Lipoxygenase of the thermophilic bacteria thermoactinomyces vulgaris—properties and study on the active site

• 1.1. A lipoxygenase activity was purified from Thermoactinomyces vulgaris and some of its properties were characterized.
• 2.2. The enzyme showed a temperature activity range of 40–55°C with still significant activity over 60°C.
• 3.3. The pH of activity on linoleic acid had a broad range with an optimum at pH 6.0 and a weaker one at pH 11.0.
• 4.4. On arachidonic acid the pattern was narrow bell-shaped with an optimum at pH 6.5.
• 5.5. The purified lipoxygenase from Th. vulgaris showed an apparent K_m of 1 mM and V_max of 0.84 μmol diene/min/mg protein.
• 6.6. It was inhibited by the oxidation products, 9-HPOD and 13-HPOD.
• 7.7. A 160,000 Da molecular weight of the enzyme was determined by molecular filtration. Methionine, tyrosine, tryptophan and cysteine are apparently involved in its activity.

     

A thioredoxin from the extreme thermophilic Archaeon Sulfolobus solfataricus

• 1.1. A purification procedure for a thioredoxin from the extremophilic archaeon Sulfolobus solfataricus is described.
• 2.2. The thioredoxin is active in the dithiothreitol-dependent reduction of insulin disulfide bonds.
• 3.3. The thioredoxin is a monomer of 24,800 Da; it is an acidic protein with a pi of 4.5.
• 4.4. The protein is stable to heating for 3 hr at 90°C.
• 5.5. The amino acid composition of S. solfataricus thioredoxin is reported.

     

Amino acid composition and the protein form of procarboxypeptidase a purified from the pancreas of the sei whale Balaenoptera bolealis

• 1.1. Procarboxypeptidase (W-PCPA) was purified from the pancreas of the sei whale Balaenoptera bolealis.
• 2.2. W-PCPA was obtained as a homogeneous protein in polyacylamide gel disc electrophoresis.
• 3.3. W-PCPA has a molecular weight of 75,000.
• 4.4. Amino acid composition of W-PCPA was compared with that of bovine procarboxypeptidase as A S5 (PCPA-S5).
• 5.5. W-PCPA may be two subunits, and the aggregate form may resemble PCPA-S5.

     

Analysis of postembryonic development of locomotor activity rhythm by corpora allata implantation in the cricket gryllus bimaculatus

• 1.1. Male crickets Gryllus bimaculatus show a drastic change in circadian rhythm from nymphal diurnality to adult nocturnality, in association with an increase in activity level several days after the imaginai moult.
• 2.2. The corpora allata implantation into male 7th or 8th instar nymphs produced supernumerary instar nymphs in about 30% of the implanted animals, but did not affected the normal development in the remaining animals.
• 3.3. The majority of the supernumerary instar nymphs were diurnal and sexually inactive, although their internal reproductive organs appeared to be fully mature.
• 4.4. The supernumerary instar nymphs became nocturnal with an increase in activity level several days after the imaginai (9th) moult.
• 5.5. The roles of the nervous system in the regulation of the rhythm reversal are discussed.

     

IDentification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system

• 1.1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes.
• 2.2. The maximum pH of the reaction in the liver microsomes was 7.6.
• 3.3. The K_m and V_max values for all-trans, 9-cis and 13-cis-retinals were determined.
• 4.4. The reaction proceeded in the presence of NADPH and molecular oxygen.
• 5.5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation.
• 6.6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and antiNADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b₅ IgG.
• 7.7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm.
• 8.8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra.
• 9.9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or β-naphthoflavone.
• 10.10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.

     

The lysozyme of the plaice Pleuronectes platessa L.

Lysozyme from the serum of the plaice, Pleuronectes platessa L., has been purified 78-fold with chitin coated cellulose.

• 1.2. Further purification on CM-cellulose yielded a single band on acrylamide electrophoresis, exhibiting lysozyme activity.
• 2.3. The quantitative amino acid composition of plaice serum lysozyme is reported.
• 3.4. The mol. wt is identical with hen egg white lysozyme.
• 4.5. A method is described for identifying fractions with lysozyme activity in polyacrylamide gels.

     

Acid and alkaline phosphomonoesterase activities in snake venoms

• 1.1. Acid and alkaline phosphatase activities of eight different snake venoms were determined quantitatively by using synthetic substrates, o-carboxyphenylphosphate and p-nitrophenylphosphate respectively.
• 2.2. It was found that most of Elapidae venoms investigated had both acid and alkaline phosphatase activities.
• 3.3. Three Crotalidae venoms investigated did not show any alkaline phosphatase activity.
• 4.4. The strength of venom acid phosphatase activity is as follows: Agkistroden acutus > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca.
• 5.5. The strength of venom alkaline phosphatase activity by using p-nitrophenylphosphate is in the order of Naja hannah > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca.When o-carboxyphenylphosphate was used as a substrate, the order of enzyme activity is Naja hannah > Naja haje > Naja naja samarensis > Naja melanoleuca > Naja naja atra.
• 6.6. Acid phosphatase activity of all the Elapidae venoms was inhibited completely by fluoride. The alkaline phosphatase activity of Elapidae venoms was not inhibited by fluoride either using p-nitrophenylphosphate or o-carboxyphenylphosphate.
• 7.7. The acid phosphatase of all the Elapidae venoms was not inhibited by zinc ion. However, most of the venom alkaline phosphatases were inhibited by zinc ion.
• 8.8. Ethylenediaminetetraacetic acid (EDTA) had inhibitory action on venom phosphatase activity. However, tris-(hydroxymethyl)-aminoethane had a counter effect on the inhibitory action of EDTA.
• 9.9. Optimum pH studies of the snake venom phosphatases showed that the acid phosphatases of the snake venoms had their highest activity in the range of pH 4–5. The alkaline phosphatases of the snake venoms had their optimum pH at 9.
• 10.10. Comparable experiments were also conducted by using chicken intestine alkaline phosphatase and wheat germ acid phosphatase.

     

The characterization of the linoleic acid desaturation and elongation system in ovine placental tissue

• 1.1. The desaturation and elongation of linoleic acid has been studied in homogenates and in subfractions of ovine placental tissue.
• 2.2. The reaction was characterized in terms of pH and temperature optima, time course and protein concentration.
• 3.3. Activity was found to be confined to the 11,000g supernatant fraction of the tissue and the results suggest that the enzymes are membrane bound.
• 4.4. The cytosolic fraction and ATP were required for full activity and the reaction was inhibited by cyanide.
• 5.5. The properties of the reaction are compared with those of other desaturation systems and their implications with regard to possible reaction mechanisms are discussed.

     

Phosphatase activity in the hepatopancreas of Helix aspersa

• 1.1. Phosphatase acid (PhA) activity in the digestive gland (hepatopancreas) of the common garden snail Helix aspersa has been investigated using cytochemical methods.
• 2.2. All the cells composing this gland show PhA activity, the distribution pattern differing according to the cell type.
• 3.3. The digestive cells show the most widely distributed reaction product (brush border, phagolysosomes, multivesicular bodies and autophagic vacuoles).
• 4.4. In the excretory cells this activity appears in large sacs, while in the calcium cells the reaction product is abundant in the calcium granules.
• 5.5. Cellular digestion processes performed by each of these cell types is discussed together with their role in the detoxification of heavy elements derived from the environment.

     

Biosynthesis of membrane and a membrane glycoprotein in Tetrahymena pyriformis

• 1.1. A membrane glycoprotein was isolated from Tetrahymena surface membrane with properties similar to mammalian erythrocyte glycoprotein.
• 2.2. This membrane glycoprotein had a specific acitivity less than whole membrane at various time intervals up to 40 hr.
• 3.3. In double labelling experiments, glucosamine contributed a greater degree of the labelling of this glycoprotein than did amino acids.
• 4.4. Greater incremental increases in the glucosamine radioactivity over amino acids at the longer time intervals suggests that the carbohydrate and protein moieties may have independent metabolic pathways.
• 5.5. When Tetrahymena was grown in proteose peptone-tryptone in the presence of labelled amino acid, the specific activity of the surface membrane was less than one-tenth of the specific activity obtained in defined growth media. Under these same conditions, there were only slight changes in the specific activities of glucosamine in the membrane.
• 6.6. Data is presented suggesting independent synthesis of the protein and oligosaccharide from different pools.

     

Protein,fatty acids,calcium and phosphate absorption along the gastrointestinal tract of the young turkey

• 1.1. The net absorption of protein, fatty acids, calcium and phosphate along the small intestine of the turkey (Meleagris gallopovo) was evaluated with the aid of ⁹¹Y as a reference substance.
• 2.2. About 85% of the ingested protein was absorbed, with most of the absorption occurring in the duodenum and upper jejunum.
• 3.3. The overall lipid absorption coefficient was around 90%, and was inversely related to the fatty acid chain length and saturation.
• 4.4. Most of the lipid absorption occurred in the duodenum and jejunum.
• 5.5. Calcium absorption also occurred in the duodenum and jejunum: its fractional rate decreased with calcium intake.
• 6.6. Phosphate absorption occurred mostly in the duodenum and jejunum and its efficacy was only slightly affected by dietary phosphate intake.
• 7.7. The high nutrient absorption in the turkey duodenum, relative to that of the chick (Gallus domesticus), was discussed.

     

Characterization of the Melanoplus sanguinipes hemolymph after infection with Beauveria bassiana or wounding

• 1.1. The phenoloxidase activity, protein and carbohydrate levels were studied for 24 hr in the hemolymph of the migratory grasshopper, Melanoplus sanguinipes after artificial wounding of the insect cuticle or the injection of Beauveria bassiana conidia.
• 2.2. Injection or wounding induced a primary response and phenoloxidase activity was found to increase within 10–60 min. The values for phenoloxidase activity in viable B. bassiana-injected insects exhibited a secondary response, i.e., an increase 24 hr after injection.
• 3.3. In wounded insects and those injected with inactivated conidia, the phenoloxidase activity receded after the initial increase and remained at low levels.
• 4.4. Protein concentrations in the hemolymph increased immediately after infection and wounding and returned to basal levels during the course of the experiment.
• 5.5. Injection of viable B. bassiana resulted in a gradual increase in the protein concentrations between 12 and 24 hr.
• 6.6. There was no apparent change in the carbohydrate levels in either B. bassiana-infected or wounded insects.
• 7.7. These results are discussed in relation to their possible role(s) and interrelationships in the immune response to infection or wounding. Furthermore, we suggest that a “factor” is released after mechanical injury of the integument.

     

Evidence of presence of a low molecular weight,non-metallothionein-like metal-binding protein in the marine gastropod Nassarius reticulatus L.

• 1.1. A low molecular weight metal-binding protein was found in the snail Nassarius reticulatus cytosol, which was induced in heavy metal contaminated environments.
• 2.2. In our sodium dodecyl sulfate-mercaptoethanol polyacrylamide gel systems it behaved as a protein of 19 kDa mol. wt.
• 3.3. Amino acid composition studies definitely established this protein not to be metallothionein (Mt) like, because it had a much lower level of cysteine and substantial amounts of aromatic amino acids and histidine.
• 4.4. The metal-binding strength of this protein was concluded to be much weaker than that of Mt.
• 5.5. In the crustacean Pagurus bernhardus L. such a protein could not be demonstrated.
• 6.6. In both the snail and the crustacean Zn may inhibit the accumulation of Hg. The premise for studying the induction of the metal-binding Nassarius protein as a supplement to environmental metal monitoring purposes is briefly discussed.

     

Purification,isolation and characterization of a phosphoglycolate phosphatase isoenzyme from human erythrocytes

• 1.1. Preparation, purification and characterization of a phosphoglycolate phosphatase (PGP)3 isoenzyme from human erythrocytes was achieved by DEAE-Sepharose CL.-6B chromatography and isoelectric focusing using carrier ampholytes. pH 4–6.
• 2.2. The isoenzyme has an isoelectric point of 5.00 ± 0.05 and could be purified 33.000 fold to a specific activity of 32.7 U/mg of protein. It represents the PGP phenotype 1 consisting of a single isoenzyme.
• 3.3. The enzyme is composed of two subunits (mol. wt 35,000) which are identical and not connected by SS-bridges.
• 4.4. At 4°C the isoenzyme is more stable in the pH range of 7–9 than at acid pH values.
• 5.5. Incubation at 30 and 40°C for 4 hr does not affect the activity of the isoenzyme.
• 6.6. It has a K_m-value of 0.28 mM for phosphoglycolate (PG) as substrate.

     

Phosphonomonoesterase activity in Metridium senile L.

• 1.1. An esterase which hydrolyzes 4-nitrophenyl(phenyl)phosphonic acid (4-NPPP) was purified from M. senile (sea anemone).
• 2.2. The enzyme showed no 5′-nucleotide phosphodiesterase activity with 5′-(4-nitrophenyl) TMP or phosphomonoesterase activity with 4-nitrophenylphosphate.
• 3.3. Addition of excess Zn²⁺ restored activity after inactivation by EDTA.
• 4.4. Thiol reagents and phenylmenthanesulfonylfluoride did not inactivate, whereas, dithiothreitol inactivated.
• 5.5. Aminoethylphosphonic acid (AEP) was a competitive inhibitor of 4-NPPP indicating possible activity with phosphonomonoesters of AEP.

     

Identification of cathepsin L-like proteinase and aminopeptidase in yolk granules of the sand worm,Nereis diversicolor

• 1.1. Two proteinases have been identified in yolk granules of Nereis diversicolor mature oocytes, an aminopeptidase and an acid cysteine proteinase.
• 2.2. The aminopeptidase was identified as a metallo-enzyme having a molecular weight of about 260 kDa.
• 3.3. Except that the acid cysteine proteinase is a high molecular weight protein (200 kDa) and has a very low pH optimum (3.0), the enzyme possesses properties resembling those of mammalian cathepsin L.
• 4.4. The cathepsin L-like proteinase was found to be liable to the in vitro proteolysis of the yolk granule proteins and is therefore suggested to be involved in yolk protein processing.

     

Effect of conjugation on the incorporation of glycine into Tetrahymena

• 1.1. Conjugation of Tetrahymena enhanced the incorporation of glycine into the nuclear fraction by 500%.
• 2.2. Incorporation of glycine into the microsomal supernatant was augmented by almost 500% by conjugation.
• 3.3. Mitochondrial incorporation was stimulated nearly 3-fold in the conjugating strains while the incorporation of glycine into the microsomes was enhanced approximately 2.5 times.
• 4.4. In the whole cell, glycine incorporation was increased nearly 2-fold by conjugation.
• 5.5. Strong nuclear involvement was indicated by elevated metabolic activity and incorporation of glycine into RNA and DNA.
• 6.6. Stimulation of the metabolism of Tetrahymena by cell communication suggests that the contents of a cell can have a synergistic effect on another cell.
• 7.7. Augmentation of the biosynthetic capacities of cells by fusion is a demonstration of the dominant role of the cell membrane in the regulation and control of cells.
• 8.8. Enhancement of biosynthesis of nuclear proteins in conjugating strains of cells indicates that fusion gives rise to the synthesis of new protein from previously existing protein or protein procursors.
• 9.9. The specific activities of the subcellular fractions after the incorporation of glycine into 2 separated starved strains of Tetrahymena followed the usual pattern of nucleus less than whole cells, whole cells less than mitochondria, mitochondria less than microsomes, but with the microsomal supernatant being much greater than that of the microsomes.

     

Phosphorylation of Escherichia coli proteins during the SOS response

• 1.1. The phosphorylation of Escherichia coli proteins was analyzed comparatively before and after induction of the SOS response in a temperature-sensitive mutant strain.
• 2.2. The presence of phosphorylated proteins was evidenced by gel electrophoresis and autoradiography after labelling with radioactive orthophosphate in vivo or radioactive adenosine triphosphate in vitro.
• 3.3. Significant changes in the intensity of protein labelling were observed upon induction of the SOS functions: six proteins were found to be more phosphorylated while two others were less phosphorylated. Moreover, five additional proteins appeared to become phosphorylated exclusively during the SOS response. The molecular mass and isoelectric point of these various proteins were determined.
• 4.4. For most proteins, the changes in the pattern of protein phosphorylation were concomitant with variations in the amount of protein synthesized.
• 5.5. The changes in the pattern of phosphoproteins observed during the SOS response were not due to the temperature shift required experimentally for expressing the SOS phenotype.
• 6.6. Phosphorylation was found to be catalyzed by protein kinases that modify amino acid residues at hydroxyl groups in protein substrates.
• 7.7. Both in vivo and in vitro studies brought evidence that neither RecA nor LexA, the two key regulatory proteins of the SOS functions, were capable of undergoing phosphorylation.

     

Amino acid activating enzymes in Sarcophaga bullata

• 1.1. The fat-body soluble fraction from two-day Sarcophaga bullata larvae contain amino acid activating enzymes for nineteen amino acids.
• 2.2. The level of activity varies with the amino acid substrate.
• 3.3. The total ³²PP-ATP exchange activity of the pupae decreased with age for the first 6 days, then increased to a maximum one or two days prior to emergence of the adults.
• 4.4. The free amino acid concentration in the pupae decreased during the period when the amino acid activating activity increased.

     

Isolation and characterization of lipoamide dehydrogenase from mackerel dark muscle

• 1.1. Lipoamide dehydrogenase was purified 1500-fold from mackerel dark muscle.
• 2.2. The enzyme was homogeneous as judged by acrylamide gel electrophoresis in the presence and absence of SDS.
• 3.3. Molecular weights of 102,000 and 55,000 were estimated for the native and denatured enzyme, respectively.
• 4.4. Optimal activity for the enzyme was obtained at around pH 5.7 and enhanced with citri acid.
• 5.5. Loss of activity was less than 5% by incubating the enzyme at 70°C for 20 min.
• 6.6. An apparent K_m of 3.1 × 10⁻³ M was obtained for dl-lipoic acid and 1.5 × 10⁻⁵ M for NADH.
• 7.7. The properties of lipoamide dehydrogenase from mackerel dark muscle observed in this investigation were very similar to those reported for the enzyme from other sources.