Diversity of native myosin and myosin heavy chain in fish skeletal muscles

• 1.1. Polymorphism of native myosin and myosin heavy chain (MHC) of fish skeletal muscles was analysed by pyrophosphate and SDS-gel electrophoreses.
• 2.2. Depending on the species, three or four myosin isoforms were detected in the white muscle, one or two isoforms in the pure red muscle, and four isomyosins were found in the red muscle composed of red and pink (intermediate) fibres.
• 3.3. It is suggested that all main types of fish muscle fibre (red, intermediate and white) differ in myosin isoform content.
• 4.4. Myosin heavy chain of the red muscle is a distinct protein from that of the white muscle. However, structural differences between these proteins vary among species.

     

Thermal stability of fish myosin

• 1.1. Thermal stability of fish myosin has been studied by using differential scanning calorimetry (DSC) and circular dichroism (CD).
• 2.2. The temperature range of the sharp decrease in α-helical content agreed very closely with that of the endothermic peaks.
• 3.3. There was a high correlation between the enthalpy of denaturation (ΔH) and the decreasing quantity in α-helicity (Δh).
• 4.4. The structure of fish myosins was much more unstable than that of rabbit.
• 5.5. The instability of fish myosins was reflected in its rod moiety.

     

Electrophoretic comparison of myosin light chains and parvalbumins of trunk and head muscles from two barbel (Barbus barbus) populations

• 1.1. Myosin light chains and parvalbumins have been compared in several trunk and head muscles from small and large size barbels (Barbus barbus) living in river or reared in hatchery.
• 2.2. These proteins isolated from white, red and ventricle fibres exhibit identical electrophoretic characteristics in the four batches.
• 3.3. The slow fibre content and the parvalbumin distribution are generally similar in river and hatchery barbels of the same size but differ between small and large barbels within a population.
• 4.4. Alterations of the mode of fertilization and breeding conditions do not modify the differentiation of myosin and parvalbumins in barbels.

     

The enzymatic properties of smooth muscle myosin B from dog colon

• 1.1. Smooth myosin B and myosin A were prepared from dog colon and their enzymatic properties were studied.
• 2.2. Colonic myosin B with two light chain corresponding to L₂ and L₃ in skeletal myosin showed much lower ATPase activities than rabbit skeletal myosin B.
• 3.3. The Mg²⁺-ATPase of myosin B was activated at high magnesium concentrations with the maximum activation between 10⁻³ and 10⁻²M and showed only a slight dependence on KCl concentration. On the other hand, Mg²⁺-ATPase activity of myosin A decreased with decreasing KCI concentration, suggesting the activation by actin of colonic myosin ATPase as much as skeletal myosin ATPase.
• 4.4. The pH dependence of Ca²⁺-ATPase showed a U-shaped curve although above pH 8.5 the activity was suppressed rapidly. The activity-ionic strength curve indicated that Ca²⁺- and ethylenediamine-tetraacetic acid (EDTA)-ATPase activities increased with increasing KCI concentration.
• 5.5. Mg²⁺-ATPase was fairly stable to urea treatment, whereas EDTA- and Ca²⁺-ATPase were activated by a low concentration of urea, followed by an inhibition.
• 6.6. These results were discussed as compared with those of skeletal myosin B.

     

Complement-like protein from the phylogenetically primitive vertebrate,Eptatretus Stouti,is a Humoral Opsonin

• 1.1. Serum from the Pacific hagfish,Eptatretus stouti,contains a complement-like protein (CLP).
• 2.2. CLP from unfractionated hagfish serum and from affinity-purified preparations binds to yeast cell surfaces.
• 3.3. Incubation with CLP enhances the phagocytosis of yeast by hagfish leukocytes.
• 4.4. CLP-mediated opsonization can be inhibited by anti-CLP antibody, EDTA, d(+)mannose and l(+)rhamnose.
• 5.5. Additional opsomic factors are also in hagfish serum.

     

Changes in myosin heavy chain isoform expression of overloaded rat skeletal muscles

• 1.1. The effect of functional overload produced by tenotomy of synergistic gastrocnemius muscle on the expression of myosin heavy chain (MHC) isoforms in the plantaris and soleus muscles of the rat was studied using gradient sodium dodecyl sulfate-acrylamide gel electrophoresis.
• 2.2. Five weeks tenotomy, the plantaris and soleus muscle weights induced by tenotomy of the gastrocnemius muscle were 44.3% (P < 0.005) and 37.4% (P < 0.005), respectively, heavier than the contralateral control muscles.
• 3.3. Although four types of MHC isoforms were observed in both control and experimental plantaris, the percentage of MHC isoforms in the control and experimental muscles differed; the hypertrophied plantaris muscle contained more HCI (P < 0.05), HCIIa and HCIId (P < 0.05) and less HCIIb (P < 0.05) than the control muscle.
• 4.4. The control soleus muscle contained two MHC isofonns, HCI and HCIIa. However, there was only a single HCI isoform in the hypertrophied soleus muscle.
• 5.5. These results indicate that overloading a skeletal muscle by removing its synergists produces not only the muscle hypertrophy but also the changes in the expression of MHC isofonns.

     

Locust vitellogenin receptor: an acidic glycoprotein with N- and O-linked oligosaccharides

• 1.1. The locust vitellogenin (VTG) receptor which is embedded in oocyte plasma membranes is a glycoprotein.
• 2.2. With various lectins oligosaccharide units have been identified, among them neuraminic acid linked to Gal or GalNAc, mannose chains, Gal linked to GalNAc or GlcNAc and fucose linked to GlcNAc.
• 3.3. With specific enzymes it could be shown that mannose and most other oligosaccharides are O-linked while others like fucose are N-linked.
• 4.4. Enzymatic removal of all O-linked carbohydrates resulted in a drop of the molecular mass of the receptor protein from 200,000 to 110,000.
• 5.5. A total of N- and O-linked oligosaccharides of 54% was calculated.
• 6.6. The isoelectric point of the receptor was found to be at pH 3.4 increasing slightly after removal of neuraminic acid.
• 7.7. Removal of neuraminic acids destroyed the binding ability for VTG.

     

Proteomics Reveals Multiple Phenotypes Associated with N-linked Glycosylation in Campylobacter jejuni

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Highlights

• •Protein N-glycosylation is essential for nitrate reductase (Nap) activity in C. jejuni.
• •Removal of N-glycosylation results in a metabolic switch from Asp to Pro uptake.
• •N-glycosylation is required for optimal chemotaxis towards several substrates.
• •Loss of N-glycosylation reduces survival following temperature and osmotic shock.

     

Preparation and characterization of biotinylated probes for the β-interferon receptor

• 1.1. Recently we described the isolation of the β-interferon receptor [Zhang et al. (1986) J. biol. Chem. 261, 8017–8021]. A highly purified product was obtained but in low quantities.
• 2.2. The use ofbiotinylated β-interferon as a ligand represents an alternate approach to receptor isolation.
• 3.3. We have prepared and characterized the derivatives N-(biotinyl)- and N-(biotinyl-ϵ-aminocaproyl)-recombinant human [Ser¹⁷-interferon β (B- and BC-recHulFNβ).
• 4.4. Biotin incorporation does not result in any loss of antiviral activity, demonstrating the recognition of the derivative by the cell receptor.
• 5.5. The biotinylated recHuIFNβ binds specifically and reversibly to succinoylavidin or guanidine thiocyanate-stripped succinoylavidin linked to a Sepharose matrix.
• 6.6. Comparison of the competition curves obtained with [¹⁴C]biotin and [³H]biotinyl recHuIFN, in the presence of increasing concentrations of biotin suggests that the IFN moiety of the derivative has little effect on the affinity of biotin for avidin.
• 7.7. Biotinylated recHuIFNβ derivatives represent useful probes for the β-IFN receptor.

     

Protease,amylase and lipase activities in the midgut and hindgut of the cricket,Gryllus rubens and mole cricket,Scapteriscus acletus

• 1.1. Midgut is the major source of protease, amylase and lipase in a cricket, Gryllus rubens and in a mole cricket, Scapteriscus actetus.
• 2.2. Hindgut makes a significant contribution, and possibly even the major contribution, to digestion in both crickets, with enzyme activities from 20% (amylase and lipase) to 30% (protease) of midgut level, and a pH favorable to action of all three.
• 3.3. Ingested food helps regulate digestive enzyme levels, and crickets starved for 5 days had only 50–60% of normal levels of enzyme activity.

     

Cumene hydroperoxide-induced chemiluminescence in human erythrocytes: Effect of antioxidants and sulfhydryl compounds

• 1.1. The time-course of cumene hydroperoxide-induced changes in lipid peroxidation, protein sulfhydryl groups and chemiluminescence intensity was determined in human erythrocytes.
• 2.2. Increase in lipid peroxidation was maximal within 60 min of incubation and was paralleled by a decrease in protein sulfhydryl groups and an increase in chemiluminescence formation.
• 3.3. A standard assay system was established to investigate the protective effects of antioxidants and scavenger compounds on cumene hydroperoxide-induced chemiluminescence formation.
• 4.4. Chain-breaking antioxidants (i.e. butylated hydroxytoluene) and sulfhydryl compounds (i.e. dithiothreitol) were able to suppress chemiluminescence formation.
• 5.5. Our results suggested that secondary free radicals, as well as sulfhydryl groups of proteins are involved in cumene hydroperoxide-induced chemiluminescence formation.

     

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.

     

Inhibition of the nad-linked glutamate dehydrogenase from Trypanosoma cruzi by sulfhydryl reagents

• 1.1. The NAD-linked glutamate dehydrogenase (EC 1.4.1.2) partially purified from epimastigotes of Trypanosoma cruzi was strongly inhibited by the sulfhydryl reagents fluorescein mercuric acetate (FMA), p-chloromercuribenzoate (p-CMB), 5,5′ dithiobis (2-nitrobenzoate) (DTNB), N-ethylmaleimide (NEM), o-iodosobenzoate (IBz) and iodoacetamide (IAm).
• 2.2. The [I]₅₀ values (concentration of inhibitor for 50% inhibition) were 0.12, 1, 20, 80 μM, 1.2 and 25 mM, respectively, and the inhibition was nearly complete. Iodoacetate was practically ineffective.
• 3.3. The inhibition by p-CMB or FMA, and to some extent that by DTNB, but not that by NEM or IBz, could be partially reversed by addition of β-mercaptoethanol.
• 4.4. The enzyme partially modified by preincubation with p-CMB or IBz presented the same apparent K_m values for α-oxoglutarate, NADH and NH₄Cl, with a decreased apparent V_max.
• 5.5. The results suggest that one or more sulfhydryl groups, at or near the active site, are required for the activity of this glutamate dehydrogenase, which seems to be the most sensitive to thiol reagents among the similar enzymes studied so far.

     

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.

     

Purification and some properties of apurinic/apyrimidinic dna endonucleases in rat liver

• 1.1. Three kinds of apurinic/apyrimidinic (AP) DNA endonucleases, APcI, APcII, APcIII were purified from rat liver chromatin.
• 2.2. Molecular weights of APcI, APcII and APcIII were 30,000, 42,000 and 13,000 Da, which have isoelectric points of 7.2, 6.3 and 6.2, respectively.
• 3.3. Mg²⁺ was essential for the activities of these 3 enzymes, and sulfhydryl compounds (βercaptoethanol) had a stimulatory effect on the enzyme activities while N-ethylmaleimide and HgCl₂ inhibited the enzyme activity.
• 4.4. K_m values of APcI, APcII and APcIII for AP site of DNA were 0.53, 0.27 and 0.36 μM, respectively, and AMP was the most potent inhibitor to these three enzymes among nucleotides tested.

     

Multi-isotype Glycoproteomic Characterization of Serum Antibody Heavy Chains Reveals Isotype- and Subclass-Specific N-Glycosylation Profiles

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Highlights

• •nLC-MS/MS method to analyze immunoglobulin (Ig) N-glycopeptides from human serum.
• •Multi-isotype, site-specific characterization of immunoglobulin N-glycosylation.
• •IgA2 sequence and glycosylation-site variant analyses.
• •Platform to define disease-specific N-glycan signatures for different Ig isotypes.

     

Poly(adp-ribosylation) in N,N-diethylnitrosamine-treated mice

• 1.1. Liver nuclei isolated from male mice treated with the carcinogen N,N-diethylnitrosamine were examined for the homopolymer poly(adenosine diphosphate ribose) and for the activity of the conjugate polymerase.
• 2.2. At all levels of the carcinogen tested, a concomitant increase in both poly(adenosine diphosphate ribose) content and activity of the enzyme were found.
• 3.3. Both responses were transitory and dose dependent.

     

Phosphorylation of a 25,000-dalton protein in slow and fast muscles of the chicken

• 1.1. Protein phosphorylation in intact chicken latissimus dorsi muscle, slow anterior (ALD) and fast posterior (PLD), was compared.
• 2.2. A major difference in [³²P]phosphate incorporation was found between the ALD and PLD in a 25,000-dalton heat soluble protein.
• 3.3. The 25,000-dalton protein was purified from both the ALD and PLD.
• 4.4. The two proteins had similar amino acid composition and both contained approximately 1 mole phosphate per mole of protein.
• 5.5. The difference in their content of radioactive phosphate was determined to be due to faster turnover in the ALD.

     

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.

     

The inhibitory effect of tetramethylethylene diamine on water soluble and membrane bound acetylcholinesterase activity

• 1.1. The inhibitory effect of N,N,N′,N′-tetramethylethylene diamine (TEMED) on water soluble (WSAChE) and membrane bound (MBAChE) acetylcholinesterase was investigated.
• 2.2. TEMED (0.5–4.0 mM) reversibly inhibited WSAChE activity (18–62%) and MBAChE (20–61%) in a concentration dependent manner.
• 3.3. The IC₅₀ being about 2.8 mM for WSAChE and 2.6 mM for MBAChE.
• 4.4. Lineweaver-Burk plots indicated that the nature of inhibition is noncompetitive for both water soluble and membrane bound acetylcholinesterase, with K_m values 68 μM and 123 μM respectively.
• 5.5. An Arrhenius plot showed that the transition temperature (TT) is unaffected in the presence of TEMED.
• 6.6. The activation energy was increased below and above TT in the case of WSAChE only.
• 7.7. On the basis of this behaviour of TEMED with AChE. it can be proposed that it can be used as an eluting agent for the bounded AChE to affinity ligand and may have beneficial action on the reactivatability of irreversibly-inhibited AChE due to its structure.
• 8.8. Moreover there is a possibility that it can be used as a therapeutic agent for the treatment of Alzheimer's disease, myasthenia gravia and glaucoma like some other inhibitors of AChE.