Purification and partial characterization of an AMP deaminase from the marine invertebrate Palaemon serratus

• 1.1. AMP deaminase from Palaemon serratus tail muscle was partially purified by chromatography on cellulose phosphate.
• 2.2. Muscle homogenates expressed very low enzyme activities and the presence of ATP was necessary to detect AMP deaminase. The specific activity and substrate affinity of the purified enzyme were also very low.
• 3.3. The purified prawn muscle AMP deaminase was contaminated by contractile proteins, one of the major contaminants being actin.
• 4.4. The enzyme displayed a very high affinity for actomyosin which was only partially abolished by pyrophosphate.

     

Regulatory properties of rabbit liver phosphorylase kinase

• 1.1. Purified native rabbit liver phosphorylase kinase becomes activated during the assay of its activity while low molecular weight forms of the same enzyme do not.
• 2.2. The activation requires ATP and maganesium ions, suggesting the phosphorylation of the enzyme by a protein kinase as the mechanism involved.
• 3.3. The activation of the enzyme can be reverted by the action of a type 1 protein phosphatase isolated from the same tissue.
• 4.4. The activation can also be catalyzed by the catalytic subunit of cAMP-dependent protein kinase in a process that requires a much lower ATP concentration to proceed.
• 5.5. The activation is believed to be due to an autocatalytic phosphorylation of phosphorylase kinase itself. In support of this hypothesis are the regulation of the process through calcium ions, the low levels of endogenous protein kinase detected in the purified preparation, the high ATP concentrations required in the absence of cAMP dependent protein kinase and the fact that the process cannot be blocked by an excess of the heat stable inhibitor specific for the later enzyme.
• 6.6. The low molecular weight forms of the enzyme on their side are not affected by the action of neither protein phosphatase 1 nor cyclic AMP dependent protein kinase.
• 7.7. Both activated and nonactivated phosphorylase kinase are partially dependent on calcium ions, the affinity of the former being higher than that of the latter. The low molecular forms do not require calcium ions to express their activity.

     

Partial purification and characterization of cysteine proteinase from metamorphosing tadpole tails of Rana catesbeiana

• 1.1. A leupeptin-sensitive proteinase was partially purified from regressing tadpole tails by acetone factionation and column chromatography on S-Sepharose.
• 2.2. The enzyme degraded hemoglobin and myoglobin at pH 3.0. The enzyme also hydrolyzed Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA at pH 4.0.
• 3.3. The enzyme activity was inhibited by leupeptin, egg cystatin, E-64 and monoiodoacetic acid and was activated by l-cysteine.
• 4.4. The enzyme degraded myosin and actin in myofibrils of tadpole tails.
• 5.5. The enzyme belongs to the cysteine proteinase and is possibly involved in tail degradation during the metamorphosis of tadpoles.

     

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.

     

Human seminal phosphatase: Properties and comparison with plant phosphatases

• 1.1. Phosphatase activities were determined in various seminal fluid and plant tissues.
• 2.2. Human semen showed a markedly higher phosphatase activity, and some plants and mushrooms exhibited a considerably higher phosphatase activity.
• 3.3. Phosphatase purified from human seminal fluid showed a pH optimum of 5–6 and was potently inhibited by l-(+)-tartrate.
• 4.4. Tartrate could not inhibit most plant phosphatases, but inhibited the mushroom phosphatase with a one-order lower affinity than that of the seminal enzyme.

     

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.

     

Hen's egg yolk alkaline phosphatase: General characterization and kinetic stuy with inhibitors

• 1.1. The non-specific hen's egg yolk alkaline phosphatase is a metalloprotein (Zn²⁺?) composed of two identical inactive subunits.
• 2.2. A second metal site preferably binds Mg²⁺ (15-fold activation). Me(II))H₂O)H⁺, a charged arginine, and tyrosine in the active site are involved in positioning and binding of the substrate and metal ion.
• 3.3. Substrate inhibition differs with pH. This may be related to the presence of two active sites in the enzyme, one in each subunit.
• 4.4. Uncompetitive inhibition with L-phenylalanine and analogues suggests a phosphorylated intermediate.
• 5.5. Inhibition is weakly competitive with P_i strong non-competitive with PPi as compared to Mg²⁺-free PP_i, and partially competitive with arsenate.
• 6.6. The purified enzyme is stabilized and activated by amines and proteins.

     

Purification and some properties of NAD+-dependent glutamate dehydrogenase from Halobacterium halobium

• 1.1. A NAD⁺-dependent glutamate dehydrogenase (EC 1.4.1.2.) was purified 126-fold from Halobacterium halobium.
• 2.2. Activity and stability of the enzyme were affected by salt concentration. Maximum activity of the NADH-dependent reductive amination of 2-oxoglutarate occurs at 3.2 M NaCl and 0.8 M KCl, and the NAD⁺-dependent oxidative deamination of l-glutamate occurs at 0.9 M NaCl and 0.4 M KCl. The maximum activity is higher with Na⁺ than with K⁺ in the amination reaction while the reverse is true in the deamination reaction.
• 3.3. The apparent K_m values of the various substrates and coenzymes under optimal conditions were: 2-oxoglutarate, 20.2 mM; ammonium, 0.45 M; NADH, 0.07 mM; l-glutamate, 4.0 mM; NAD⁺, 0.30 mM.
• 4.4. No effect of ADP or GTP on the enzyme activity was found. The purified enzyme was activated by some l-amino acids.

     

A comparative study of nuclear and chloroplast DNA dependent RNA polymerases from wheat leaves

• 1.1. Three DNA dependent RNA polymerases have been purified from chromatin and chloroplast fractions of wheat leaves.
• 2.2. The purified enzymes were completely dependent on exogenous DNA after purification by glycerol gradient, DEAE-Sephadex and phosphocellulose chromatography.
• 3.3. The nuclear enzymes, I and II, showed a strong preference for denatured nuclear DNA, whereas the chloroplast enzyme preferred denatured chloroplast DNA.
• 4.4. The three enzymes require either Mg²⁺ or Mn²⁺ for activity.
• 5.5. α-amanitin specifically inhibited RNA polymerase II but has no effect on polymerase I and chloroplast polymerase.
• 6.6. Enzyme I is most active at very low ionic strength (0.10 mM KC1), whereas enzyme II and chloroplast enzyme show maximum activity at 150mM and 50 mM KC1 respectively.

     

Some kinetic properties of pyruvate kinase from Phycomyces blakesleeanus

• 1.1. Pyruvate kinase from mycelium of Phycomyces blakesleeanus NRRL 1555(−) has been partially purified and some kinetic properties has been investigated at pH 7.5.
• 2.2. Positive homotropic interactions were observed with phosphoenolpyruvate and Mg²⁺, showing Hill coefficient values of 2.8 and 2.5, respectively, whereas hyperbolic kinetics are found when ADP was the variable substrate.
• 3.3. Fructose 1,6-bisphosphate acts as a heterotropic allosteric activator, markedly decreasing the S_0.5 value for phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form.
• 4.4. ATP inhibits pyruvate kinase from mycelium of Phycomyces blakesleeanus. ATP appears to be a non-competitive inhibitor with respect PEP and competitive inhibitor with respect ADP.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

Age-related changes in total dna and rna and incorporation of uridine and thymidine in rat liver,kidney and spleen

• 1.1. Total content of DNA and RNA in liver, kidney and spleen were measured in young and aged rats. At the same time the incorporation of [¹⁴C]thymidine, a DNA precursor, and [³H]uridine, an RNA precursor, were also determined.
• 2.2. Changes in total organ DNA and RNA correlated with sexual maturation as did incorporation of precursors.
• 3.3. Young animals have more DNA per organ relative to RNA. with kidney and spleen DNA showing a decrease between maturity and senescence.
• 4.4. However, liver RNA increases with age. a change probably due to decreased catabolism of RNA since [³H]uridine uptake decreases.
• 5.5. Liver polyploid differentiation, and [¹⁴C]thymidine and [³H]uridine uptake, are correlated.
• 6.6. In kidney, incorporation of [³H]uridine is inversely related to [¹⁴C]thymidine incorporation.

     

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.

     

Partial purification of rat liver cytoplasmic acetyl-CoA synthetase; Characterization of some properties

• 1.1. Rat liver cytoplasmic acetyl-CoA synthetase was partially purified (purification factor = 23, yield = 30%).
• 2.2. The apparent K_ms for acetate, coenzyme A, ATP and MgCl₂ were determined and found to be 52.5 μM, 50.5 μM, 570 μM and 1.5 mM, respectively.
• 3.3. The partially-purified enzyme showed a low affinity for short-chain carbon substrates other than acetate.
• 4.4. The properties of the partially-purified enzyme were compared with those of enzymes from other sources.

     

Isolation and partial characterization of uric acid spherules obtained from cockroach tissues (Dictyoptera)

• 1.1. Urate spherules obtained from three cockroach species have been isolated, purified and examined by scanning electron microscopy, atomic absorption, i.r. and Kjeldahl analysis.
• 2.2. Purified spherules exposed to ionic environments may change from a smooth to a textured form.
• 3.3. Ion analysis confirmed that the spherules are high in K⁺ and low in Na⁺.
• 4.4. All preparations from the cockroach tissues diaplayed similar i.r. spectra, but were dissimilar to those of uric acid and urate salt standards.
• 5.5. It appears that there is a distinct pattern or mechanism by which the cockroach urates are deposited in the spherule matrix.

     

The precipitation of human IgG and its subunits with heparin

• 1.1. About 40% of IgG is precipitated when heparin is added to human plasma at pH 5.4, while only a little IgM and no IgA are precipitated.
• 2.2. The precipitation of purified IgG by heparin is pH-dependent and follows a sigmoid curve between pH 7.0 and 5.0.
• 3.3. The precipitate has a constant molar ratio heparin: IgG, independent of pH or the amount of heparin that is added.
• 4.4. The precipitate does not redissolve at high heparin concentrations.
• 5.5. The heavy chains of IgG precipitate also at pH 5.4, but this precipitate redissolves in excess heparin.
• 6.6. Light chains do not precipitate and the F_ab and F_c fragments are only partly precipitated.

     

On the comparative turnover rates of the lactate dehydrogenase isozymes in rat tissues

• 1.1. An affinity chromatography technique for the determination of turnover parameters has been utilized for the derivation of new data for lactate dehydrogenase and its isozymes in a wide variety of tissues from female rats.
• 2.2. This methodology allowed the establishment of relative turnover characteristics in all tissues examined, and the evaluation of rate constants for synthesis and degradation and half-life values in the majority of these enzyme sources.
• 3.3. Considerable variation was evident in the turnover characteristics of total protein and lactate dehydrogenase in the different tissues, with the synthesis of the enzyme being highest in reproductive tissues, heart and liver, and degradation most prominent in reproductive tissues.
• 4.4. With regard to the isozymes, marked differences in half-lives were evident not only between the same isozyme in different tissues, but also between the various isozymes in any one tissue.
• 5.5. Bi-directional trends in the half-life values for the isozyme sequences in several tissues, indicated a distinctive contribution to turnover from the different cell types present.
• 6.6. These data have been discussed in relation to the available comparisons in the literature, and the known physiological correlations of lactate dehydrogenase in these tissues.

     

Anabolic and catabolic pathways of pyrimidine metabolism in rat liver

• 1.1. Use of pyrimidine nucleotide precursors labelled in various positions on the ring shows that in rat liver, pyrimidine nucleotides formed from orotate follow anabolic pathways almost exclusively, whereas trace quantities of uridine are mostly degraded to β-alanine and its metabolites.
• 2.2. Annomalies in the ratios of [¹⁴C] and [³H] in various common nucleotide products of orotate and uridine can be accounted for on the basis of metabolic compartmentation and recycling of CO₂.

     

Intracellular localization and characterization of 3-hydroxykynureninase in human liver

• 1.1. 3-hydroxykynureninase in human liver was present in cytosol and mitoehondria.
• 2.2. The cytosolic enzyme and mitochondrial enzyme had the same physiological and enzymic properties.
• 3.3. The enzyme had a mol. wt of 130,000 by gel filtration and isoelectric point of pH 5.9.
• 4.4. The enzyme was active for 3-hydroxykynurenine and kynurenine, and its activity ratio was 15:1. The apparent K_m values of the enzyme were 7.7 × 10⁻⁵M for 3-hydroxykynurenine, 1.0×10⁻³M for kynurenine and 2.5 × 10⁻⁶M for pyridoxal 5'-phosphate with 3-hydroxykynurenine.
• 5.5. Some other properties of purified enzymes are described.

     

Human spleen cathepsin D: Its characterization and localization in human spleen

• 1.1. The cathepsin D was purified 1830-fold under mild conditions by a rapid procedure, based on two-step affinity chromatography.
• 2.2. Its molecular weight, amino acid composition and substrate specificity were shown to display minor differences from materials of other origins.
• 3.3. Inhibition with thiol compounds was found to be a specific phenomenon of the cathepsin D from the human spleen.
• 4.4. Production of antiserum specific for purified cathepsin D was demonstrated by immunodiffusion test, an immunoadsorbent column and immunoblotting of the crude enzyme in SDS gel.
• 5.5. In an immunocytochemical study, the antigenic sites for this enzyme were found to be localized in the reticuloendothelial system of the human spleen.
• 6.6. The role of this enzyme in human spleen cell was discussed.