five enzymes except DNase II can be chromatographed with almost the same or higher efficiency than DNase I, because the proteins adsorbed with no-specific affinity could be mostly eluted at lower ionic strength. On the other hand, the fact that nucleases P1 and S1 were adsorbed in spite of electrostatic repulsion suggests that these two enzymes can also be effectively chromatographed, especially when other cationic proteins are previously removed by an appropriate method such as adsorption to a typical cation exchanger.
Animals↗
Hydrophobic-ionic chromatography: its application to microbial glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase.
Glucose oxidase from Aspergillus niger, hyaluronidase from Streptomyces hyalurolyticus, and cholesterol oxidase and cholesterol esterase from Pseudomonas fluorescens were effectively adsorbed on an Amberlite CG-50 column, when the cell-free cultured medium or the cultured medium with cell extract and without cell debris was applied without desalting but at pH less than or equal to 4.5. At the acidic pH, all the ion-exchange groups (-COOH) exist in the protonated form; the adsorption is not due to electrostatic attraction, but to hydrophobic interaction. The enzymes thus adsorbed were effectively eluted by increasing pH, at which the ion-exchange groups became dissociated. This type of adsorption-elution is called hydrophobic-ionic chromatography. By a single run of chromatography, glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase were purified 30-fold, 12-fold, 45-fold, and 20-fold with yields of 82%, 83%, 80%, and 90%, respectively. This indicates that hydrophobic-ionic chromatography on an Amberlite CG-50 column is effective for the purification of various enzymes, provided that they are stable at the acidic pH.
Aspergillus niger↗
Purification and properties of chlorophyllase from greened rye seedlings.
1. Chlorophyllase [EC 3.1.1.14] was extracted from the acetone-dried powder of the chloroplasts of greened rye seedlings with 1% cholate, and purified 870-fold with a yield of about 30%. The purification procedure was composed of fractionations with acetone and ammonium sulfate, and hydrophobic chromatography on a phenyl-Sepharose CL-4B column. 2. The purified enzyme was pure as analyzed by molecular-sieve chromatography and isoelectric electrophoresis. It had an isoelectric point of 4.5 and a molecular weight of 39,000. 3. The purified enzyme was stable at pH 6-9 and 4 degrees C. At pH 7.5, it was stable in the presence and absence of 30% acetone. However, at 30 degrees C, it was not stable above a 10% concentration of acetone. 4. The purified enzyme hydrolyzed chlorophylls a and b from spinach into chlorophyllides a and b and phytols, respectively; and bacteriochlorophyll a from Rhodospirillum rubrum into bacteriochlorophyllide a and a derivative of phytol, possibly all-trans-geranylgeraniol. The hydrolysis rates were stimulated to their maxima in the presence of 30% acetone; maximum stimulation was about 50% with bacteriochlorophyll a and about 400% with chlorophyll a. 5. At pH 7.5 and 30 degrees C in the presence of 30% acetone, the Km values and specific activities were 12 microM and 480 nmol . min-1 . mg-1 for chlorophylls a, and 4 microM and 170 nmol . min-1 . mg-1 for R. rubrum bacteriochlorophyll a, respectively.
Bacteriochlorophylls↗
The mechanisms of inhibitory effect of 8-methoxypsoralen and long-wave ultraviolet light on experimental contact sensitization.
A suppressive effect of treatment with 8-methoxypsoralen (8-MOP) and long-wave ultraviolet light (PUVA) on the induction and elicitation of contact sensitivity to dinitrochlorbenzene was observed in guinea pigs. The mechanisms by which PUVA exerts its inhibitory effect seemed to be variable depending on the site and extent of the treatment. When PUVA was given at the induction site, only one treatment using topical 1% 8-MOP and 7,6 J/cm2 UVA is a small area was enough to inhibit sensitization, and the effect was long-lasting. This seemed to be caused by impairment of Langerhans cell function. On the other hand, when induction was attempted through nontreated skin, repeated exposures in larger areas were needed to suppress the challenge reactions. Nonspecific primary irritant dermatitis revealed a similar effect. This inhibitory effect was transient and possibly caused by lack of sufficient number of immune cells for the elicited reactions. An apparent enhancement of challenge reactions at the PUVA-treated site was too complicated to permit interpretation of its mechanism.
Animals↗
Formation of UV-induced apoptosis relates to the cell cycle.
The relationship of the rate of epidermal proliferation to ultraviolet (UV)-induced cell death, namely sunburn cell (SC) formation, was investigated in vivo using the guinea-pig model. The number of SCs was correlated with (1) cellophane tape stripping which stimulates the proliferation rate and (2) treatment with antimitotic and antimetabolic agents which suppress the proliferation rate. The tape stripping performed 14 h before UV irradiation significantly increased the number of SCs counted 24 h after UV irradiation from a mean of 17.4 to 62.6 per section, and the tritiated thymidine (TdR-3H)-labelling index (LI) increased from a mean of 8.1 to 27.4%. SC counts and LI were markedly decreased by the intradermal injection of colchicine, methotrexate, hydroxyurea or 5-fluorouracil given once immediately after the tape stripping. The autoradiographic study showed that 23.2% of SCs were labelled when TdR-3H was given intradermally 1 h before UV exposure, while no SCs were labelled when TdR-3H was given immediately after UV exposure. The labelled SC count (23.2%) which presumably represents S phase cells at the time of UV exposure, was higher than the LI (mean 8.1%). These results suggest that proliferating cells, possibly S phase cells, may be responsible for SC formation.
Animals↗
Sulphhydryl crosslinking in cutaneous apoptosis: a review.
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Amyloid↗
Ulcerative contact dermatitis caused by sodium silicate. Coexistence of primary irritant contact dermatitis and contact urticaria.
A 57-year-old man had had recurrent ulcerative lesions on his left hand for two years. The ulcers were associated with chronic eczematous changes resulting from primary irritant contact dermatitis to sodium silicate, as indicated by positive patch tests. The patient also had another type of cutaneous reaction to sodium silicate, ie, contact urticaria. An immediate wheal and flare reaction was found 15 minutes after application of sodium silicate to a scratch test site. This response was not seen in healthy control subjects. To our knowledge, the coexistence of primary irritant contact dermatitis and contact urticaria, both induced by sodium silicate, has not previously been described.
Dermatitis, Contact↗
Actinic reticuloid via persistent light reaction from photoallergic contact dermatitis.
Photoallergic contact dermatitis developed in a patient who had used deodorant soaps containing tribromsalan. He persistently reacted to light, since the dermatitis persisted long after cessation of contact with the causative chemical. During this period, he experienced generalized, eczematous dermatitis on several occasions. On testing, the patient showed extremely severe photosensitivity extending through the ultraviolet to the visible spectrum. At that time, a skin biopsy specimen showed the microscopic findings of eczematous dermatitis. A diagnosis of actinic reticuloid was first made histologically nine years after the initial examinations, when the patient was 56 years old. This long-term follow-up study may support the view that photoallergic contact dermatitis, persistent light reaction, and actinic reticuloid could constitute various phases of a single disease.
Chronic Disease↗
Topical methoxsalen followed immediately by long-wave UV irradiation. A preliminary report on the treatment of psoriasis.
In the treatment of psoriasis with topically applied methoxsalen, the patient is exposed to long-wave UV (UV-A) energy two hours after the application of methoxsalen. Using the guinea pig as an experimental model, we found that UV-A irradiation given immediately after the topical application of methoxsalen resulted in the notable suppression of tritiated thymidine incorporation into epidermal basal cells. This finding suggests that the rate of suppression of epidermal DNA synthesis by PUVA (methoxsalen plus UV-A light) may not correlate with the interval between methoxsalen application and UV-A irradiation. This modified method of PUVA therapy (immediate irradiation) was successfully used in the treatment of psoriasis.
Administration, Topical↗
Coexistence of Kimura's disease and lichen amyloidosus in three patients.
Three patients had Kimura's disease with multiple asymptomatic subcutaneous tumors of 11 to 20 years' duration on the head, soft palate, elbow, or inguinal regions associated with enlarged regional lymph nodes. In each patient, lichen amyloidosus had also developed on the extensor surfaces of the extremities during the past 12 to 20 years. Blood eosinophilia (26% to 75%) was present in all cases. The possible pathogenetic relationship between Kimura's disease and primary localized cutaneous amyloidosis is discussed.
Adult↗
Effect of partial hepatectomy and tumor-bearing on phosphorylation of nuclear protein in rats.
When nuclei from various tissues of rats were incubated at 25 degrees with ATP[gamma-32P], the amounts of 32P incorporated in histone and non-histone protein per mg DNA reached maxima at 1 hr (maximum phosphorylation level). H1 histone and over 30 species of non-histone protein were phosphorylated as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Only some of those species of non-histone protein, which were so low in content as to be indetectable by conventional protein staining, were prominently phosphorylated; their apparent molecular weights were 106K, 82K, 71K, 62K, 39K, 38K and 28K with brain, 39K, 38K, 32K, 31K and 22K with kidney and liver, 17.5K and 11.5K with thymus, and 11.5K with Rhodamine sarcoma (tissue-specific). In phosphorylation with liver nuclei, the tissue specificity changed neither with partial hepatectomy nor upon transplanting Rhodamine sarcoma. However, the maximum phosphorylation levels of the five species of non-histone protein increased with operation and decreased with tumor-bearing, to similar extents. On the other hand, the amounts of all the species of histone per mg DNA changed neither with operation nor with tumor-bearing, whereas the maximum phosphorylation level of H1 histone increased with operation, but not with tumor-bearing.
Animals↗
Purification and characterization of alkaline protease and neutral protease from chromatin of rats.
It was previously reported that, in addition to a known chymotrypsin-like protease capable of hydrolyzing histones with an optimum pH of 8 (neutral protease), another protease is bound to the chromatin of various rat tissues and in situ hydrolyzes casein more quickly than histones with an optimum pH of 10 (alkaline protease). In the present study, the alkaline protease was purified 14 000-fold to approx. 75% purity from the chromatin of Rhodamine sarcoma. This tumor contains both proteases at higher levels than normal tissues. For purification, affinity columns of Sepharose with bound soybean trypsin inhibitor, casein and histones were successively used. Also, the neutral protease was purified 920-fold to an apparently homogeneous state by affinity chromatography on a Sepharose column with bound soybean trypsin inhibitor under conditions, in which an excess amount of the enzyme was applied on the column so that part of the enzyme would pass through the column without adsorption and the enzyme thus adsorbed was then eluted. The purified alkaline and neutral proteases had molecular weights of approx. 18 000 and 27 000, respectively, and isoelectric points of approx. 11. The former enzyme hydrolyzed casein (100) in preference to histones (18) with an optimum pH of 9.5, whereas the latter enzyme preferred histones (100) to casein (32) with an optimum pH of 8. Their actions against other proteins and synthetic substrates were also studied.
Animals↗
The fate of experimental cutaneous candidiasis in guinea pigs under the suppressed polymorphonuclear leukocyte chemotaxis by colchicine.
Experimental cutaneous Candida albicans infections in guinea pigs are histologically characterized by intense epidermal polymorphonuclear leukocyte (PMN) accumulation. To study the role of PMNs in vivo, we injected 250 microgcolchicine/kg i.p., a strong inhibitor of PMN chemotaxis, and observed the influence of reduced PMN migration in experimental cutaneous candidiasis with special interest in the elimination course of the organisms. There was a significant delay in the clearance of the organisms in the colchicine-treated group with decreased epidermal PMN infiltration and prolonged visible scaling process. Our data suggest that the delay of epidermal PMN migration parallels the disappearance of the organisms from the infected skin and that PMNs play an important part in defense against candida infections especially in the elimination process.
Animals↗
Ferredoxin excreted from photosynthetic bacterium, Rhodospirillum rubrum: purification and properties.
When the photoheterotroph, Rhodospirillum rubrum, was grown in the light, ferredoxin was excreted from the cells in a significant amount, as well as hydrogenase. The extracellular ferredoxin was purified to a homogeneous state. The molecular weight was approximately 9,000, and the oxidation-reduction mid-potential was -0.29 V (N=1) at pH 7.0 and 25 degrees C. The amino acid composition was different from those of the intracellular ferredoxins, which were already known. The contents of non-heme iron and acid-labile sulfur were 10.6 and 7.9 mol/mol protein, respectively. The extracellular hydrogenase catalyzed the evolution of hydrogen gas from the ferredoxin in the reduced form. The Km for the ferredoxin was 4.1 micro M, one-seven hundredth as low as that for methyl viologen. There is a possibility that hydrogenase here were functional for evolution of hydrogen gas outside the cells.
Amino Acids↗
Isolation and characterization of two potent inhibitors of various NADH dehydrogenases formed during storage of NADH.
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Chromatography, DEAE-Cellulose↗
The effect of 8-methoxypsoralen and long-wave ultraviolet light on Langerhans cell.
The effect of 8-methoxypsoralen plus long-wave ultraviolet light (PUVA) on Langerhans cell was examined in guinea pigs using ATPase staining method. The number of ATPase-positive Langerhans cells gradually decreased until 5 to 7 days after PUVA treatment, when it was found to be less than 10% to 20% of the original density. Even the ATPase-positive cells exhibited abnormal appearance. The dendritic processes were shortened or disappeared and the central body became oval or round. These changes in population and morphology of Langerhans cell almost returned to normal by 14 days. The inhibitory effect of PUVA on the induction of contact hypersensitivity may be partly due to the alterations of Langerhans cells. Furthermore, the damage on the antigen-presenting cells may accelerate the development of PUVA-induced tumors.
Adenosine Triphosphatases↗