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.

     

A peptide inhibitor for S-adenosyl-l-methionine-dependent transmethylation reactions: Purification and characterization

• 1.1. A proteinaceous inhibitor for S-adenosyl-l-methionine (AdoMet)-dependent transmethylation reactions has been purified to apparent homogeneity from rat liver cytosolic fraction.
• 2.2. The peptide was made up of 29 amino acid residues with a molecular weight of 2,584. Glycine accounted for 52% of the total amino acids.
• 3.3. Employing AdoMet: protein-carboxyl O-methyltransferase (Protein methylase II) and bovine serum γ-globulin as in vitro substrate, the mode of inhibition was found to be non-competitive with K_i value of 1.9 × 10⁻⁸ M.
• 4.4. When the inhibitor was present in the reaction mixture together with S-adenosyl-l-homocysteine (AdoHcy), which is a competitive inhibitor for AdoMet, the extent of inhibition exceeded that exerted by each individual inhibitor alone, suggesting that the sites of the inhibitors on the enzyme molecule are different.
• 5.5. Almost a stoichiometric relationship exists between the enzyme and the inhibitor molecule, the ratio being approx one.

     

Purification and properties of the proteinase inhibitors from Erythrina caffra (coast erythrina) seed

• 1.1. Four proteinase inhibitors (DE-1 to DE-4) were purified from Erythrinu caffra seed by gel filtration on Sephadex G-50 followed by ion-exchange chromatography involving DEAE-cellulose and DEAE-sepharose.
• 2.2. They comprise 164–166 amino acid residues (mol. wt 18,100) including 4 half-cystine residues and resemble the Kunitz-type proteinase inhibitors.
• 3.3. The N-terminal primary structure of DE-3 revealed also homology with those of the Kunitz-type inhibitors. For DE-1, DE-2 and DE-4 no free N-terminal amino acid was found.
• 4.4. DE-1 contains a potent inhibitor for both porcine trypsin and bovine α-chymotrypsin. Whereas DE-2 inhibits a-chymotrypsin strongly and has practically no action on trypsin, DE-3 inhibits both trypsin and a-chymotrypsin strongly. DE-4 is a potent inhibitor for trypsin but it binds a-chymotrypsin only weakly.

     

Purification and properties of tetralysine endopeptidase from Escherichia coli AJ005

• 1.1. A new tetralysine endopeptidase from Escherichia coli AJ005 has been purified about 135-fold.
• 2.2. The peptidase seems to be specific to tetralysine among lysine homopolymers.
• 3.3. The optimal pH was about 7.5
• 4.4. The activity was inhibited by KCN but not inhibited by soybean trypsin inhibitor.
• 5.5. The apparent K_m value was 2.5 × 1O⁻³ M for tetralysine.

     

Kinetic study of the inhibition of rat liver ornithine decarboxylase by diamines; considerations on the mechanism of interaction between enzyme and inhibitor

• 1.1. Partially purified rat liver ornithine decarboxylase is inhibited by several diamines including putrescine, 1,3-diaminopropane, cadaverine and p-phenylenediamine.
• 2.2. The inhibition is dependent on pH, being strong at pH above 8 and negligible below pH 6.5.
• 3.3. The kinetic study of the inhibition showed that while the aromatic diamine behaved as a simple competitive inhibitor, the aliphatic diamines presented a more complex pattern of inhibition in which two molecules of inhibitor might bind to the enzyme active site.
• 4.4. The K_I values for the different inhibitors were calculated and the degree of affinity for the enzyme was p-phenylenediamine > putrescine > cadaverine > 1,3-diaminopropane.
• 5.5. A molecular mechanism explaining how one or two molecules of inhibitor can bind to the enzyme is proposed.

     

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.

     

The fate of the bowman-birk trypsin inhibitor from soybeans in the digestive tract of chicks

• 1.1. Intestinal absorption of the Bowman-Birk trypsin inhibitor from soybeans was studied comparatively in chicks by direct follow-up of absorbed radioactive inhibitor and by radioimmunological estimation of the unlabelled inhibitor.
• 2.2. Initial studies, performed in vitro by the inverted sac technique, suggested that both the native inhibitor and its degradation products were absorbed.
• 3.3. However, the results obtained for in situ and in vivo experiments indicated that the absorption of the native inhibitor is negligible.
• 4.4. Most of the inhibitor is degraded during its passage through the intestine, and the majority of the degradation products is excreted in the feces.
• 5.5. The effects of ingested inhibitor on pancreas enlargement and on secretion of pancreatic enzymes stems from an indirect effect on the intestine.

     

The induction of α1-acid glycoprotein by methylmercury

• 1.1. The incorporation of [¹⁴C]leucine into protein was measured in liver preparations and blood of rats following the s.c. administration of methylmercury hydroxide (24 mg/kg body wt) or turpentine (5.0 ml/kg body wt).
• 2.2. The translatability of the RNA obtained from polysomes in an mRNA-dependent reticulocyte lysate was elevated significantly in the preparations derived from the treated rats compared to control rats.
• 3.3. Immunoprecipitation of the labelled translation products or of serum proteins showed that the mRNA activity and the synthesis of α₁-acid glycoprotein, an acute phase reactant, was elevated by the methylmercury treatment as well as by the turpentine-induced inflammatory response.

     

The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes

• 1.1. The interaction of insulin with purified brush-border membranes from rat kidney was studied with the use of [¹²⁵I]insulin.
• 2.2. The specific binding of insulin by brush-borders could be demonstrated, and was time- and temperature-dependent.
• 3.3. [¹²⁵I]insulin was displaced by unlabelled insulin. A₁-B₂₉ dodecoyl insulin and insulin A- and B-chains in proportion to their relative bioactivity.
• 4.4. Brush-border membranes showed high insulin-degrading activity with an apparent K_m of 2.2 μM.
• 5.5. A number of proteinase inhibitors were effective in inhibiting insulin degradation but the greatest degree of inhibition was achieved by the use of thiol-blocking reagents.
• 6.6. No evidence was obtained for the involvement of the enzyme glutathione-insulin transhydrogenase.

     

Anionic trypsin-like enzymes from the crab Eriocheir japonicus De Haan active in more acidic media

• 1.1. Proteolytic enzymes from digestive fluid of the catadromous crab Eriocheir japonicus De Haan have been investigated.
• 2.2. Four types of the enzyme referred to as A₁, A₂, A₃ and A₄ are purified by gel filtration and DEAE-Sephadex column chromatography in a homogeneous state (only A₂ contaminated with minor component).
• 3.3. Molecular weight of A₂, A₃ and A₄ is approx. 29,000 on 0.1% SDS-polyacrylamide gel electrophoresis.
• 4.4. These enzymes show optimal pH in acidic and neutral media (A₁, at pH 5.8; A₂, at pH 6.3; A₃, at pH 7.0; A₄, at pH 7.5) and high stability in more alkaline media at pH 5 or above.
• 5.5. A₁ is inhibited with phenylmethylsulfonyl fluoride, diisopropylphosphoryl fluoride, and soy bean trypsin inhibitor.
• 6.6. A₂, A₃ and A₄ are inhibited with tosyl-l-lysine chloromethyl ketone, diisopropylphosphoryl fluoride, leupeptin, soy bean trypsin inhibitor and potato protease inhibitor II-a and II-b.
• 7.7. A₂, A₃ and A₄ act on synthetic lysyl and arginyl derivatives, but not on aromatic amino acid derivatives.
• 8.8. From the findings including electrophoretic behavior, A₂, A₃ and A₄ are anionic trypsin-like enzyme and different from A₁.

     

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.

     

Isolation and partial characterization of a porcine low molecular weight protein occurring in plasma and urine

• 1.1. A low molecular weight (LMW) glycoprotein was isolated in the pig from urine produced after the induction of proximal tubular damage and uremia by maleic acid.
• 2.2. The purification steps included ultrafiltration, gel chromatography on Sephadex and anion exchange chromatography.
• 3.3. The molecular weight, determined by SDS-polyacrylamide electrophoresis was 12,500. The protein appeared heterogeneous in agarose gel electrophoresis. Immunoelectrophoresis and crossed immuno-electrophoresis demonstrated 2 major zones in the α-region, a minor in the early α₁- and one in the β-region.
• 4.4. Like the human LMW proteins it appeared in trace amounts in normal plasma and urine but its characteristics were unlike any of the known human plasma LMW proteins.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Amino acid accumulation in kidney cortex of lambs and piglets as affected by insulin

• 1.1. Insulin stimulated intracellular accumulation of α-amino-isobutyric acid (AIB) in kidney cortex slices from young lambs and piglets.
• 2.2. The effect was similar in the absence or presence of glucose.
• 3.3. The induction of the stimulatory effect on renal AIB transport was blocked by cycloheximide. an inhibitor of protein synthesis.
• 4.4. The insulin stimulation of intracellular AIB accumulation is due to an increased influx and not to a reduced efflux of AIB.
• 5.5. Analysis of transport kinetics for AIB showed that insulin increased V_max but did not change K_m.
• 6.6. It is concluded that insulin stimulates uptake of certain neutral amino acids into kidney cortex cells in young animals.
• 7.7. The effect on renal amino acid transport appears to be mediated through increased synthesis of a membrane carrier.

     

Detergent solubilisation of rat liver particulate cyclic AMP phosphodiesterase

• 1.1. Detergent solubilisation of particulate rat liver low K_m cyclic AMP phosphodiesterase in the presence of protease inhibitors yields a form of the enzyme with a larger molecular weight than the form solubilised by protease treatment.
• 2.2. The detergent solubilised enzyme could be partially purified by anion exchange chromatography.
• 3.3. It displayed a marked tendency to precipitate from solution when detergent was removed.

     

Effect of aminooxyacetate and α-ketoglutarate on glutamate deamination by rat kidney mitochondria

• 1.1. Glutamate dehydrogenase flux by rat kidney mitochondria incubated with 1 mM glutamine plus 2–3 mM glutamate was stimulated by aminooxyacetate. This effect was inhibited by α-ketoglutarate.
• 2.2. Studies with intact mitochondria and mitochondrial sonicates revealed a linear inverse relationship between glutamate deamination and α-ketoglutarate levels.
• 3.3. The data revealed that α-ketoglutarate is a competitive inhibitor of glutamate dehydrogenase with an apparent K_i of 0.6mM.
• 4.4. The data suggest that aminooxyacetate stimulates glutamate deamination by a mechanism mediated by α-ketoglutarate.

     

The subunit structure of Daphnia pulex hemoglobin

• 1.1. The extracellular hemoglobin of Daphnia pulex has an apparent molecular weight of 430,000–470,000 by gel chromatography and an S_20,w = 16.9 at pH 7.0.
• 2.2. Purified hemoglobin contains one heme per 18,000–20,000 g protein. The polypeptide chains are heterogeneous with mol. wts between 31,000–37,000. Some high mol. wt (M_r = 53,000–86,000) material is also present.
• 3.3. The hemoglobin dissociates at pH 10.5 in EDTA into 3S material which can be digested with subtilisin into 16,000 mol wt heme-containing polypeptide chains.
• 4.4. The amino acid composition of the intact hemoglobin is identical to that of the heme-containing fragments generated by proteolytic digestion of the 3S material.
• 5.5. These results are consistent with the hypothesis that D. pulex hemoglobin is composed of subunits containig two heme groups per 35,000 mol. wt polypeptide chain.

     

Cyclic AMP stimulation and prostaglandin inhibition of Na transport in freshwater mussels

• 1.1. Pondwater acclimated Carunculina texasensis and Ligumia subrostrata experienced a 230% increase in Na influx when injected with dibutyryl cyclic AMP (0.4mM/l blood).
• 2.2. Theophylline, a phosphodiesterase inhibitor, or indomethacin, an inhibitor of prostaglandin (PG) synthetase, caused a dose dependent stimulation of Na transport.
• 3.3. Prostaglandin E₂ injected into mussels caused an inhibition of Na influx. Arachidonic acid, the precursor of PGE₂, inhibited Na influx or stimulated Na efflux depending on the animal's acclimation conditions.
• 4.4. Chloride transport was unaffected by the drugs used in this study.

     

Myo-inositol in the reproductive tract of the female rat

• 1.1. myo-Inositol concentrations in oviduct, ovary and uterus were many-fold those of blood serum during all four stages of the estrous cycle of the female rat.
• 2.2. Inositol concentration was higher in oviduct than in ovary or uterus and was lower in uterine fluid than in uterus.
• 3.3. Estrus uteri had higher inositol concentrations than uteri in other phases of the cycle.
• 4.4. In order to measure dynamic aspects of the distribution of inositol. the distribution of radioactivity among organs of the reproductive tract of mature female rats was measured 45 min after i.p, injection of [2-³H]myo-inositol.
• 5.5. These organs concentrated inositol from the blood, and the tissue radioactivity (expressed as dpm/mg wet wt of tissue) increased in the sequence: vagina < cervix < uterus < ovary < oviduct.
• 6.6. The uterus and ovary concentrated myo-inositol more strongly during proestrus than during metestrus. diestrus or estrus.
• 7.7. The contents of proestrus follicles were more highly radioactive than was the ovary itself, whereas proestrus uterine fluid was less radioactive than the uterine tissue.

     

A novel inhibitor of glycogen phosphorylase from crayfish hepatopancreas

• 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
• 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
• 3.3. The inhibitor is a small protein (M_r = 23,000) which did not show proteolytic activity.
• 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
• 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
• 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.