Lysosomal acid deoxyribonuclease from vervet monkey livers—II: Enzymic properties

• 1.1. Primate liver lysosomal acid DNase is an endonucleolytic enzyme.
• 2.2. The enzyme has both 3'- and 5'-nucleotidohydrolase activities.
• 3.3. The oligonucleotides produced by DNase are polymers mainly about 30 mononucleotides long.
• 4.4. The Arrhenius plot shows a discontinuity with a transition temperature at 47°C, with an activation energy of 107 kJ/mol below and 67 kJ/mol above this temperature.
• 5.5. The activation enthalpy is 104kJ/mol and the entropy −0.498 kJ/mol/K.
• 6.6. The enzyme is subject to substrate inhibition and the K_m value is 159 × 10⁻³mM DNA-P.

     

Nucleoside monophosphoramidate hydrolase from rat liver: Purification and characterization

• 1.1. Adenosine 5'-phosphoramidate hydrolase of 29 kDa was isolated from rat liver cytosol.
• 2.2. It consisted of two subunits of 14 kDa.
• 3.3. It hydrolyzed nucleoside 5'-monophosphoramidates into nucleoside 5'-monophosphates and ammonia, while it did not hydrolyze adenylyl phosphoramidate, adenylyl imidodiphosphate and N-phosphorylated compounds like phosphocreatine, N^ω-phosphoarginine, 6-phospholysine and 3-phosphohistidine.
• 4.4. Divalent cations and cyclic AMP had no effect on the hydrolytic activity.

     

Transamination and transsulphuration of l-cysteine in ehrlich ascites tumor cells and mouse liver: The nonenzymatic reaction of l-cysteine with pyruvate

• 1.1. The activity of cysteine aminotransferase (CAT), 3-mercaptopyruvate sulfurtransferase (MPST) and rhodanese is much lower in Ehrlich ascites tumor cells (EATC) than in mouse liver.
• 2.2. Contrary to mouse liver homogenate, no synthesis of sulphane sulphur-containing compounds from L-cysteine is observed in EATC homogenate.
• 3.3. 2-Methyl-thiazolidine-2,4-dicarboxylic acid (CP), 2-methyl-thiazolidine-4-carboxylic acid (CA) and thiazolidine-4-carboxylic acid (CF) can be used as sources of low molecular-weight thiol compounds both in EATC and mouse liver homogenate.
• 4.4. Pyruvate formed from phosphoenolopyruvate (PEP) in EATC homogenates reacts with L-cysteine (l-CYS) to CP.

     

Comparison of minerals in milk of four species

• 1.1. Milk samples of 4 ml or more were obtained from guinea pigs (Cavia porcellus), dairy cattle (Bos taurus), horses (Equus caballus) and humans (Homo sapiens). The milks were analysed for minerals including calcium (Ca), phosphorus (P), magnesium (Mg), potassium (K) and sodium (Na) by Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES).
• 2.2. Calcium was approximately twice as concentrated in guinea pig milk as in cows' milk, which was twice as much as the level in mares' milk, and this, in turn, was twice as concentrated as human milk.
• 3.3. The ratio of Ca to P in guinea pig milk was 1.66:1, while it was 1.24:1 in cows' milk, 1.56:1 in mares' milk and 2.07:1 in human milk.
• 4.4. Potassium was the most abundant mineral in the milks of cows, mares and humans, but not in guinea pig milk, and was much higher in cows' milk than in others.
• 5.5. Sodium was highest in guinea pig milk with cows' milk being a close second.
• 6.6. Magnesium was one-tenth as concentrated as Ca.

     

Distribution of beta-adrenergic binding in fractionated porcine adipocytes

• 1.1. The subcellular distribution of the porcine adipocyte beta-adrenergic receptor was studied in fractionated adipocytes.
• 2.2. The 30,000 g pellet obtained from hypotonically lysed cells contained membrane vesicles and mitochondria; it yielded approx 200–300 fmol dihydroalprenolol-bound receptors/mg protein.
• 3.3. Activity was increased to about 1000 fmol/mg protein after isolation of a plasma membrane fraction on a Percoll gradient.
• 4.4. The 5'-nucleotidase, succinate dehydrogenase and lactate dehydrogenase activities were usually enriched in compartments different from the ligand-binding activity.
• 5.5. Activity of porcine adipocyte 5'-nucleotidase, a purported plasma membrane marker enzyme, was not distributed in the same manner as the beta-adrenergic receptor.

     

Miniature gel filtration: a simple and inexpensive method for the measurement of protein- and non-protein-bound cortisol: application to guinea pig plasma

• 1.1. A method is described for the accurate and rapid measurement of protein- and non-protein-bound cortisol by miniature gel filtration in small volumes of plasma, e.g. of rodents.
• 2.2. Binding of cortisol by guinea pig plasma proteins is strongly reduced at elevated temperature (4°C: 102 ± 12ng/ml; 40°C: 5 ± 2 ng/ml).
• 3.3. Incubation of guinea pig plasma with 1–5000 ng cortisol resulted in a dose-dependent increase in cortisol bound to proteins (specific binding by corticosteroid binding globulin: 230 ± 12 ng/ml).
• 4.4. Administration of 20 IU (1–24)ACTH induced a significant increase of total protein-bound and non-protein-bound cortisol.
• 5.5. Values reported in this study agree well with those of previous investigations, in which bound and non-bound glucocorticosteroids were separated by gel filtration on large Sephadex® columns.

     

Mixed-function oxygenase in juvenile rainbow trout exposed to hexachlorobenzene or 3, 3′, 4, 4′-tetrachlorobiphenyl

• 1.1. 3,3',4,4'-tetrachlorobiphenyl (PCB 77), but not hexachlorobenzene, induced liver micro-somal cytochrome P-450 (Cyt P-450), ethoxycoumarin-O-deethylase (ECOD) and ethoxyresorufin-O-deethylase (EROD) in rainbow trout. Maximum induction was observed in a PCB 77 injected group of fish (1.0mg/kg, i.p. injection) 13 days after the injections being 2, 10 and 50 times the values of non-induced fish, respectively.
• 2.2. The apparent K_m value of ethoxyresorufin of this induced group of fish differed only slightly from that of non-induced fish. The apparent V_max value (EROD) was 50 times higher.
• 3.3. Freezing small pieces of liver in liquid nitrogen did not produce cytochrome P-420.
• 4.4. Fluorimetric and spectrophotometric measurements of EROD correlated.

     

Comparative study of ovalbumins from various avian species by Con A/Sepharose chromatography

• 1.1. Ovalbumin samples from 7 chicken subspecies and other avian species were subfractionated on Con A/Sepharose column.
• 2.2. The ovalbumins from chicken subspecies were separated into 4 fractions, OA, OB, OC and OD without exceptions, although the fractional ratio (OA:OB:OC:OD) was varied from one sample to another. Similarly, turkey ovalbumin also could be separated into the 4 fractions.
• 3.3. Quail ovalbumin was tightly bound to Con A/Sepharose column and effectively eluted as a new characteristic peak (OQ) behind OD from the column at room temperature, while goose ovalbumin was totally eluted as unadsorbed fraction from the column.

     

Temperature acclimation in respiratory and cytochrome c oxidase activity in common carp (Cyprinus carpio)

• 1.1. Common carp (Cyprinus carpio) exposed to experimental temperatures of 12, 18, 24, 30 or 36°C for a 4-week period were used to investigate the effect of temperature acclimation on the frequency of opercular movement (FOM), growth and cytochrome c oxidase (CCO) activity in heart, liver and muscle.
• 2.2. An exponential relationship between FOM and temperature after the first week (10₁₀ =1.76) disappeared after the second week.
• 3.3. The initially high FOM at temperatures of 30 or 36°C and the low FOM at 18 or 12°C changed over 4 weeks to approach the FOM of fish at 24°C.
• 4.4. This change in the relationship of FOM to temperature from highly dependent to independent appeared to be thermal compensation.
• 5.5. Heart and liver CCO activities were significantly affected by temperature, with the lowest activity at the approximate optimum temperature for growth, 24°C.
• 6.6. Highest CCO activities for heart and liver occurred at both the highest and lowest temperatures.
• 7.7. Among the three tissues, heart CCO activity was generally the highest and most affected by acclimation temperature.
• 8.8. Muscle tissue had the lowest CCO activity and was unaffected by temperature.
• 9.9. The high CCO activity at a cold acclimation of temperature 12°C was probably due to thermal compensation and the high activity at 36°C may have been a result of thermal stress.

     

The formation of a dimeric product of raffinose by the action of galactose oxidase

• 1.1. Galactose oxidase is known to catalyze the oxidation of the C-6 hydroxymethyl group of galactose to an aldehyde group.
• 2.2. When the products of a galactose oxidase-catalase treatment of raffinose were examined by gel filtration and ion exchange chromatography, we found that in addition to the expected 6'-aldehydoraffinose, two other components were present.
• 3.3. One component was neutral and had an elution volume close to that of maltopentaose and on treatment with sodium borohydride or hypoiodite, this component was converted to raffinose or 6'-carboxyraffinose, respectively.
• 4.4. Fast atom bombardment mass spectroscopy in the negative mode indicated that the major molecular ion had an M/Z of 1003.
• 5.5. These data are consistent with this component being a dimer of 6'-aldehydoraffmose.

     

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.

     

Intracellular localization and characterization of 3-hydroxykynureninase in human liver

• 1.1. 3-hydroxykynureninase in human liver was present in cytosol and mitoehondria.
• 2.2. The cytosolic enzyme and mitochondrial enzyme had the same physiological and enzymic properties.
• 3.3. The enzyme had a mol. wt of 130,000 by gel filtration and isoelectric point of pH 5.9.
• 4.4. The enzyme was active for 3-hydroxykynurenine and kynurenine, and its activity ratio was 15:1. The apparent K_m values of the enzyme were 7.7 × 10⁻⁵M for 3-hydroxykynurenine, 1.0×10⁻³M for kynurenine and 2.5 × 10⁻⁶M for pyridoxal 5'-phosphate with 3-hydroxykynurenine.
• 5.5. Some other properties of purified enzymes are described.

     

Evidence that the 5' end of rabbit globin mRNA is hybridized with its 3' end

• 1.1. A radiopolyadenylated rabbit globin mRNA was treated with different concentrations of ribonuclease V₁ from cobra venom.
• 2.2. The enzymatic digests were chromatographed on an aminophenylboronate-agarose column, which specifically captured the cap structure i.e. n⁷G(5') ppp (5') NmP.
• 3.3. When the capture fragment was chromatographed on a Sephadex G-100 column, its size was smaller than the native molecule and also bore radioactivity, i.e. a poly(A) tail.
• 4.4. These results provide evidence that the 5' end (which encompasses the cap structure) of rabbit globin mRNA is hybridized and in close proximity to its 3' end.
• 5.5. We conclude that this conformation is required for messenger translation efficiency.

     

Thermoreceptor distribution in different skin layers and its significance for thermoregulation

• 1.1.|The activity pattern of 50 cold receptors of the rabbit nose back skin was investigated.
• 2.2.|The latency of the response of individual cold receptors to identical cold stimuli varied between 0.8 ± 0.3 to 29.4 ± 4.5 s; maximal firing rates are attended after 5.5 ± 0.5 to 72.2 ± 6.2 s. Characteristic phasic responses are only demonstrated by short latency receptors.
• 3.3.|The results suggest that cold receptors are distributed throughout the skin of the rabbit's nose.
• 4.4.|Changes of temperature gradients between different skin layers were measured at different ambient temperatures.
• 5.5.|It is suggested that cold receptors might indicate heat flow through the skin.

     

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.

     

Influence of environmental factors on the deep and skin temperature in the European bison,Bison bonasus (L.)

• 1.1. In 43 European bison divided into three groups (Group A, 3–8-month-old calves; Group B, 18-month-7-year-old young bison; Group C, 12–24-year-old bison) the rectal, humerus region and abdomen region temperatures were measured.
• 2.2. The experiments were carried out in winter months, from mid-December to mid-March.
• 3.3. The mean rectal temperatures changed from 38.55°C in calves to 38.15°C in the oldest bison.
• 4.4. The mean temperatures of the humerus region changed from 20.69°C in calves to 21.49°C in older bison.
• 5.5. The mean temperatures of the abdomen region changed from 20.79°C in calves to 22.17°C in older bison (Gr. B).
• 6.6. The cluster analysis divided the bison into four groups named hot, warm, cool and cold bison.
• 7.7. Only air temperature measured 2 m above the ground and snow cover influenced the integrated bison temperature. Age, sex and mass as well as some environmental factors had no influence.
• 8.8. Measurements made 1 to nearly 4hr after a bison's death showed a drop in rectal temperature and mostly increases in temperatures of the humerus and abdomen regions.

     

Effects of milk fat,unhydrogenated and partially hydrogenated vegetable oils on serum lipoproteins in growing pigs

• 1.1. Crossbred Yorkshire (Yorkshire × Landrace) pigs were fed butter oil, cream, low erucic acid rapeseed oil, sunflower oil and partially hydrogenated sunflower oil in amounts representing 30% of energy for periods of up to 13 weeks.
• 2.2. After 13 wk of feeding serum total cholesterol levels of pigs fed milk fat were significantly higher than of pigs fed vegetable oils.
• 3.3. The difference in cholesterol was mainly due to an increase in the density range of 1.063–1.125 g/ml containing pig LDL₂ and some HDL.
• 4.4. A shift towards smaller LDL particle size was apparent in pigs fed milk fat.
• 5.5. The effects of dietary trans fatty acids did not differ from cis polyunsaturated or monounsaturated fatty acids.

     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

Identification and characterization of vitellin in a hermophrodite shrimp,Pandalus kessleri

• 1.1. A female specific protein (FSP, vitellogenin) in hemolymph and its related ovarian protein (vitellin) of Pandalus kessleri were studied by means of electrophoretical and immunological procedures.
• 2.2. The vitellin was purified from vitellogenic ovaries using hydroxylapatite, DEAE cellulose and Sepharose 6B columns, consecutively.
• 3.3. The vitellin had a molecular weight of approximately 560 kD and was composed of two subunits, 81 and 110 kD, respectively.
• 4.4. The vitellogenin concentrations in the hemolymph increased as vitellogenesis in the ovarian oocytes advanced and dropped markedly after the release of mature eggs.

     

Copurification and separation of β-galactosidase and sialidase from porcine testis

• 1.1. A soluble sialidase was copurified apparently as an enzyme complex with acid β-galactosidase from porcine testis.
• 2.2. The sialidase exhibited its maximum activity at acidic pH. It was efficiently active towards 4-methylumbelliferyl-α-d-N-acetyl-neuraminic acid and sialyllactose, relatively inactive towards glycoproteins, and had little activity towards glycolipids.
• 3.3. The complex could be separated by sucrose gradient centrifugation or isoelectric focusing.
• 4.4. The separated enzymes had molecular weights about 600,000 for β-galactosidase and more than about 1,000,000 for sialidase by Sepharose 4B gel filtration.
• 5.5. SDS-polyacrylamide gel electrophoresis of the β-galactosidase showed three protein bands with molecular weights of 63,000, 31,000 and 20,000.