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.

     

The isolation and partial characterization of α2-macroglobulin from the serum of the ostrich (Struthio camelus)

• 1.1. α₂-Macroglobulin (α₂M) activity is present in the serum of the ostrich, Struthio camelus. The chromogenic synthetic peptide substrates BAPNA and ATNA were hydrolysed by trypsin and chymotrypsin, respectively, in the presence of ostrich serum and the α₂M in ostrich serum protected trypsin from being inhibited by soybean trypsin inhibitor. Ostrich α₂M proved to be a potent inhibitor of bovine pancreatic trypsin and chymotrypsin.
• 2.2. α₂M was purified to apparent homogeneity by PEG precipitation, DEAE-Toyopearl 650M, Bio-Gel A-5m and Zn²⁺-affinity chromatography.
• 3.3. Ostrich α₂M migrated as a single band (M_r 779,000) during non-denaturing gradient gel electrophoresis and showed increased mobility after reaction with trypsin. Denaturation dissociated ostrich α₂ M into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits.
• 4.4. Isoelectric focusing revealed a pI of 5.3.
• 5.5. The amino acid composition of ostrich α₂M is typical of an α₂M, comparing favourably with those of other animal species. The carbohydrate composition of the purified protein, in percentage dry weight of the molecule, was galactose: mannose (1:1), 4.55; N-acetylglucosamine, 2.35; N-acetylneuraminic acid, 0.58; and fucose, 0.77.
• 6.6. α₂M was assessed immunologically by Ouchterlony double-diffusion and Western blot analysis with polyvalent antisera directed against ostrich α₂M.
• 7.7. Ostrich α₂M seems to show many physical, chemical and kinetic properties similar to those of other known α₂Ms, but is expected to differ from other αMs when considering the primary structure of the bait region, the area differing among α Ms from different species and determining its specificity.

     

Effect of bovine milk antigens and egg lysozyme on the binding of 59Fe-lactoferrin to platelet plasma membranes

• 1.1. Platelets bind specifically to lactoferrin. A significant similarity between human lactoferrin and some bovine milk proteins has been established.
• 2.2. Because of the structural homology of lactoferrin and cows milk proteins they are able to influence lactoferrins regulatory function on the level of its binding to membrane receptors on platelets.
• 3.3. An inhibitory effect of bovine α-lactalbumin and of β-lactoglobulin on lactoferrin-receptor interaction was shown.
• 4.4. Bovine α-lactalbumin competes with lactoferrin for the binding sites.
• 5.5. Scatchard plot analysis of data shows one binding site for lactoferrin in the presence of α-lactalbumin with an affinity constant, Ka = 0.46 × 10⁹ mol/1 and 335 receptors/cell.
• 6.6. The inhibitory effect of β-lactoglobulin reaches 62% and is different for the common fraction ⨿-lactoglobulin and the genetic variants β-lactoglobulin A and B.
• 7.7. β-lactoglobulin does not compete with lactoferrin for the membrane receptors.
• 8.8. Bovine casein and egg lysozyme stimulate ⁵⁹Fe-lactoferrin binding to the receptors. The mechanism of these effects is still unknown.
• 9.9. Tested alimentary antigens are able to interact with lactoferrin and also with some platelet membrane structures.
• 10.10. Established changes in lactoferrin binding to the platelet membrane might be in relation to lactoferrins regulatory function and (or) eliminating mechanisms of these alimentary antigens.

     

Evidence for a molecular polymorphis of cholinesterase in Sepia officinalis (cephalopoda: decapoda)

• 1.1. Three forms of cholinesterase were sequentially extracted from head and tentacles of Sepia officinalis and noted as low-salt (LSS), detergent (DS) and high-salt (HSS) soluble. They represent about 24, 30 and 46% of total activity.
• 2.2. All enzyme forms seem to be amphiphilic proteins with hydrophobic domains interacting with non-ionic detergent (Triton X-100) and giving self-aggregation (LSS form).
• 3.3. The DS form is membrane-anchored by a phosphatidylinositol, while the HSS form is likely linked to some proteoglycan molecule of the extracellular matrix by ionic interactions.
• 4.4. According to V_max/K_m values, all the enzymes are acetylcholinesterases, even if hydrolyze propionylthiocoline at the highest rate.
• 5.5. Some kinetic and molecular properties of the studied enzymes are compared with those of other cholinesterases from vertebrates and invertebrates. Possible phylogenic and adaptive features are discussed.

     

Chemical intermediates in α-methylnoradrenaline oxidation by tyrosinase—II. Kinetic study of process

• 1.1. The pathway for α-methylnoradrenaline oxidation to α-methylnoradrenochrome by tyrosinase. has been studied as a system of various chemical reactions coupled to an enzymatic reaction.
• 2.2. A theoretical and experimental kinetic approach was proposed for such a system, we named this type of mechanism as a mechanism enzymatic-chemical-chemical (E₂CC).
• 3.3. Rate constants for the implied chemical steps at different temperature and pH values, were evaluated from measurement of the lag period, arising from the accumulation of aminochroine, that took place when α-methylnoradrenaline was oxidized at acid pH.
• 4.4. The thermodynamic activation parameters of the chemical steps, the deprotonation and the internal cyclization of o-quinone into leukoaminochrome, were also calculated.

     

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.

     

Detection of 180 kDa proteins in electroplax sodium channel preparations

• 1.1. Co-isolating proteins (M_r 170,000–220,000) from sodium channel preparations made from the electric organ of the electric eel (Electrophorus electricus) were detected on Western blots using monoclonal a antibodies.
• 2.2. Similar protein patterns were seen on immunoblots containing immunoprecipitated protein from eel muscle and brain tissues but not heart.
• 3.3. These co-isolating proteins could be separated from the mature TTX-sensitive channel protein (M_r 280,000) using a lentil lectin-Sepharose column.
• 4.4. The 180 kDa proteins do not appear to be channel-related and can be detected as contaminants in electroplax sodium channel preparations using the monoclonal antibodies described here.

     

The isolation and partial characterization of α1-proteinase inhibitor from the serum of the ostrich (struthio camelus)

• 1.1. Native and cleaved α₁-proteinase inhibitor was purified from ostrich serum using Sepharose-blue dextran chromatography, ammonium sulfate precipitation and ion exchange chromatography on DEAE-Toyopearl 650 M at pH 8.8 and 6.5.
• 2.2. Ostrich α₁PI displayed M_r values of 68,100 using gradient PAGE and 66,200 using Ferguson plots.
• 3.3. Isoelectric focusing of ostrich α₁-PI in the pH range 3–10 revealed pi values of 4.84 and 4.91, and in the pH range 4–6 the characteristic microheterogeneity observed for mammalian α₁-PIs was displayed.
• 4.4. The presence of sialic acid, hexoses and hexosamines was detected using chemical methods, but were found in much lower quantities as compared to α₁-PIs of other species.
• 5.5. Western blot analysis demonstrated a positive reaction between the native and cleaved ostrich α₁-PIs and the antibodies to the ostrich α₁-PIs raised in rabbits. No cross-reactivity was demonstrated by Western blot analysis between human α₁-PI and antibodies to ostrich α,-PI.
• 6.6. The inhibitory effect of α₁-PI on elastase and chymotrypsin was also investigated.

     

Structure of heparan sulfate from the fresh water mollusc Anomantidae sp: Sequencing of its disaccharide units

• 1.1. The disaccharide sequences of a heparan sulfate isolated from Anomantidae sp. was determined with the aid of heparitinase I, heparitinase II from Flavobacterium heparinum, mollusc β-glucuronidase and α-N-acetylglucosaminidase besides nitrous acid degradation and chemical analyses.
• 2.2. Like the mammalian heparan sulfates the mollusc heparan sulfate is composed of different oligosaccharide blocks of N-acetylated disaccharides, N-sulfated disaccharides and N,6-sulfated disaccharides and has in its nonreducing end the monosaccharide glucosamine 2,6-disulfate.
• 3.3. The oligosaccharides produced by heparitinase I degradation contain at their reducing ends a N-acetylated, 6-sulfated disaccharide.
• 4.4. These and other results lead to the conclusion that the general structure of the heparan sulfate is maintained through evolution.

     

Current developments in stepwise edman degradation of peptides and proteins

• 1.1. Since Edman's (1950, 1956) first publications about 30 years ago, the stepwise degradation of proteins and peptides is universally performed by protein chemists. We review the mechanism of the chemical reactions, and the different special problems encountered, during degradation and different manual methods of degradation.
• 2.2. We take one example of an alternative method using DABITC manually for the degradation of peptides in order to illustrate the evolution of manual degradation techniques (Chang, 1983).
• 3.3. Possibilities and limits of the liquid phase sequenator of Edman and Begg (1967), solid phase sequencer of Laursen (1975) and gas-liquid sequenator of Hewick et al. (1981) and those of Hunkapiller et al. (1983) are considered in detail.
• 4.4. We describe different procedures for identification of PTH-AA or DABTH-AA: thin layer chromatography, gas chromatography, high performance liquid chromatography, etc., in order to illustrate the evolution of the procedures of identification.
• 5.5. We conclude by taking two manual examples and two automatic procedures of degradation to underline the progress over the last decade.

     

Changes in the activities of lysosomal enzymes in the fat body and midgut of two lepidopteran insects (Mamestra brassicae and Pieris brassicae) during metamorphosis

• 1.1. The activities of three lysosomal enzymes (acid phosphatase, β-galactosidase, catepsin D) was observed during metamorphosis in the fat body and midgut cells of two insects (Mamestra brassicae and Pieris brassicae).
• 2.2. The activities increased slightly during the feeding period and showed a sharp rise at the beginning of the wandering period.
• 3.3. Subsequently, a decrease was observed during the pre-pupal stage and pupation.
• 4.4. The activities increased again 2 days after the larval-pupal moult.
• 5.5. We suggest that an inhibitory mechanism works in the studied cells before pupation to protect the stored proteins from the degradation until the beginning of differentiation of imaginai cells in the pupal stage.

     

Characteristics of Crassostrea virginica crystalline style chitin digestion

• 1.1. Fundamental chitin digestion characteristics of Crassostrea virginica crystalline style were investigated.
• 2.2. Optimum temperature and pH were 34°C and 4.8. respectively.
• 3.3. The colloidal regenerated chitin (0.56mol/0.5 ml: GlcNAc equivalents) was saturating under all enzyme levels encountered.
• 4.4. There was no evidence of end product inhibition, even after 100 hr incubation.
• 5.5. Calculated K_m for the chitinase complex was 1.19mM when determined using a 30 min assay, but was only 0.70 mM when determined using a 4.6 hr assay.
• 6.6. Both K_m values are lower than reported for similar assays in other molluscs and for most bacteria.
• 7.7. Effect of substrate preparation on the kinetics are discussed.
• 8.8. Eight peaks of chitinase activity were resolved by DEAE-Fractogel ion exchange chromatography.

     

Hyaluronidase degradation of hyaluronic acid from different sources: Influence of the hydrolysis conditions on the production and the relative proportions of tetra- and hexasaccharide produced

• 1.1. Hyaluronic acid (HA) can be digested with a Streptomyces hyaluronidase.
• 2.2. The rate of production and the ratio of tetrasaccharide (T) and hexasaccharide (H), studied by HPLC, varied with the temperature and duration of hydrolysis.
• 3.3. The rates of production and the respective amounts of the two oligosaccharides depended on the rheological properties of the HA from different sources.
• 4.4. A close relationship was found between the initial rate of hydrolysis and the intrinsic viscosity of the HA (η_i).
• 5.5. Our data suggest that enzymatic degradation at a given pH value, temperature, and duration of hydrolysis is dependent on the conformation of HA.
• 6.6. Moreover, under given conditions, the relative proportions of the two oligosaccharides depend on the η_i and may also reflect the degree of hydrolysis of the substrate.

     

Antagonist like action of synthetic α2-adrenoceptor agonists on contractile response to catecholamines in smooth muscle strips isolated from rainbow trout stomach (Salmo gairdneri)

• 1.1. The effects of some synthetic α₂-adrenoceptor agonists on the mechanical activity and on contractile responses to catecholamines were examined in smooth muscle strips isolated from rainbow trout stomach.
• 2.2. Contractile responses to noradrenaline and adrenaline in the rainbow trout stomach strips were due to α₂-adrenoceptor activation.
• 3.3. Clonidine, p-aminoclonidine, naphazoline and guanabenz caused no mechanical response but concentration-dependently inhibited the contractile responses to noradrenaline and adrenaline without affecting the responses to acetylcholine, carbachol, 5-hydroxytryptamine and methionine-enkephalin. The order of potency was naphazoline >p-aminoclonidine > clonidine > guanabenz.
• 4.4. It is suggested that in the smooth muscle preparation of the trout stomach, some synthetic compounds (clonidine, p-aminoclonidine, naphazoline and guanabenz), which act on mammalian preparations as α₂-adrenoceptor agonists, show an antinoradrenaline (-adrenaline) effect; those compounds can be classified as α₂-adrenoceptor antagonists.

     

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.

     

A simple procedure for evaluating the protein degradation rate in mussel (Mytilus galloprovincialis Lam.) tissues and its application in a study of phenanthrene effects on protein catabolism

• 1.1. An improved, simple method for the evaluation of the protein catabolic rate in the tissues of the lamellibranch mollusc Mytilus galloprovincialis Lam. is presented.
• 2.2. This procedure, which utilizes the technique of the decay curve of a labeled amino acid (¹⁴C-leucine) in the tissues, exploits the capacity of these organisms to rapidly take up soluble compounds from sea-water.
• 3.3. When mussels are exposed to ¹⁴C-leucine in the sea-water, the labeled amino acid is rapidly accumulated into the cell proteins.
• 4.4. A further addition of unlabeled leucine to the sea-water drastically decreases the specific activity of soluble amino acids into the cells, so that the reincorporation of the labeled leucine into the proteins becomes negligible, allowing a correct estimation of the degradation rate of the proteins.
• 5.5. This procedure was utilized to evaluate the effect of phenanthrene on the rate of catabolism of cytosolic proteins in the digestive gland of mussels, and to study the relationship between the protein degradation rate and the activity of lysosomes, which play a well-established role in the catabolism of macromolecules.

     

Effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail

• 1.1. The effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail were investigated.
• 2.2. The level of liver NAD was not affected by niacin deficiency whereas the level of pectoral muscle NAD was markedly reduced.
• 3.3. In all dietary treatments the liver had the highest turnover rates of proteins, heart and brain had intermediate rates, and pectoral muscle had the lowest rates.
• 4.4. Relative turnover rates of proteins in all tissues (particularly pectoral muscle) of the niacin deficient group were significantly higher than those of pair-fed control group, although there were no significant differences in turnover rate between pair-fed control and control groups.
• 5.5. The high turnover rate of proteins in niacin deficiency was primarily attributed to enhanced degradation rate of proteins rather than enhanced synthesis rate of proteins.
• 6.6. Optical density scanning (or densitometric) of water-soluble pectoral muscle proteins separated by isoelectric focusing revealed several additional minor protein bands between major protein bands in the niacin deficient group which were more pronounced in the acidic region of the gel.
• 7.7. These results suggest that proteins with a low pI value in pectoral muscle of the niacin deficient animal are highly sensitive to protein degradation.

     

Chemical intermediates in α-methylnoradrenaline oxidation by tyrosinase—I. Spectral properties and stoichiometry

• 1.1. A pathway for a-methylnoradrenaline oxidation to α-methylnoradrenochrome, by tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometry determination have both been performed.
• 2.2. It has been possible to detect spectrophotometrically o-quinone-H⁺ as the first intermediate in this pathway after oxidizing α-methylnoradrenaline with mushroom tyrosinase or sodium periodate in a pH range from 5 to 6.
• 3.3. The steps for α-methylnoradrenaline transformation into its aminochrome could be: α-methylnoradrenaline → o -α-methylnoradrenaline — H⁺ → o — α -methylnoradrenalinequinone → leuko — α — methylnora — drenochrome→α-methylnoradrenochrome.
• 4.4. No participation of oxygen was detected in the conversion of leuko-α-mehtylnoradrenochrome into α -methylnoradrenochrome.
• 5.5. Matrix analysis of the spectra obtained with a rapid scan spetrophotometer verified that o-quinone-H⁺ was transformed into aminochrome in a constant ratio.
• 6.6. The stoichiometry for this conversion followed the equation: 2 α-methylnoradrenalinequinone-H⁺ → α-methylnoradrenaline + α-methylnoradrenochrome.

     

Effect of tumour promoting agents on protein phosphorylation in human placenta

• 1.1. The effects of okadaic acid (OA) and phorbol-12-myristate-13-acetate (PMA) on protein phosphorylation were studied in human term placentas.
• 2.2. When samples treated with tumour promoters were compared with untreated samples, the phosphorylation of a 135 kDa protein was significantly decreased; OA also produced a decrease in phosphorylation of a 24 kDa protein.
• 3.3. Both substances produced an alteration in the proportions of bands of masses 170, 65 and 24 kDa, relative to total phosphorylation: PMA treatment also affected the band of mass 135 kDa.
• 4.4. Placental cell extracts were also subjected to Western blotting with a protein kinase C (PKC) antibody, reportedly specific for the α- and β-isoforms.
• 5.5. Two immunoreactive proteins were detected; an 80 kDa band, presumably corresponding to the α- or β-PK.C, and a 64 kDa protein, which could be a degradation production of the 80 kDa protein or it could correspond to another form of the enzyme. The expression of PKC did not change on treatment with PMA.

     

Lipid components and utilization in consumers of a seagrass community: An indication of carbon source

• 1.1. The lipid components of three animals, the rock crab Nectocarcinus integrifons, the rock flathead Platycephalus laevigatus and the southern garfish Hyporhamphus melanochir, feeding in the seagrass beds at Corner Inlet, Victoria, Australia have been examined in detail in order to provide further information on seagrass community structure.
• 2.2. Biological marker compounds detected within animal gut content material were used to recognize dietary sources and then utilized by community members.
• 3.3. Both H. melanochir and N. integrifons have been shown to ingest and to varying degrees incorporate seagrass lipid material, thus further confirming the importance of seagrass carbon in the Corner Inlet environment.
• 4.4. The southern sea garfish H. melanochir is observed to remove C₁₈ PUFAs (polyunsaturated fatty acids) from ingested seagrass material.
• 5.5. Seagrass sterols are altered during incorporation into the lipids of this fish.
• 6.6. Lipid-rich digestive juices play a role in the digestive processes of all three animals.
• 7.7. Components tentatively identified as (NMI) (non-methylene interrupted) fatty acids have been detected in the lipids of the garfish H. melanochir and the crab N. integrifons.
• 8.8. The fecal material of all three animals represent possible sources of these lipids (NMI acids) in Corner Inlet sediments.
• 9.9. Based on lipid compositional data, N. integrifons feeds on Posidonia australis detritus and associated epiphyte material.
• 10.10. The removal of both plant and epibiota cellular lipids along the digestive tract of the crab was observed, although structural components such as long chain mono- and α,ω-dicarboxylic acids, which have been previously recognized as seagrass marker lipids are not directly absorbed.