Further purification and characterization of diacetyl reductase from pigeon liver

• 1.1. Electrophoretically pure preparation of an enzyme from pigeon liver which is classified in the I.U.B. Enzyme List as a specific diacetyl reductase (Acetoin: NAD oxidoreductase. EC 1.1.1.5) have been obtained.
• 2.2. This enzyme has been characterized as an α-dicarbonyl reductase (L(+)-α-hydroxicarbonyl: NAD(P) oxidoreductase, EC 1.1.1.…) similar to the one recently discovered from beef liver by the authors' group.
• 3.3. Although differences in mol. wt among these two reductases had been reported, both of them are oligomers of 25,000–26,000 dalton subunits.

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

Agaricus bisporus tyrosinase—II. Characterization of hydroxylase and dehydrogenase activities

• 1.1. Preparative Isoelectric focusing (PIEF) was used to isolate hydroxylasic and dehydrogenasic activities, at different pI.
• 2.2. The fraction at pI 4.7 and 4.9 displays a pure dehydrogenase activity (substrate l-DOPA).
• 3.3. This fraction did not react with tyrosine, either in the spot-test or in absorption spectra (200–620 nm), and did not exhibit any oxygen consumption.
• 4.4. The fraction at pI 4.1 and 4.3 reacted with both l-DOPA and tyrosine as substrate, showing dehydrogenase and hydroxylase activity.
• 5.5. The latter activity was confirmed by the oxygen consumption test, showing that molecular oxygen is used to ortho-hydroxylate tyrosine.

     

Main serum protein of rainbow trout (Salmo gairdnerii): its biological properties and significance

• 1.1. Main serum α₁-protein (α₁P) of rainbow trout was purified and its biochemical and physico-pathological properties were studied.
• 2.2. α₁P was suggested to be a primitive protein having both properties of albumin and AFP in serum proteins of mammals according to the following results.
• 3.3. Molecular weight (75,000), two kinds of molecules (pI 4.55 and 5.05) and amino acid composition.
• 4.4. Dye- or ConA binding activity.
• 5.5. Estrogen binding activity and inhibitory effect on lymphoblastoid-forming activity.
• 6.6. Possible osmotic regulator.
• 7.7. Significant elevation of blood α₁P level in the course of hepatoma induction.

     

Purification and characterization of camel liver glutathione S-transferase

• 1.1. Glutathione S-transferases have been purified (18-fold) in 65–70% yield from the liver of one humped camel using affinity chromatography on glutathione-linked agarose.
• 2.2. Chromatofocusing technique resolves the glutathione S-transferases into seven distinct isoenzymes with apparent pI of 8.7, 8.4, 8.0, 7.8, 7.3 and 6.5.
• 3.3. The major isoenzyme (pI 8.7) which accounted for over 95% of the total activity was composed of two identical subunits of molecular mass 24,000 and was immunologically similar to the other six isoenzymes.
• 4.4. The substrate specificities and the effect of various inhibitors on the activity of the abundant camel liver isoenzyme were also examined.

     

Multiplicity of dog kidney high-Km aldose reductase and conversion mechanism into aldose reductase

• 1.1. High-K_m, aldose reductase purified from dog kidney inner medulla was easily converted into aldose reductase by incubation in the neutral buffer solution.
• 2.2. High-K_m, aldose reductase was found to be in multiple forms, and was separated into three kinds of species designated as a-, b- and c-forms by HPLC.
• 3.3. The a-form observed as a single peak by HPLC was assumed to be present in three forms (al-, a2- and a3-forms), one was aldose reductase (a 1-form) and the others were the precursors of aldose reductase (a2- and a3-form).
• 4.4. The b-form was rapidly converted into the a3-form, followed slowly by the a2-form and finally into the a 1-form.
• 5.5. The c-form was either directly converted into the al-form, or indirectly into the a2-form followed by the al-form.
• 6.6. Four kinds of species (a2-, a3-, b- and c-forms) of high-Ap, aldose reductase were finally converted into aldose reductase (al-form).

     

Fluorescence studies on the interaction of muscle M-line proteins,creatine kinase and the 165,000 dalton component,with each other and with myosin and myosin subfragments

• 1.1. The binding of 8-anilino-l-naphthalene sulfonate (ANS) by M-line creatine kinase (CPK) and the 165,000 dalton protein component was studied by fluorescence.
• 2.2. One mole of ANS binds to a mole of each M-protein and the binding site on these two M-proteins is hydrophobic in nature.
• 3.3. M-line proteins labeled with ANS were used to demonstrate their interaction with myosin and myosin subfragments 1 and 2.
• 4.4. A unique finding of this study, that labeled M-line CPK binds to subfragment 2 of myosin, is of significance from a structural view point, since only the rod portions of myosin molecules are exposed at the M-line and therefore able to interact with CPK.
• 5.5. The use of a sulfhydryl fluorescent probe, N-(2-iodoacetyl)-N-(5-sulfo-l-napthyl) ethylene diamine, (1,5-AEDANS), has shown that the essential sulfhydryl group on CPK is very important for its interaction with both myosin and the 165,000 dalton M-line protein component.

     

Properties of ferredoxin reductase and ferredoxin from the bovine corpus luteum

• 1.1. Ferredoxin reductase and ferredoxin were purified from the bovine corpus luteum and their properties compared to the corresponding adrenal proteins.
• 2.2. The luteal and adrenal proteins had similar absorbance spectra and molecular weights.
• 3.3. Evidence was obtained from spectrophotometric titrations for formation of 1:1 complexes between luteal ferredoxin reductase and ferredoxin and between ferredoxin and cytochrome P-450_scc.
• 4.4. Adrenal ferredoxin reductase and ferredoxin were equally as effective as luteal ferredoxin reductase and ferredoxin in supporting cholesterol side-chain cleavage by luteal cytochrome P-450_scc.

     

Comparative examination of the soluble muscle proteins of two species of angler-fish (Lophiidae): Lophius piscatorius (L.) and Lophius budegassa (Spinola)

• 1.1. A comparative examination of sarcoplasmic proteins of the two nominal European species of angler-fish, Lophius piscatorius and L. budegassa was carried out using isoelectric focusing techniques.
• 2.2. Two protein bands differing in isoelectric point proved diagnostic for L. budegassa (pI 4.40 and pI 5.75) while a third characterized L. piscatorius (pI 4.65).
• 3.3. These species-specific protein profiles provide a method of species discrimination independent of morphological criteria.
• 4.4. Within-species heterogeneity of banding pattern suggested the presence of polymorphic gene loci of potential use in studies of population structure.

     

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. the cytosolic enzyme has a novel 3α/17β-hydroxysteroid dehydrogenase activity

• 1.1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively.
• 2.2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low K_m values.
• 3.3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase.
• 4.4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

     

On the synthesis and incorporation of catalase and urate oxidase into the peroxisomes of mouse liver

• 1.1. The processes associated with the biogenesis of peroxisomes in mouse liver have been studied by following the incorporation of radiolabelled leucine into major enzymic components of this organelle.
• 2.2. Maximal incorporation of label into peroxisomal catalase and urate oxidase occurred within 2 hr, with the urate oxidase being labelled before catalase, but subsequent to the incorporation of phospholipid into this organelle.
• 3.3. Subsequently, immunoprecipitation of catalase from the large granular fraction of mouse liver was shown to result in the isolation of a catalase molecule which had lost a peptide of approx. 2000 dalton from each subunit by comparison with the newly-synthesized enzyme.
• 4.4. It was observed that the modification of catalase was obviated by the presence of leupeptin and iodoacetamide and this information has enabled the purification of both modified and unmodified forms of the enzyme.
• 5.5. The possible significance of these data has been discussed and the major features incorporated into a working model of peroxisomal biogenesis.

     

Properties of enzymes hydrating epoxides in human epidermis and liver

• 1.1. Cytosolic and microsomal epoxide hydrolyzing enzymes of human skin and liver were compared and found to be different.
• 2.2. Epidermal and hepatic cytosolic epoxide hydrolases were different in terms of substrate selectivity, pI, inhibitor sensitivity and affinity Chromatographic properties.
• 3.3. Microsomal epoxide hydrolases had the same pIs but different substrate selectivities.
• 4.4. Cytosolic epoxide hydrolase from adults had higher specific activity than that from neonates or cultured epidermis, but lower activity than adult hepatic enzymes.
• 5.5. The sizes of cytosolic epoxide hydrolase from epidermis and liver were similar and lower than that from cultured fibroblasts.
• 6.6. Cytosolic epoxide hydrolase from all sources shared similar antigenic determinants.

     

Characterization of subfractions of β2-glycoprotein I: Evidence for sialic acid microheterogeneity

• 1.1. β₂-Glycoprotein I is a sialic acid microheterogeneous protein and contains on the average 11 mol sialic acid/mol.
• 2.2. Linear correlation was found between sialic acid content and pI of isolated subfractions.
• 3.3. Asialo-β₂2-glycoprotein I consists of 2 isoforms. Each of which can originate from the same subfraction.
• 4.4. The isolated subfractions exhibited almost the same amino acid composition.

     

Esterases in quail (Coturnix coturnix). Physicochemical and developmental aspects

• 1.1. Soluble esterases of digestive system organs of various developmental stages in the quail (Coturnix coturnix) were resolved by polyacrylamide gel electrophoresis into several molecular forms which were characterized as carboxylesterases, acetylesterases, cholinesterases and esterases sensitive to eserine.
• 2.2. The pI of the majority of esterasic activity in several quail and chicken tissues was observed in the range of 5.1–5.6, while the apparent molecular weight in liver extracts was 60,000.
• 3.3. The expression of the esterase multiple molecular forms was found to be both tissue- and developmental stage-specific, with electrophoretic patterns becoming more complex in number and/or staining intensity upon hatching and thereafter, especially in liver and intestine.

     

Conversion of a NADPH-dependent aldehyde reducing enzyme into aldose reductase

• 1.1. Aldose reductase, aldehyde reductase and high-K_m, aldose reductase were purified from the inner medulla of dog kidney.
• 2.2. Compared with aldose reductase, high-K_m aldose reductase had a lower isoelectric point, a lower activity for aldo-sugars and a lower sensitivity for aldose reductase inhibitors, and it was not activated by sulfate ions. Both reductases had the same molecular weight (38,500) and immunochemical properties.
• 3.3. High-K_m aldose reductase was easily converted into an aldose reductase-like enzyme, namely a generated reductase upon incubation in neutral buffer solution.
• 4.4. The generated reductase was identical with aldose reductase with respect to the isoelectric point, substrate specificity, activation by sulfate ions and IC₅₀ values for aldose reductase inhibitors. The generated reductase revealed immunochemical identity with aldose reductase as well as high-K_m aldose reductase.

     

Effects of inhibition of nadph: cytochrome p-450 reductase on benzo(a)pyrene metabolism in mouse liver microsomes

• 1.1. Effects of antioxidants (butylated hydroxytoluene and nor-dihydroguaiaretic acid), vitamin K-related quinones (vitamin K₁ and coenzyme Q₁₀) and inorganic copper (CuSO₄), in concentrations inhibiting NADPH: cytochrome P -450 reductase, were re-examined on benzo(a)pyrene metabolism in mouse liver uninduced microsomes.
• 2.2. It was found that all these compounds decrease production of the two-electron oxygenation products of benzo(a)pyrene (monophenoles, diols) and the amounts of glucuronides in a manner parallel to their inhibitory potency against NADPH: cytochrome P-450 reductase.
• 3.3. No correlation was found between amounts of one-electron oxidation products of benzo(a)pyrene and inhibition of NADPH: cytochrome P-450 reductase.
• 4.4. Without added UDPGA the compounds studied decreased protein associated benzo(a)pyrene metabolites in parallel to the decreased overall metabolism of this polyaromatic hydrocarbon.
• 5.5. The mode of action of the studied compounds is discussed.

     

Isolation and characterization of the plasma membrane from slime-forming,encapsulated Streptococcus cremoris

• 1.1. The plasma membrane of slime-forming, encapsulated Streptococcus cremoris from “viili” was isolated in hypotonie conditions in the presence of lysozyme (EC 3.2.1.17) using density gradient centrifugation as the last purification step.
• 2.2. The membrane yield was 15.8% of wet weight cells and the preparation contained 64.4% protein. 19.1% carbohydrate, 5.8% aminosugars, 5.1% RNA and 0.07% DNA.
• 3.3. Buffered 1% (w/v) Triton X-100 solubilized 33.6% of membrane proteins. The number of polypeptides detected by SDS-polyacrylamide gel electrophoresis was 59 when the membrane was isolated without a protease inhibitor and 44 in the presence of a protease inhibitor.
• 4.4. The molecular weights of the polypeptides varied from 13,500 to 100,000.
• 5.5. Ultrathin-layer electrofocusing analysis revealed the range of protein pi values to be between 3.50 and 5.85 concerning 77.3% of proteins and between pI 5.85 and 8.15 concerning 18.2% of proteins.
• 6.6. The isoelectric point of the only basic protein component was 9.3.

     

Induction of a 60-kDa heat shock protein in rat pancreas by water-immersion stress

• 1.1. In this study, expression of a 60-kDa heat shock protein in rat pancreas was investigated before and after water-immersion stress, which has been known as an exacerbation factor of caerulein-induced pancreatitis in rats, by Western blot.
• 2.2. A 60-kDa heat shock protein increased after water-immersion stress in both soluble and insoluble fractions of the pancreas.
• 3.3. Serum amylase level and pancreas weight did not increase after water-immersion.
• 4.4. No pathologic alteration was observed in the pancreas after water-immersion.

     

Variation in turtle myoglobins (subfamily emydinae: Testudines) examined by isoelectric focusing

• 1.1. Myoglobins from heart and skeletal muscle of turtles were analyzed by thin-layer isoelectric focusing.
• 2.2. Within the subfamily Emydinae, variation in the occurrence of two myoglobin electromorphs (pI 6.8 and 6.9) was detected.
• 3.3. Patterns of myoglobin polymorphism support dividing the Emydinae into two subfamilies and help resolve controversial theories on relationships of the genus Deirochelys.
• 4.4. Possible adaptive significance of the myoglobin variants (isoforms) remains to be determined.

     

Purification and characterization of a blue-colored red-fluorescent protein from the silkworm (Bombyx mori L.) larvae

• 1.1. A red-fluorescent blue protein (P600) was purified from the digestive juice of the silkworm (Bombyx mori L.) larvae raised on mulberry leaves.
• 2.2. The purified protein was electrophoretically homogeneous and showed the absorption maxima at 601.5 nm and 278 nm, and the fluorescence maximum at 621 nm.
• 3.3. The molecular weight was estimated to be 540,000 by gel filtration on Sepharose CL-6B. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggests that the protein consists of two heterogeneous polypeptide subunits with a mol. wt of 15,000 and 18,000.
• 4.4. The P600 contains excess acidic amino acid residues over basic groups. The polarity and pI were 45.5% and 4.6, respectively.
• 5.5. The production of H₂O₂ was observed in the presence of P600 upon illumination.