Soluble cyclic amp-dependent protein kinases from chick liver: Effects of NaCl and heat-stable modulator

• 1.1. Two cyclic AMP-dependent protein kinases—Fraction I and II—have been isolated from chick liver soluble preparation on DEAE-cellulose.
• 2.2. Both fractions have an apparent K_m for ATP of 2 × 10⁻⁶M, are stimulated maximally by 5 × 10⁻⁸ M cyclic AMP and phosphorylate mainly basic proteins—histone and protamine.
• 3.3. They exhibit various pH values for optimal activity and show differences with respect to both sensitivity to NaCl and substrate specificity.
• 4.4. The heat-stable protein modulator inhibits the cyclic AMP-dependent protein kinase activity of both fractions, but with cyclic GMP one kinase is stimulated and the other inhibited.
• 5.5. Slight differences in histone triggered holoenzyme dissociation as well as the lack of difference between their ability for subunit reassociation do not allow to classify these isozymes as protein kinases of Type I and II, according to Corbin et al. (1975).

     

Binding proteins for cyclic AMP in mammalian tissues

• 1.1. The distribution and physicochemical properties of proteins known to bind cyclic AMP in vitro and methodological aspects of their interaction with ligands is reviewed.
• 2.2. The interaction between such proteins and cyclic AMP is discussed, the allosteric binding of the nucleotide to cyclic AMP dependent protein kinase type I being considered in detail.
• 3.3. The use of naturally occurring binding proteins in assays for cyclic AMP is briefly reviewed.
• 4.4. Finally, some aspects of the control of cyclic AMP binding in the intact cell are considered.

     

Identification and characterization of a third form (D3) of cyclic 3'5'-nucleotide phosphodiesterase from rhizobwm fredii

• 1.1. A third form (D3) of cyclic nucleotide phosphodiesterase from Rhizobiumfrediiv/as detected and characterized for the first time.
• 2.2. The enzyme could hydrolyse both cyclic AMP and cyclic GMP with apparent K_m for cyclic AMP of approx. 0.2 μM.
• 3.3. D3 cyclic nucleotide phosphodiesterase had a pH optimum of about 6.0 when hydrolysing cyclic AMP.
• 4.4. The enzyme lost almost all its activity when heated to 60°C for 20 min.
• 5.5. Gel filtration with Sephadex G-100 gave a mol. wt of approx. 42.5 kD for the native enzyme.

     

A simple and reliable method for the purification of Pseudomonas aeruginosa phospholipase C produced in a high phosphate medium containing choline

• 1.1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-P_i basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite.
• 2.2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate.
• 3.3. The peak containing the PLC activity revealed a single protein after SDS-PAGE.
• 4.4. The method could also be applied to purify PLC produced in a low-P_i complex medium. The resultant preparation was not homogeneous.
• 5.5. The molecular weight for both PLC preparations was about 70 kDa.
• 6.6. Both PLC used phosphatydilcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent K_M of 25 mM for p-nitrophenylphosphorylcholine.
• 7.7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100.
• 8.8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HP_l-BSM plus choline but not the enzyme from the LP_l-CM.

     

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.

     

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).

     

Purification and characterization of haemagglutinin in the haemolymph of the silkworm,Bombyx mori

• 1.1. Changes in haemagglutinating activity in haemolymph during 4th and 5th larval instars of Bombyx mori were stage specific. The activity increased in time concomitant with an increase in the secretory activity of prothoracic glands.
• 2.2. The protein with haemagglutinating activity was purified by ammonium sulfate fractionation, gel-filtration on Sephacryl S-300 and affinity chromatography using either glucuronic acid or galacturonic acid as a ligand.
• 3.3. Western blotting analysis using antibody raised against this protein revealed that Bombyx haemagglutinin is a tetramer composed of two different subunits with mol. wts of ca 88,000 and 90,000.

     

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.

     

Phosphofructokinase from the anterior byssus retractor muscle of Mytilvs edulis: Modification of the enzyme in anoxia and by endogenous protein kinases

• 1.1. Anoxia exposure resulted in a stable modification of the kinetic properties of 6-phosphofructo-1-kinase (PFK) from the anterior byssus retractor muscle (ABRM) of the sea mussel Mytilus edulis L.
• 2.2. Compared to the aerobic enzyme, the anoxic form of PFK. showed a reduced affinity for both substrates, fructose-6-phosphate (F6P) and ATP, and an increased sensitivity to inhibition by phosphoenolpyruvate.
• 3.3. To analyze the involvement of protein kinases in the modification of PFK, extracts from aerobic or anoxic muscle were incubated with ATP and Mg²⁺ plus protein kinase second messengers cyclic 3',5'-adenosine monophosphate (cAMP), cyclic 3',5'-guanosine monophosphate (cGMP) or Ca²⁺ plus phorbol 12-myristate 13-acetate (PMA).
• 4.4. Both forms of the enzyme responded to the presence of cAMP with a strong increase in affinity for F6P.
• 5.5. In response to cGMP affinity of the aerobic enzyme for F6P decreased whereas that of the anoxic enzyme form was not affected (at 0.5 mM ATP) or increased (at 3 mM ATP).
• 6.6. Incubation with Ca²⁺ + PMA had only a limited effect on PFK kinetics but appeared to enhance the response to cGMP when the three compounds were given together.
• 7.7. Treatment of PFK-aerobic with alkaline phosphatase resulted in a strong decrease in enzyme activity and affinity for F6P; subsequent treatment with cAMP reversed the effect on S_0.5 F6P.
• 8.8. The data indicate that PFK activity is altered during the aerobic-anaerobic transition by a change in the phosphorylation state of the enzyme and that cAMP and cGMP act oppositely to regulate PFK activity, and thereby alter glycolytic rate, during this transition.

     

Sex-specific androgen binding in spotted hyaena (Crocuta crocuta) plasma

• 1.1. A charcoal adsorption assay demonstrated a large variance in androgen binding ability in female spotted hyaenas.
• 2.2. A positive correlation between plasma androgen binding ability and ovarian steroid concentrations was demonstrated in adult females.
• 3.3. The strong plasma binding affinity for testosterone and dihydrotestosterone (DHT) (nM) together with the lack of cortisol and weaker oestradiol-17β binding suggests that a specific androgen binding substance, possibly a protein, is present in adult females of this species.
• 4.4. The lack of high affinity binding in male spotted hyaenas is unusual and deserves further investigation.
• 5.5. Some androgen binding in all, including males and immature animals suggests that albumin may bind some plasma androgens in this species.

     

Oxidation of methionine residues in equine growth hormone by chloramine-T

• 1.1. Reactivity of methionine residues towards Chloramine-T was studied in the equine growth hormone.
• 2.2. With a 20.0-fold molar excess of reagent over methionine, full oxidation of the four residues of the protein is achieved.
• 3.3. Methionine 4 is the most reactive group, followed by methionines 72 and 178—methionine 123 being the less reactive residue.
• 4.4. As judged by circular dichroism spectra and binding assays, protein conformation and binding capacity to specific receptors remains unchanged even after full oxidation of all four methionine residues.
• 5.5. Results agree with data previously obtained with bovine growth hormone.

     

The measurement of glutamate dehydrogenase activity in Praunus flexuosus and its role in the regulation of ammonium excretion

• 1.1. A strong correlation (r² = 0.916) was found between glutamate dehydrogenase (GDH) activity and ammonium excretion in Praunus flexuosus suggesting a key regulatory role for this enzyme.
• 2.2. The high level of GDH activity found in this mysid was sufficient to account for all the ammonium excreted.
• 3.3. Activator-inhibitor studies imply that GDH may regulate energy production and growth, in addition to ammonium excretion.
• 4.4. The GDH assay presented here appears useful as a technique for quantifying zooplankton ammonium excretion impacts in the world oceans.

     

The binding curves in relation to protein concentration. The interaction of human serum albumin with phthalein dyes and azo-dyes

• 1.1. The binding curves of three phthalein dyes and two azo-dyes were measured in a concentration range of human serum albumin from 0.1 to 10%.
• 2.2. Contrary to other authors' findings, the courses of the binding curves appeared independent of protein concentration in all cases.
• 3.3. This discrepancy may be due to precision of the experimental techniques.

     

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.

     

A Platform for Extracellular Interactome Discovery Identifies Novel Functional Binding Partners for the Immune Receptors B7-H3/CD276 and PVR/CD155

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Highlights

• •New platform for high throughput detection of transient interactions between membrane proteins.
• •IL20RA is a receptor for the orphan checkpoint inhibitor B7-H3.
• •The natural killer cell protein KIR2DL5A binds the immune receptor PVR.
• •KIR2DL5 binding to PVR regulates natural killer cell cytotoxicity and inhibits tumor cell killing.
• •Elucidation of receptor interactomes to gain insights into extracellular protein biology.

     

δ-Aminolevulinic acid dehydratase inactivation by uroporphyrin I in light and darkness

• 1.1. The action of uroporphyrin I on erythrocytic ALA-D activity under dark and light conditions was examined.
• 2.2. Photo and non-photoinactivation of ALA-D induced by uroporphyrin I were observed.
• 3.3. Both effects were dependent on uroporphyrin concentration, temperature and time of exposure of the protein to the porphyrin.
• 4.4. Light-dependent effect of uroporphyrin I is related with the phototoxicity of porphyrins and could be produced by primary amino acid photooxidation followed by secondary cross-linking of the protein.
• 5.5. Light-dependent effect of uroporphyrin I could be ascribed to a direct enzyme inhibition due to binding of the porphyrin to the protein inducing structural changes at or near its active site.

     

Chasing Tails: Cathepsin-L Improves Structural Analysis of Histones by HX-MS,

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Highlights

• •Cathepsin-L is introduced as a novel protease for HX-MS studies.
• •Cathepsin-L improves resolution of traditionally challenging histone tails.
• •Cathepsin-L can be readily combined with pepsin for improved protein coverage.
• •In-solution dynamics of the H3.1 and H4 monomers reveal extensive EX1 kinetics.

     

Identification of a glycoprotein complex containing a glycogen synthase isozyme in Ascaris Suum obliquely-striated muscle

• 1.1. A glycogen/protein complex which contains the major portion of glycogen synthase activity in Ascaris suum muscle has been purified.
• 2.2. The complex contains two proteins which can be dissociated from a glycoprotein component.
• 3.3. The glycoprotein contains glycogen-like domains and is resistant to trypsin digestion.
• 4.4. The glycogen synthase activity in the purified complex catalyzes glycogen synthesis in the absence of exogenous glycogen, but demonstrates an absolute glucose 6-phosphate requirement for activity.
• 5.5. The data support the hypothesis that this isozyme of glycogen synthase is significantly different from the cyclic AMP-regulated enzyme.

     

Respiratory function and nucleotide composition of erythrocytes from tropical elasmobranchs

• 1.1. Oxygen carrying capacity and parameters of erythrocyte-oxygen binding are similar for a range of elasmobranchs with markedly different swimming behaviour.
• 2.2. Erythrocyte nucleotide components in sharks include the allosteric hemoglobin modifiers GTP and ATP in similar ratios, and the total pool appears independent of locomotory activity. A rhinobatoid ray had no detectable erythrocyte trinucleotides, but had an appreciable pool of AMP together with IMP.
• 3.3. There was no evidence for either urea or NaCl modulation of hemoglobin function in erythrocytes from a carcharhinid shark.
• 4.4. These observations lead to the conclusion that parameters of the oxygen transport system in elasmobranchs are highly conserved.

     

Identification and characterization of vitellin in a hermophrodite shrimp,Pandalus kessleri

• 1.1. A female specific protein (FSP, vitellogenin) in hemolymph and its related ovarian protein (vitellin) of Pandalus kessleri were studied by means of electrophoretical and immunological procedures.
• 2.2. The vitellin was purified from vitellogenic ovaries using hydroxylapatite, DEAE cellulose and Sepharose 6B columns, consecutively.
• 3.3. The vitellin had a molecular weight of approximately 560 kD and was composed of two subunits, 81 and 110 kD, respectively.
• 4.4. The vitellogenin concentrations in the hemolymph increased as vitellogenesis in the ovarian oocytes advanced and dropped markedly after the release of mature eggs.