l-lysinamidase from Cryptococcus laurenti 112. Purification and properties

• 1.1. The purified enzyme hydrolyzes the linear l-lysinamide and the cycle amide of l-lysine—l-α-amino-ϵ-caprolactam.
• 2.2. The apparent relative molecular mass is 180,000. The enzyme consists of four subunits and the molecular mass of a single subunit was found to be 47,000.
• 3.3. The coefficient of molecular sedimentation equals 8.3 S, the isoelectric point was determined to be pH 4.3
• 4.4. The enzyme is not a glycoprotein. p-Mercuribenzoate binds 10 SH-groups of the native enzyme molecule and 20 SH-groups in the presence of 0.7% SDS.
• 5.5. pH- optimum for the hydrolysis of l-lysine amides was observed to be 7.5–7.7. The enzyme is strictly dependent on Mn²⁺ and Mg²⁺.
• 6.6. The kinetic parameters for the hydrolysis of l-lysinamide where K_m = 3.8 mM and k_cat = 3000 sec⁻¹ For the hydrolysis of cyclic L-lysinamide K_m = 4.8 mM and k_cat = 2600 sec⁻.

     

Ostrich (Struthio camelus) carboxypeptidase A: Purification,kinetic properties and characterization of the pancreatic enzyme

• 1.1. Carboxypeptidase A and carboxypeptidase A,-type from the pancreas of the ostrich were purified by water extraction of acetone powder, aminobenzylsuccinic acid affinity and hydroxylapatite chromatography.
• 2.2. The final preparations were homogeneous when subjected to SDS-PAGE and PAGE. The M, values obtained from SDS-PAGE for CPAp and CPA,-type were 34,600 and 34,400, respectively.
• 3.3. The effects of inhibitors (1,10 phenanthroline and indole-3-acetic acid), pH and temperature on CPA activity were examined. K_i-values for CPI, PPA, D-phe, D-trp and aminobenzylsuccinic acid were determined.
• 4.4. K_m, k_cat and k_cat/K_m values were determined for hipp-phe, cbz-gly-phe, cbz-(gly)₂-phe, cbz-gly-leu, cbz-(gly)₂-leu and cbz-(gly)₂-val.
• 5.5. N-terminal sequencing and amino acid analysis were performed for CPA_β and CPA_τ-type.

     

Temperature-dependence of spectroscopic and catalytic properties of the eel (Anguilla anguilla) liver mitochondrial F1-ATPase

• 1.1. F₁-ATPase from eel liver mitochondria at low concentrations preserves unaltered the enzymatic activity for more than 20 min over a temperature range of 6–36°C.
• 2.2. The Arrhenius plot of ATP hydrolysis at saturating substrate concentration appears biphasic with a break-point at 16°C and activation energies of 14.4 and 56.1 kJ/mol.
• 3.3. The ultraviolet, fluorescence and circular dichroism spectra of the enzyme, below and above 16°C, have been recorded; the fluorescence emission spectra of F₁-ATPase excited at 275 nm, and the circular dichroism spectra, are different at the two temperatures examined.
• 4.4. It is concluded that temperature induces two different conformational states of F₁-ATPase with different catalytic properties.
• 5.5. Ultraviolet spectroscopic features and temperature-dependence of eel liver mitochondrial F₁-ATPase are discussed in relation to mammalian F₁-ATPases.

     

Purification and some properties of elastase from hepatopancreas of king crab Paralithodes camtschatica

• 1.1. Elastase has been purified from the hepatopancreas of the king crab (Paralithodes camtschatica). Specific activity of the enzyme measured toward Suc-(Ala)₃-pNA and Boc-(Ala)₃-pNA was 926 and 3700 mUnits per mg of protein, respectively.
• 2.2. The enzyme is an anion protein (pI 4.5) with an approximate mol.wt of 28.5 kDa.
• 3.3. The enzyme exhibited a bell-shaped pH-dependence for the hydrolysis of Suc-(Ala)₃-pNA with a maximum at 8–8.5. Under these conditions the values of K_m and k_cat of the crab elastase are 4 mM and 4.75 s⁻¹, respectively.
• 4.4. The serine elastase is effectively inhibited by elastinal and diisopropylfluorophosphate.
• 5.5. It is shown that some salts except HgCl₂ activate the protease. In the presence of HgCl₂ with concentrations of 10 mM and higher, the crab elastase is inactive. SDS and Triton X-100 have no any effect on the activity of crab elastase.

     

The possibility that Ca2+-ATPase from the plasma membrane-rich fraction of bovine parotid gland is ecto-Ca2+-dependent nucleoside triphosphatase

• 1.1. Parotid plasma membrane nonpump low-affinity Ca²⁺-ATPase, which possesses high-affinity (Ca²⁺ + Mg²⁺ )-ATPase activity, was characterized.
• 2.2. Purified Ca²⁺-ATPase hydrolyzed the nucleoside triphosphates, GTP, ITP, CTP, UTP, TTP (67–93% of ATP) and nucleoside diphosphates, ADP. GDP, IDP, CDP, TDP (12–40% of ATP) but not AMP and p-NPP.
• 3.3. The maximum activities of Ca²⁺- and (Ca²⁺ +Mg²⁺ )-ATPases were obtained in the presence of 1 mM and 0.13 μ M Ca²⁺, respectively.
• 4.4. The K_m values for Ca²⁺ in Ca²⁺- and (Ca²⁺+ Mg²⁺ )-ATPases were 0.2 mM and 22 nM. respectively.
• 5.5. The activities of both Ca²⁺- and (Ca²⁺ + Mg²⁺ )-ATPases were found in the right-side-out-vesicles obtained from the plasma membrane-rich fraction.
• 6.6. These features suggest that Ca²⁺-ATPase is an ecto-Ca²⁺-dependent nucleoside triphosphatase.

     

Mg2+-dependent (Na+ + K+)- and Na+-ATPases in the kidneys of the gilthead bream (Sparus auratus L.)

• 1.1. The (Na⁺ + K⁺)- and Na⁺-ATPases, both present in kidney microsomes of Sparus auratus L., have different activities and optimal assay conditions as, in the first of the two stocks of fish used (A), the spec. act. of the former is 51.7 μmol P_i mg prot⁻¹ hr⁻¹ at pH 7.5, 100 mM Na⁺, 10 mM K⁺, 17.5 mM Mg²⁺, 7.5 mM ATP and that of the latter is 6.5 μmol P_i mg prot⁻¹ hr⁻¹ at pH 6.5, 40 mM Na⁺, 4.0 mM Mg²⁺, 2.5 mM ATP.
• 2.2. Ouabain and vanadate specifically inhibit the (Na⁺ + K⁺)-ATPase but not the Na⁺-ATPase that is preferentially inhibited by ethacrynic acid.
• 3.3. While the (Na⁺ + K⁺)-ATPase is strictly specific for ATP and Na⁺, Na⁺-ATPase can be activated by various monovalent cations and, apart from ATP, hydrolyses CTP, though less efficiently.
• 4.4. The second stock B, subjected to higher salinity than A, shows an acidic shifted Na⁺-ATPase optimal pH, opposed to the stability of that of the (Na⁺ + K⁺)-ATPase, a decreased (Na⁺ + K⁺)-ATPase and a strikingly depressed Na⁺-ATPase.
• 5.5. The results are compared with literature data and discussed on the basis of the presumptive different roles as well as functional prevalence in various salinities of the two ATPases.

     

The flow theory of enzyme kinetics: Role of solid geometry in the control of reaction velocity in live animals

• 1.1. When blood flows, membranes are bombarded with ions etc., whose entry creates an ATP demand proportional to flow rate. Also proportional to flow rate is ATP production from oxidation of substrates [S] from the same blood volume.
• 2.2. O₂ is limiting and reaction velocity at rest (metabolic rate) is determined by flow rate, F, but not by [S].
• 3.3. Since resting blood O₂ A-V difference is about 5 vol%, 11 circulated produces about 0.25 kcal in mammals, birds or warm reptiles.
• 4.4. Where O₂ is not limiting, as in most amino acid deaminations, V = K F[S] with K a constant unrelated to K_m.
• 5.5. At equal blood vol/kg, solid geometry dictates that the average cross-sectional area of major vessels/kg will be an inverse function of body mass. The smaller the animal, the shorter the vessels, the “thicker” the vessels/kg body wt, and at any one blood pressure, the higher the flow/kg/hr. If a man's major vessels were equal in cross-section/kg to those of a shrew, it would take 2241 of blood to fill them.
• 6.6. Growth decreases flow/kg (and therefore metabolic rate), by decreasing vessel cross-section/kg without changing blood pressure or linear velocity of flow.
• 7.7. Surface area/g, body wt to some power, average vessel length/kg, circulation time and average major vessel cross-sectional area are all related mathematically.

     

Purification and characterization of casein kinase II from lactating rabbit mammary gland

• 1.1. Highly purified 200 kDa casein kinase II from rabbit lactating mammary gland (MG-CK II) was obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono Q steps.
• 2.2. Its K_m for ATP was 2.22 μM and 0.57 mg/ml and 0.13 mg/ml for partially dephosphorylated casein and phosvitin respectively. Stathmine was also suitable as substrate. 2-aminopurine and 6-dimethylaminopurine inhibited efficiently MG-CK II K_i = 5 and 1 mM respectively).
• 3.3. MG-CK II autophosphorylated on its α-, α '- and β-subunits. The β-subunit auto-phosporylation was enhanced in presence of exogenous substrate. Its modulation was highly dependent on ATP concentration.
• 4.4. The effects of basic compounds which affected dramatically the phosphorylation of dephosphorylated casein in presence of various ATP concentrations were reported.

     

Effects of adenine nucleotides on low Km 5′ nucleotidase from human seminal plasma

• 1.1. Low K_m 5' nucleotidase purified from human seminal plasma has been used in this study to investigate the response of the enzyme to adenine nucleoside di- and triphosphates in the presence of AMP and IMP as substrates.
• 2.2. In the presence of AMP, the addition of 0.5 mM ATP to the enzyme Mg-free results into the highest V_max/K_m ratio value and other experimental combinations of effectors tested cause variation of the kinetic parameters of the enzyme, indicating a control of AMP dephosphorylation by adenine nucleotides.
• 3.3. In the presence of IMP, ATP and ADP activate the enzyme but the response to various experimental combinations of effectors shows no significant difference in the kinetic properties of the enzyme, indicating a different control of the dephosphorylation of IMP.

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

Ouabain sensitive Na+/K+-transporting ATPase from the brain of Poekilocerus bufonius

• 1.1. The properties of Na⁺/K⁺-transporting ATPase in microsomal fractions from the nervous tissue of the grasshopper, Poekilocerus bufonius were investigated.
• 2.2. Two components of ATPase activity are present.
• 3.3. Inclusion of 1 mM ouabain in the incubation media reduced the activity of total and Na⁺/K⁺-ATPase by 57 and 79%, respectively.
• 4.4. The maximum velocity (V_max) was decreased by the addition of 1 mM ouabain, whereas the apparent K_m value was not affected indicating a non-competitive type of inhibition.
• 5.5. The calculated value of the pI₅₀ was 6.4 (I₅₀ = 3.98 × 10⁻⁷M) for ouabain inhibition of the enzyme showing great sensitivity to the cardiac glycoside ouabain.
• 6.6. The present results show that the physicochemical properties of Na⁺/K⁺-transporting ATPase from the brain of P. bufonius are essentially the same as for the enzyme prepared from the excretory system of the insect which has been previously investigated.
• 7.7. Dissimilarities were also observed between these tissues in the way that the enzyme from the brain was sensitive to ouabain inhibition with a non-competitive type rather than a ouabain-resistance and a competitive type of inhibition for the enzyme from the excretory system.
• 8.8. These dissimilarities are probably due to different isoenzyme patterns available in the same insect.

     

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.

     

Ostrich fructose-1,6-bisphosphatase: Kinetic characterization of the liver isoenzyme

• 1.1. Purified ostrich (Struthio camelus) liver fructose-1,6-bisphosphatase exhibited an absolute requirement for Mg²⁺.
• 2.2. The enzyme catalyzed the hydrolysis of fructose-1,6-bisphosphate, sedoheptulose-l,7-bisphosphate and ribulose-l,5-bisphosphate.
• 3.3. S_0.5 for substrate was 1.4 μM.
• 4.4. AMP was a potent non-competitive inhibitor with respect to substrate (K_i of 25 μM).
• 5.5. Fructose-2,6-bisphosphate was a potent competitive inhibitor of the enzyme (K_i of 4.8 μM).

     

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.

     

Modulation of active potassium transport by aldosterone

• 1.1. The role of aldosterone on active potassium transport across lizard colon under voltage-clamped conditions has been investigated.
• 2.2. Control colons exhibited no net potassium flux (J^k_net) despite of the existence of active opposite unidi ectional fluxes.
• 3.3. An important net secretory potassium flux was found in short-circuited aldosterone-stimulated colons.
• 4.4. Mucosal amiloride did not change (J^k_net) either in control or aldosterone-stimulated colons.
• 5.5. Luminal barium alters K ⁺ transport in a manner consistent with the presence of barium-sensitive conductances at the apical membrane of both control and aldosterone-treated colons.
• 6.6. The effects of ouabain and barium on control and aldosterone-induced potassium flows were consistent with a model involving basolateral uptake by an Na ⁺-K ⁺-ATPase and conductive exit across the apical membrane.
• 7.7. The stimulatory effect of aldosterone on potassium secretion is associated with parallel increases of both basolateral K ⁺ entry and the apical conductive pathway.

     

Isolation and characterization of glutamine synthetase from the diazotroph Azospirillum brasilense

• 1.1. Glutamine synthetase was purified from the diazotroph Azospirillum brasilense.
• 2.2. The holoenzyme with a M_r of 630,000 is composed of 12 subunits of M_r 52,000.
• 3.3. A modified subunit of M_r 53,000 was also found by electrophoresis under denaturing conditions.
• 4.4. It is shown that the M_r 53,000 species is the adenylylated subunit.
• 5.5. The apparent K_m values for glutamate, ATP and ammonia were 2.5 ± 0.3 mM, 200 ± 20 μM and42 ± 2 μM, respectively.
• 6.6. Levels of glutamine synthetase activity in A. brasilense cells varied by a factor of 8 depending on the nitrogen source and its concentration in the growth medium.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

Conversion of a NADPH-dependent aldehyde reducing enzyme into aldose reductase

• 1.1. Aldose reductase, aldehyde reductase and high-K_m, aldose reductase were purified from the inner medulla of dog kidney.
• 2.2. Compared with aldose reductase, high-K_m aldose reductase had a lower isoelectric point, a lower activity for aldo-sugars and a lower sensitivity for aldose reductase inhibitors, and it was not activated by sulfate ions. Both reductases had the same molecular weight (38,500) and immunochemical properties.
• 3.3. High-K_m aldose reductase was easily converted into an aldose reductase-like enzyme, namely a generated reductase upon incubation in neutral buffer solution.
• 4.4. The generated reductase was identical with aldose reductase with respect to the isoelectric point, substrate specificity, activation by sulfate ions and IC₅₀ values for aldose reductase inhibitors. The generated reductase revealed immunochemical identity with aldose reductase as well as high-K_m aldose reductase.

     

Gastric acid secretion in the chicken: Effect of histamine H2 antagonists and H+/K+-ATPase inhibitors on gastro-intestinal pH and of sexual maturity calcium carbonate level and particle size on proventricular H+/K+ ATPase activity

• 1.1. Cimetidine was more potent 4hr after a single injection of 25 or lOOmg/kg body wt in increasing gastric pH than other H₂ receptor antagonists, ranitidine and famotidine but was less efficient than H⁺/K⁺-ATPase inhibitors. Omeprazole rose proventricular and gizzard pH at a lower dose than SCH 28080 and Ro 18-5364 (30, 50 and 200 mg/kg body wt, respectively).
• 2.2. Proventricular and gizzard pH values were maximal 1 and 4 hr after a single injection of 7.5 μmol/kg body wt omeprazole. Inhibition of acid secretion was maintained for 24 hr after an injection of 100 μmol/kg.
• 3.3. H⁺/K⁺-ATPase activity in vitro was 10μimol P_i/hr/mg protein in the microsomal fractions of the proventriculus. It was doubled by nigericine and inhibited by SCH 28080. However, western blots by high specific H⁺/K⁺-ATPase monoclonal antibody 95-A3 and 95–111 recognized a 42kDa band but hardly exhibited the specific 95 kDa band recognition.
• 4.4. Chickens and immature pullets showed a higher H⁺/K⁺ -ATPase activity than laying hens. Calcium level of the diet did not affect the enzyme activity but coarse particles of calcium fed to pullets or laying hens enhanced the H⁺/K⁺-ATPase activity when compared with ground particles.

     

Multiplicity of dog kidney high-Km aldose reductase and conversion mechanism into aldose reductase

• 1.1. High-K_m, aldose reductase purified from dog kidney inner medulla was easily converted into aldose reductase by incubation in the neutral buffer solution.
• 2.2. High-K_m, aldose reductase was found to be in multiple forms, and was separated into three kinds of species designated as a-, b- and c-forms by HPLC.
• 3.3. The a-form observed as a single peak by HPLC was assumed to be present in three forms (al-, a2- and a3-forms), one was aldose reductase (a 1-form) and the others were the precursors of aldose reductase (a2- and a3-form).
• 4.4. The b-form was rapidly converted into the a3-form, followed slowly by the a2-form and finally into the a 1-form.
• 5.5. The c-form was either directly converted into the al-form, or indirectly into the a2-form followed by the al-form.
• 6.6. Four kinds of species (a2-, a3-, b- and c-forms) of high-Ap, aldose reductase were finally converted into aldose reductase (al-form).