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.

     

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.

     

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.

     

Regulation of gluconeogenesis from propionate and propanol in the perfused guinea pig liver

• 1.1. Gluconeogenesis from propanol in perfused guinea pig liver is stimulated by aspartate, glutamate and phenazine-methosulfate, and inhibited by octanoate and by aminooxyacetate.
• 2.2. Gluconeogenesis from propionate is inhibited by octanoate and by NH₄⁺, and stimulated by ethanol.
• 3.3. Phosphoenolpyruvate formation from propionate in isolated guinea pig liver mitochondria is inhibited by octanoate and by NH₄⁺ or by both.
• 4.4. The data are discussed in terms of regulation of gluconeogenesis from both substrates in guinea pig liver.

     

The major serum protein of fetal and newborn pigs: Biochemical properties and identification as a fetal form of α1-acid glycoprotein

• 1.I. The major protein component of fetal pig serum, has been immunologically identified as α₁-acid glycoprotein (orosomucoid).
• 2.2. Amino acid composition and total carbohydrate content (around 38% by weight) were similar in the adult and fetal forms of α₁-acid glycoprotein. These forms differ, however, in the proportion of individual monosaccharides.
• 3.3. Fucose, represented the 1.5% (by weight) in the fetal protein, and the 2.5% in its adult counterpart. The latter was more susceptible to ncuraminidase and also possesses a higher mannose/galactose ratio than the fetal form.
• 4.4. Insolubilized Concanavalin A (Con A) retained 80%, of the adult protein, whereas the fetal form was mostly Con A-non reactive. The proportion of this -non reactive fraction, as revealed by crossed immuno-affino-electrophoresis experiments, was age-dependent and varied from 62% at fetal age of 50–60 days to 80% at birth.

     

Ostrich α-amylase: Purification and characterization of the pancreatic isoenzymes

• 1.1. Four ostrich pancreatic α-amylase isoenzymes were isolated by isoelectric focusing, following affinity chromatography on cyclohepta-amylose-Sepharose 4B.
• 2.2. Amino acid compositions of the four isoenzymes are very similar with only one charged amino acid (Arg) being significantly different.
• 3.3. The molecular weights, as determined by SDS-PAGE and amino acid composition, are nearly identical (52–53 kDa) for all four isoenzymes.
• 4.4. The four α-amylase isoenzymes appear to be kinetically distinct enzymes with a requirement for calcium.
• 5.5. Ostrich α-amylase isoenzymes appear to be non-glycosylated and contain one free thiol group.

     

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.

     

Purification and some properties of isocitrate dehydrogenase of a blowfly Aldrichina grahami

• 1.1. NADH-dependent isocitrate dehydrogenase has been purified 110-fold from the crude extract of the flight muscle mitochondria of Aldrichina grahami.
• 2.2. The purification procedure involved Triton X-100 treatment of isolated mitochondria, column chromatography on DEAE-cellulose, Affi-gel blue, and P-cellulose.
• 3.3. The purified enzyme was homogeneous by criteria of the polyacrylamide gel electrophoresis.
• 4.4. The enzyme of the blowfly contains more acidic amino acids and less hydrophobic amino acids than that of pig heart.
• 5.5. The molecular weight was determined to be 330,000 daltons. The subunit construction differs from ghat of mammalian isocitrate dehydrogenase.

     

Purification of endo- and exolaminarinase and partial characterization of the exoacting form from the copepod acartia clausi (Giesbrecht, 1889)

• 1.1. The copepod Acartia clausi exhibited two laminarinases (exo- and endo-acting forms) purified by gel chromatography followed by affinity chromatography. Specific antibodies have been raised against the purified exolaminarinase antigen.
• 2.2. A single band of protein appeared on a polyacrylamide disc gel electrophoresis; its mol. wt is 21,000.
• 3.3. Biochemical properties of the purified enzyme showed a maximum activity at pH 5.2 and a temperature of 40°C with laminarin as substrate. The thermal stability of the enzyme and the effect of various cations on its activity were examined. The enzyme hydrolyses specifically the β(1–3) linked polysaccharides and had no activity against the α(1–4) or β(1–4) disaccharides or polysaccharides.
• 4.4. The kinetic parameters V_m and K_m vary with the temperature; the affinity constant (K_a) was maximum between 25–30°C. The Arrhenius plot defined two values of energy of activation: 7980 cal/mole and 17,506 cal/mole.
• 5.5. From the purification scheme the exoacting form appears to be largely dominant over the endoacting form.

     

Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

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.

     

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.

     

Purification of the flavin-containing monooxygenase from mouse and pig liver microsomes

• 1.1. The microsomal flavin-containing monooxygenase has been purified from mouse and pig liver utilizing Cibacron-Blue Sepharose, Procion-Red agarose, and 2'5'-ADP Sepharose.
• 2.2. The enzymes had a final specific activity of 1200 and 954 nmol/min/mg protein from mouse and pig liver respectively.
• 3.3. The enzyme from both mouse and pig liver displayed typical flavoprotein spectra and appeared homogeneous by denaturing polyacrylamide gel electrophoresis.

     

Reducing equivalent transport by substrate shuttles in rat liver and colon

• 1.1. The observed level and subcellular distribution of the α-glycerophosphate and malate-aspartate substrate shuttle enzymes in liver and colon were consistent with their proposed roles in reducing equivalent transport.
• 2.2. K_m value determinations of shuttle enzymes were performed.
• 3.3. Substrate shuttles were reconstructed from isolated liver and colon mitochondria which displayed satisfactory respiratory control and P:O ratios.
• 4.4. The results obtained suggest that while the malate-aspartate shuttle is the primary means of reducing equivalent transport in the liver, the α-glycerophosphate shuttle predominates in the colon.

     

Trypsin inhibitors in serum of adult and suckling rats and in rat milk

• 1.1. Trypsin inhibitors in serum from adult and suckling rats and in rat milk were studied by gel filtration and electrophoresis in casein-agarose gels. In addition trypsin binding properties and inhibiting activity in the presence of low mol. wt substrates were studied.
• 2.2. In adult rat serum 6 trypsin inhibitors were found, most of these having not been described before. One inhibitor, di-macroglobulin is a homologue of human α₂-macroglobulin. Two inhibitors in the α₁- and α₂-regions of the electropherogram had a mol. wt of about 200,000. The third group of inhibitors was eluted together with albumin and had electrophoretic mobilities in the di-α₂- and β-region.
• 3.3. In the serum of the newborn rat the inhibitors of the third protein peak dominated, especially that in the α₂-region. In later developmental stages the inhibitor pattern became increasingly similar to that of the adult. A specific inhibitor, α₂-acute phase globulin, was found in the neonatal rat but disappeared in later developmental stages.
• 4.4. The trypsin inhibitors in rat milk were dominated by inhibitors in the third protein peak after gel nitration and had the same electrophoretic mobilities as the inhibitors of the corresponding fraction in serum. No low mol. wt trypsin inhibitors specific for milk were found.
• 5.5. Rat milk trypsin inhibiting activity seems to have a higher resistance to low pH-values than the inhibiting activity of rat serum.
• 6.6. The trypsin inhibitor pattern in the serum of the suckling rat is similar to that of milk, indicating an absorption of inhibitors from milk. The milk inhibitors may have the function of preventing protein breakdown in the intestine of the suckling rat.

     

Comparison of fibrinogenases isolated from copperhead venoms

• 1.1. Fibrinogenase activity from fractions obtained by ion-exchange chromatography of whole copperhead (Agkistrodon contortrix) venoms were further purified by molecular sieve chromatography.
• 2.2. All four enzymes had apparent mol. wt of 23,000–24,000, both non-reduced and reduced as determined by sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography.
• 3.3. All four enzymes rapidly degraded the α-chain of the fibrinogen while apparently leaving the β- and γ-chains intact.

     

Immunological reactivities of rat and human α-lactalbumin

• 1.1. Antisera to rat α-lactalbumin immediately displayed precipitation bands to human, bovine, pig and goat α-lactalbumin, whereas antisera to human α-lactalbumin displayed similar cross reactivities only at a later time.
• 2.2. Cross reactivity of the antisera was determined by radioimmune assays to rat and human α-lactalbumin.

     

Isolation of heart specific isophosphorylase by affinity chromatography on 5′-AMP sepharose from pig heart

• 1.1. 5′-AMP Sepharose was used for adsorption and separation of the isophosphorylases from pig heart.
• 2.2. The heart specific isophosphorylase was selectively eluted by glucose-6-phosphate from the 5′-AMP Sepharose.
• 3.3. This preparation was homogeneous, the homogeneity was tested by SDS-gel electrophoresis and immunotitration using skeletal muscle anti-phosphorylase.

     

Sarcolemmal membranes from hamster and chicken skeletal muscle: Isolation and chemical composition

• 1.1. Plasma membranes were obtained from hamster (Mesocricetus auratus Wateth.) and chicken (Callus gallus L.). Skeletal muscle was isolated by muscle homogenization, protein extraction by inorganic salt solutions (0.4 M LiBr and 0.6 M KCl) and differential centrifugation. After purification on a discontinuous sucrose gradient, several fractions were obtained. The upper fraction (20% sucrose, w/w) yielded in the form of vesicles by electron microscope examination.
• 2.2. (Na⁺ + K⁺ )-ATPase and 5'-nucleotidase as plasma membrane markers were found to be concentrated in the upper fraction. Practically no succinate dehydrogenase activity was detected.
• 3.3. By means of polyacrylamide gel electrophoresis it was possible to separate 7–8 protein bands the molecular weights of which range from 26,000 to 200,000 daltons; and 4 bands for glycoproteins.
• 4.4. The ratio lipid: protein and the molar ratio cholesterol :phospholipid were found to be 0.93–0.94 and 0.25–0.38, respectively.
• 5.5. Glucose, mannose, galactose and fucose, hexosamines and sialic acids were determined in these preparations of membranes.