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.

     

Antioxidant activity of neuston and plankton extracts from vostok bay,sea of Japan

• 1.1. Antioxidant activity of chloroform-methanol extracts of biomass of the oceanic surface film (neuston) is ascertained.
• 2.2. Antioxidant activity of extracts is due mainly to zooforms and directly depends on their quantitative content in the total neuston composition.
• 3.3. Antioxidant activity of neuston extracts is due mainly to specific individual phospholipids.

     

Ribosome inactivating protein-like activity in seeds of diverse Cucurbitaceae plants

• 1.1. Seed extracts of 20 plants species belonging to the family Cucurbitaceae were examined for their ability to inhibit protein synthesis in rabbit reticulocyte lysate and induce mid-term abortion in mice.
• 2.2. Eleven extracts were found to inhibit protein synthesis by about or over 90%, seven extracts produced about 80% inhibition, one caused about 70% inhibition and one brought about approx. 40% inhibition, when the extracts were tested at a final concentration of 10 μg per ml.
• 3.3. All of the seed extracts possessed potent mid-term abortifacient activity.
• 4.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the seed extracts disclosed the existence of a Coomassie Blue-stainable band with a mol. wt of ca 30,000 Da. This band probably accounts for the protein synthesis inhibiting and mid-term abortifacient activities.
• 5.5. There was a similarity in the electrophoretograms of seed extracts of plants belonging to the same genus.

     

Prostaglandin E2/parathyroid hormone-induced suppression of alkaline phosphatase activity is mediated by protein kinase C

• 1.1. Bone resorptive factors, prostaglandin E2 and parathyroid hormone are shown to suppress alkaline phosphatase activity in a rat osteoblastic cell line.
• 2.2. Phorbol myristate acetate, but not dibutyryl cAMP or calcium ionophore can suppress alkaline phosphatase activity.
• 3.3. The protein kinase C inhibitors (H89, staurosporine) are able to block the suppression of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone.
• 4.4. These data suggest that protein kinase C is involved in the inhibition of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone.

     

Ca2+-dependent stimulation of muscle and liver phosphorylase kinase by heparin

• 1.1. Heparin stimulates the activity of nonactivated and activated skeletal muscle phosphorylase kinase in a Ca²⁺-dependent manner.
• 2.2. The stimulatory effect of heparin on the activity of nonactivated phosphorylase kinase is also expressed in the presence of calmodulin and glycogen. Heparin acted in synergism with glycogen.
• 3.3. Heparin increases the affinity of phosphorylase kinase to Ca²⁺ 5–12 fold depending upon the activation conditions.
• 4.4. Ca²⁺ influences the stimulation of liver phosphorylase kinase by heparin in a similar way.

     

The role of Zn2+ in pyridoxal phosphate mediated regulation of glutamic acid decarboxylase in brain

• 1.1. γ-Aminobutyric acid, a major inhibitory neurotransmitter in the CNS, is synthesized by glutamic acid decarboxylase which demonstrates an absolute requirement for pyridoxal phosphate.
• 2.2. At physiological concentrations, zinc stimulates the activity of pyridoxal kinase, enhancing the formation of pyridoxal phosphate, which in turn stimulates the activity of glutamic acid decarboxylase.
• 3.3. At pharmacological concentrations, zinc inhibits the activity of glutamic acid decarboxylase without inhibiting pyridoxal kinase.
• 4.4. These results suggest that zinc may play a role in pyridoxal phosphate-mediated regulation of glutamic acid decarboxylase.

     

Creatine kinase from the electric organ of Electrophorus electricus (L.)—isozyme analysis

• 1.1. Creatine kinase (CPK) isozymes of extracts from the electric organ, dorsal muscle and brain of Electrophorus electricus (L.) were analysed with Cellogel electrophoresis. A single component corresponding to the MB-form was obtained for both electric organ and the dorsal muscle. The BB-form was present in the brain extract.
• 2.2. Upon acetone fractionation of the aqueous extract of electric organ, the final fraction was submitted to gel filtration and presented a single peak of CPK activity.
• 3.3. Characterization of this fraction by thin-layer gel filtration indicated an apparent molecular weight of 80,000 which corresponds to the enzyme dimeric structure.
• 4.4. The implications of this finding with the muscular origin of the electric organ are discussed.

     

Properties of enzymes hydrating epoxides in human epidermis and liver

• 1.1. Cytosolic and microsomal epoxide hydrolyzing enzymes of human skin and liver were compared and found to be different.
• 2.2. Epidermal and hepatic cytosolic epoxide hydrolases were different in terms of substrate selectivity, pI, inhibitor sensitivity and affinity Chromatographic properties.
• 3.3. Microsomal epoxide hydrolases had the same pIs but different substrate selectivities.
• 4.4. Cytosolic epoxide hydrolase from adults had higher specific activity than that from neonates or cultured epidermis, but lower activity than adult hepatic enzymes.
• 5.5. The sizes of cytosolic epoxide hydrolase from epidermis and liver were similar and lower than that from cultured fibroblasts.
• 6.6. Cytosolic epoxide hydrolase from all sources shared similar antigenic determinants.

     

Activation of neutrophils NADPH oxidase by PMA: Cytosol activity is translocated in phorbol-primed neutrophils

• 1.1. Translocation of cytosol activity in phorbol-primed neutrophils was studied.
• 2.2. Prior exposure of PMA or FMLP could potentiate the oxidative response by subsequent heterogeneous stimulus, FMLP or PMA.
• 3.3. In FMLP-primed neutrophils, the cytosol had almost the same activity as resting one and cytosol activity was not eluted from the membrane.
• 4.4. In PMA-primed neutrophils, however, the cytosol had less activity and cytosol activity was correspondingly eluted from the membrane.
• 5.5. These observations suggested that cytosol activity was translocated in PMA-primed cells.

     

Purine metabolism in cotyledons and embryonic axes of black gram (Phaseolus mungo L.) Seedlings

• 1.1. The metabolism of purine bases and nucleosides in cotyledons and embryonic axes of black gram (Phaseolus mungo L.) was studied.
• 2.2. A large portion of absorbed [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]adenosine was salvaged in nucleotide and nucleic acid synthesis.
• 3.3. Most of the radioactivity of [8-¹⁴C]hypoxanthine and [8-¹⁴C]inosine was incorporated into allantoin and allantoic acid.
• 4.4. Activity of adenine phosphoribosyltransferase in enzyme extracts was much higher than that of hypoxanthme and guanine phosphoribosyltransferase(s).
• 5.5. Apparent activity of adenosine kinase was higher than that of inosine kinase. 6. NAD⁺-dependent xan thine dehydrogenase was detected in both cotyledons and embryonic axes of the seedlings.
• 6.7. The capacity of purine salvage was higher m 24 hr old cotyledons than 24 and 48 hr old embryonic axes. The reverse was observed concerning that of purine degradation.

     

Digestion of protein in different marine species

• 1.1. The digestion proteases in five marine species (Atlantic halibut, Hippoglossus hippoglossus (L); Dover sole, Solea solea (L); turbot, Scophthalmus maximus, (L); European lobster, Hommarus gammaarus (L); and the giant prawn, Penaeus monodon) have been compared by biochemical methods.
• 2.2. The pH profiles for the hydrolysis of casein by extracts from the digestive systems of each species showed different characteristics; extracts from adult halibut, turbot and sole exhibited strong pepsin-like activity; whereas this enzyme was absent in P. monodon and in sole larvae.
• 3.3. Although lobster extracts, from either the hepatopancreas or the stomach, showed peaks at pH values of 5.8 and 2.5, this latter activity did not hydrolyse a specific substrate for pepsin.
• 4.4. Halibut and turbot digestive extracts contained an activity optimal at pH values in the region of 5.0 resembling a cathepsin-like enzyme; an activity which was not evident in the other species under similar experimental conditions.
• 5.5. Although all species possessed trypsin-like activity, the pH profiles of activity in the neutral to alkaline region were unique to each species.
• 6.6. The significance of these results is considered with respect to the anatomical differences in the alimentary systems of these species.

     

Rat liver endoplasmic reticulum protein kinases

• 1.1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography.
• 2.2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca²⁺/calmodulin-dependent protein kinase and casein kinases.
• 3.3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis.
• 4.4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca²⁺ /calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).

     

Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts

• 1.1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri).
• 2.2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots.
• 3.3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions.
• 4.4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.

     

Effects of high dietary methionine on activities of selected enzymes in the liver of kittens (felis domesticus)

• 1.1. Growing male kittens were fed an 18% casein diet supplemented with 2, 3, or 4% l-methionine (MET) for 6 weeks.
• 2.2. Free MET concentration in liver increased 30-fold and cystathionine two- to three-fold; the activity of adenosyl-MET transferase and cystathionase also increased but remained lower than previously found in rats.
• 3.3. Taurine concentration in liver decreased in cats fed excess MET and appeared to depend on taurine intake.
• 4.4. Alanine aminotransferase activity was high in all groups while serine dehydratase activity was very low.
• 5.5. Pyruvate kinase and malic enzyme activities which are normally low in cat liver increased after excess MET. Also, glucose 6-phosphate and 6-phosphogluconate dehydrogenases increased.
• 6.6. Cat liver metabolism showed limited adaptation to an excess dietary intake of methionine compared to that found in rats.

     

Lactate dehydrogenase isozymes of the leopard danio,Brachydanio nigrofasciatus: their characterization and ontogeny

• 1.1. The tissue specific patterns and ontogeny of lactate dehydrogenase (LDH) are reported.
• 2.2. While all tissues (eye, brain, heart, intestine, liver, ovary and skeletal muscle) show isozymes of A and B subunit composition, only liver extracts possess isozymes resulting from C subunit synthesis.
• 3.3. The A₄ homopolymer appears simultaneously with initial muscle contractility and is correlated with the physiological function of muscular contraction.
• 4.4. The activation of the Ldh-C locus is correlated with the first functioning of liver. It is suggested that the state of differentiation of liver cells may be the stimulus required for C locus expression.

     

Amino acid substitutions within the analogous nucleotide binding loop (P-loop) of aminoglycoside 3'-phosphotransferase-II

• 1.1. Oligonucleotide-directed mutagenesis of APH(3')-II was used to investigate the functions of key amino acids in the P-loop analogous motif of the enzyme.
• 2.2. The mutations of Gly205 → Glu, Gly210 → Ala and Arg211 → Pro considerably reduced the resistance of the resulting strains to KM and to related drugs, e.g. G418.
• 3.3. Similarly, enzyme activity in the crude extracts of these mutants was substantially reduced as well as the enzyme's affinity for Mg²⁺ ATP.
• 4.4. Alternatively substitutions at a highly conserved basic residue (Arg211 → Lys and Arg211 → His) were not sufficient for the enzyme to sustain the activity at a level comparable to that of the wildtype.
• 5.5. Moreover, an Arg211 → His mutation drastically reduced affinity of the enzyme for Mg²⁺ ATP.
• 6.6. This argues the importance of Arg211 residue in contributing to the formation of the P-loop structure in addition to its involvement in phosphoryl transfer reaction.
• 7.7. Computer analysis of the secondary structure predicted that the APH(3')-II loop connects a β -strand to an α-helix and that the above mutations caused varying degrees of structural distortions at the corresponding regions of the protein.

     

Effect of inorganic phosphate on synthesis of 5-phosphoribosyl-1-pyrophosphate in cultured plant cells

• 1.1. Inorganic phosphate (Pi) was absorbed rapidly by suspension-cultured cells of Catharanthus roseus which had previously been cultured in Pi-free Murashige Skoog medium.
• 2.2. The intracellular levels of ATP, ADP and 5-phosphoribosyl-l-pyrophosphate (PRPP) increased markedly during the 24 hr which followed the addition of Pi (1.25mM).
• 3.3. Availability of PRPP in vivo, estimated by the measurement of nucleotide synthesis from [8-¹⁴C]adenine, was also increased by addition of Pi.
• 4.4. Only a 20% increase in the maximum catalytic activity of PRPP synthetase was observed in extracts of cells, prepared 24 hr after addition of Pi.
• 5.5. In contrast to results for mammalian PRPP synthetase, the activity of PRPP synthetase, partially purified from Catharanthus roseus, was inhibited by concentration of Pi greater than 5mM.
• 6.6. The mechanisms involved in the increased availability of PRPP and the synthesis of adenine nucleotides in the plant cells cultured in Pi-containing medium are discussed.

     

Proteolytic system in Babesia hylomysci

• 1.1. Babesia hylomysci has an aminopeptidase and an acid endoprotease
• 2.2. The amino-peptidase has properties very similar to the aminopeptidase in Plasmodium yoelii nigeriensis and P. chabaudi.
• 3.3. The acid endoprotease is specific towards haemoglobin and practically has no action on bovine serum albumin.
• 4.4. In mouse normal red blood cells we find an acid protease having physico-chemical properties similar to the enzyme present in B. hylomysci extracts.
• 5.5. The similarity of electrophoretic velocity between acid protease in B. hylomysci and non-infected red blood cells leads us to think that the acid protease of parasitic extracts comes from the host-cell.
• 6.6. The proteolytic system of Babesia and Plasmodium are similar.

     

Investigation of ribosome inactivating protein-like activity in tissues of cucurbitaceae plants

• 1.1. Extracts of roots, seeds and fruits of seventeen plant species belonging to Family Cucurbitaceae were examined for the ability to inhibit protein synthesis in rabbit reticulocyte lysate and induce mid-term abortion in mice.
• 2.2. Out of the 22 tissue extracts examined, 16 were found to inhibit protein synthesis by >90%, three caused 65–85% inhibition and 3 caused <25% inhibition.
• 3.3. In general, there was a close correlation between protein synthesis inhibiting activity and mid-term abortifacient activity of the tissue extracts.
• 4.4. SDS-PAGE of the tissue extracts revealed the presence of a Coomassie Blue-stainable band with a mol. wt of ca 30,000. The data suggest that this band is responsible for the protein synthesis inhibiting and mid-term abortifacient activities.