Effects of 6-aminonicotinamide on levels of soluble proteins and enzyme activities in various tissues of Japanese quail

The effects of 6-aminonicotinamide (6-AN) on the levels of soluble proteins and enzyme activities in various tissues of Japanese quail were investigated. SDS-polyacrylamide gel electrophoresis showed that the soluble proteins with molecular masses corresponding to 160.4 and 52.5 kDa were either missing or present at lower concentrations in the brain of the 6-AN treated group compared to those in the control group. The soluble liver proteins with molecular masses 200, 120 and 70.5 kDa were missing in the treated group compared to those in the control while those of a molecular mass 15.1 kDa were found to be present at higher concentrations. Similarly, treatment with 6-AN decreased the concentration of soluble proteins in pectoral muscle with molecular masses 92.3, 54.5, 43.5, 41.2, 34.5, 27.5, 20.1 and 17.5 kDa and increased those with molecular masses 96.5, 37.7, 25.0, 19.3, 16.6, 13.8 and 10.8 kDa. In the heart, soluble proteins with molecular mass 84.6 kDa were increased. There was a marked reduction in the treatment group in the concentration of NAD in pectoral muscle but not in other tissues. A similar observation was also made with total RNA levels. The specific activity of malic enzyme was markedly increased by 6-AN treatment in the kidney and pectoral muscle but reduced in the liver. 6-Phosphogluconate dehydrogenase and lactate dehydrogenase activities were markedly reduced in the liver. Glyceraldehyde-3-phosphate dehydrogenase activity was significantly decreased in liver and pectoral muscle. NAD glycohydrolase activity was markedly decreased in pectoral muscle. Acetylcholinesterase activity was markedly reduced in liver but was enhanced in pectoral muscle. The results suggest that the metabolic actions of 6-AN are specific for certain proteins in the liver and muscle with the effect being most pronounced in muscle. The effects are also quite distinct from those shown by its analogue 3-acetylpyridine.     

A re-evaluation of the molecular size of duck epsilon-crystallin and its comparison with avian lactate dehydrogenases

A biochemical comparison of epsilon-crystallin isolated from the duck lens and lactate dehydrogenases of chicken heart has been made in order to establish the structural and functional identities of these two proteins. The native molecular weight of epsilon-crystallin was re-examined by combining sedimentation and gel-filtration data. It was found that epsilon-crystallin is 150 kDa in contrast to the 120 kDa reported previously for this crystallin. Subunit cross-linking experiments corroborated that lactate dehydrogenase and epsilon-crystallin both exist as tetramers of four identical subunits in their native quaternary structures. Amino acid compositions plus N-terminal analyses revealed no differences between the two proteins. Duck epsilon-crystallin exhibited high enzymatic activity of lactate dehydrogenases even after a long period of storage, and showed characteristic thermostability at 50 degrees C for several hours. Comparison of the enzyme activity of duck lens homogenate with those of heart, liver and muscle tissues revealed that duck lens is a much richer source than other tissues for the isolation and characterization of this important enzyme which appears also as a structural protein in the lens.     

A re-evaluation of the molecular size of duck ε-crystallin and its comparison with avian lactate dehydrogenases

A biochemical comparison of ε-crystallin isolated from the duck lens and lactate dehydrogenase of chicken heart has been made in order to establish the structural and functional identities of these two proteins. The native molecular weight of ε-crystallin was re-examined by combining sedimentation and gel-filtration data. It was found that ε-crystallin is 150 kDa in contrast to the 120 kDa reported previously for this crystallin. Subunit cross-linking experiments corroborated that lactate dehydrogenase and ε-crystallin both exist as tetramers of four identical subunits in their native quaternary structures. Amino acid compositions plus N-terminal analyses revealed no differences between the two proteins. Duck ε-crystallin exhibited high enzymatic activity of lactate dehydrogenases even after a long period of storage, and showed characteristic thermostability at 50°C for several hours. Comparison of the enzyme activity of duck lens homogenate with those of heart, liver and muscle tissues revealed that duck lens is a much richer source than other tissues for the isolation and characterization of this important enzyme which appears also as a structural protein in the lens.     

Physical and chemical properties of lactate dehydrogenase in Homarus americanus.

Two isoenzymes of lactate dehydrogenase have been purified from Homarus americanus: One is found predominantly in the tail muscles; the other, in the walking leg muscles. This is the first demonstration of multiple forms of l-specific lactate dehydrogenase in an invertebrate organism. These proteins contain four essential sulfhydryl groups titratable by p-hydroxymercuribenzoate and 5,5′-dithiobis(2-nitrobenzoic acid). The molecular weights of these isoenzymes are dependent upon ionic strength. The native tetramer (M_r 145,000) exists in low ionic strength solutions; the active dimer (M_r 75,000), in high ionic strength solutions; this is the only example of lactate dehydrogenase disaggregation without concomitant loss in enzymatic activity. Microcomplement fixation studies suggest that there may be less than 4% difference in the primary structures of these two proteins.     

Enzymatic activities in slow and fast denervated old rat muscles

The activities of five enzymes have been studied quantitatively in denervated extensor digitorum longus, gastrocnemius and soleus muscles of 24-month-old rats. The results have been compared with those obtained from normal muscles of a similar age group of rats. Three weeks after denervation, the activity of hexokinase was increased in gastrocnemius and extensor digitorum longus. Phosphofructokinase, lactate dehydrogenase, malate dehydrogenase and 3-hydroxyacyl-CoA-dehydrogenase showed decreased activities. These results suggest that enzyme which represents glucose uptake increased its activity in fast muscles and that enzymes for anaerobic glycolysis, lactate fermentation, citric acid cycle and beta-oxidation had a decreased activity in slow and fast muscles.     

Purification of the human placental NAD-linked 15-hydroxyprostaglandin dehydrogenase

An NAD-linked 15-hydroxyprostaglandin dehydrogenase has been purified 13,100-fold from human placental tissue. The specific activity of the purified enzyme ranges from 6900 to 8300 mU/mg protein depending on the method used to determine the protein concentration. On discontinuous electrophoresis in sodium dodecyl sulfate more than 95% of the protein migrates as a single band; its estimated molecular weight is 25.5-26.0 kDa. This is half the value obtained when the molecular weight is estimated under non-denaturing conditions and suggests that the enzyme is composed of two identical or nearly identical subunits.     

Formate dehydrogenase activity in cells and outer membrane blebs of Desulfovibrio gigas

Formate dehydrogenase in Desulfovibrio gigas was measured by following the release of 14CO2 from radiolabeled formate. Experiments with whole cells using sulfate as the electron acceptor revealed optimal formate dehydrogenase activity at pH 7.0 and formate utilization followed saturation kinetics. While formate dehydrogenase was constitutively produced in pyruvate or lactate media, the formate dehydrogenase activity was markedly increased in cells grown with formate as the electron donor. In cell-free experiments with methyl viologen or 2,6 dichlorophenolindophenol, about 1% of the cellular formate dehydrogenase activity was present in blebs from the outer membrane. Electron microscopy revealed that these blebs were closed structures with diameters ranging from 80-800A and were not induced by changes in osmotic pressure or cellular autolysis. Analysis of blebs revealed the presence of lipopolysaccharides and two proteins with molecular masses of 70 and 53 kDa.     

Purification and properties of NADP-dependent isocitrate dehydrogenase from the bovine adrenal cortex

NADP-dependent isocitrate dehydrogenase (EC 1.1.42) was isolated and 430 times purified from the hyaloplasm fraction of bull adrenal cortex using fractionation by ammonium sulphate and acetone, heat treatment, chromatography on DEAE-Sephadex A-50, gel-filtration on Sephadex G-200 and affinity chromatography on 2',5'-ADP-sepharose 4B. The specific activity of homogeneous enzyme is 60 units per 1 mg of protein at 30 degrees C, yield--34%, pH optimum--8.0, molecular weight, determined by gel filtration on Sephadex G-200, is 96 kDa. The preparation electrophoresis in PAAG in the presence of DS-Na reveals one protein fraction with the mobility corresponding to that of protein having molecular weight of 46 kDa. The data obtained evidence for a dimer structure of the isocitrate dehydrogenase molecule from bull adrenals.     

Purification of the human placental NAD-linked 15-hydroxyprostaglandin dehydrogenase

An NAD-linked 15-hydroxyprostaglandin dehydrogenase has been purified 13, 100-fold from human placental tissue. The specific activity of the purified enzyme ranges from 6900 to 8300 mU/mg protein depending on the method used to determine the protein concentration. On discontinous electrophoresis in sodium dodecyl sulfate more than 95% of the protein migrates as a single band; its estimated molecular weight is 25.5–26.0 kDa. This is half the value obtained when the molecular weight is estimated under non-denaturing conditions and suggests that the enzyme is composed of two identical or nearly identical subunits.     

Kinetic Properties of Purified Pseudomonas aeruginosa Phosphorylcholine Phosphatase Indicated That This Enzyme May Be Utilized by the Bacteria to Colonize in Different Environments

A protein oligomer with an approximate molecular weight of its 37-kDa monomer form was purified from the cell envelope fraction of Vibrio damsela cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with phosphatidyl choline. The functional properties for the 37-kDa protein suggest that it is a nonspecific or general porin, with an apparent pore size of 1.6 nm. This porin allows penetration of a variety of hydrophilic solutes according to their molecular mass. After electroelution, the oligomer was partially dissociated into monomers, whereas treatment with EDTA did not affect its dissociation. The monomers of the 37-kDa protein were not active in the reconstitution assay. The effect of culture media on the composition of the outer membrane protein of V. damsela was examined. Only one outer membrane protein with an apparent molecular weight of 37 kDa (37-kDa protein) was formed in cells grown in 3% NaCl–BHI broth and in 3% NaCl–nutrient broth with the addition of 2% glucose. Three outer membrane proteins, with apparent molecular weights of 37 kDa, 40 kDa, and 46 kDa, were produced in cells grown in 3% NaCl–nutrient broth. An additional outer membrane protein with an apparent molecular weight of 44 kDa (44-kDa protein) was found in cells grown in 3% NaCl–nutrient broth with the addition of 2% maltose. This protein was found to exhibit specificity to maltose derivatives. The results obtained in this study confirm the porin-like character of discussed proteins and give a basis for advanced study of those proteins. Received: 16 June 1998 / Accepted: 18 July 1998     

Organ culture of frog muscle: maintenance of mass, enzyme levels, and contractile force

This paper describes an organ culture system that maintains frog sartorius muscles in good condition for 5 days. In the absence of serum and insulin, muscles maintained at approximately 93% of resting length atrophied with significant decreases in dry weight, protein content, and contractile force, and in the levels of activity of citrate synthase, lactate dehydrogenase, and creatine kinase. Inclusion of 1.0 mU/ml of insulin in the culture medium prevented the decreases in muscle mass, twitch tension, and citrate synthase activity and minimized the decreases in lactate dehydrogenase, creatine kinase, and tetanic tension. Inclusion of 10% serum, in addition to 1 mU/ml insulin, in the medium did not have clear cut additional benefits. Stretching muscles to 110% of resting length (L0) resulted in marked deterioration with decreases in total protein, enzyme levels, and contractile force. Keeping muscles at approximately 93% L0 was as effective as maintenance at L0 in preventing atrophy and loss of contractile force and enzyme activities. This organ culture procedure, which maintains frog sartorius muscle in good condition without serum for at least 5 days, may provide a useful model for studying the regulatory mechanisms responsible for a variety of adaptations in muscle.     

Testosterone and muscle hypertrophy in female rats

The effects of chronic treatment with testosterone propionate on compensatory muscle hypertrophy secondary to synergist removal were studied in female rats. Synergist removal resulted in a significant (2-fold) increase in muscle wet weight, with no changes in protein concentration. As reported previously, oxidation of [2-14C]pyruvate to 14CO2 was significantly decreased in hypertrophic muscles. In addition, malate dehydrogenase and lactate dehydrogenase activities were significantly decreased in overloaded muscles on a wet weight basis but not on the basis of noncollagen protein. These data suggest that specific metabolic adaptations may occur in response to overload of muscle. Administration of testosterone propionate in subcutaneously implanted Silastic capsules resulted in a 20-fold increase in serum testosterone levels. This treatment had no effect on body weight, muscle weight, pyruvate oxidation, or malate and lactate dehydrogenase activities in both control and hypertrophic muscles, although there was an effect on the noncollagen protein content of overloaded muscles. These results do not support the hypothesis that androgens, in conjunction with weight-bearing exercise in female subjects, are effective in increasing muscle mass or function in female subjects.     

The foetal development of lactate dehydrogenase isoenzymes, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from human striated muscle

1. Human foetal skeletal muscles involved in support and in periodic contractility were studied for their content of total extractable lactate dehydrogenase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities as well as for the relative distribution of lactate dehydrogenase isoenzymes. 2. During foetal development a linear steady increase in total lactate dehydrogenase activity as well as a linear decrease in the H/M sub-unit ratio of the isoenzymes was found. 3. No significant changes were found in the activities of the enzymes of the hexose monophosphate shunt (C-6 oxidation). 4. The changes found suggest a steady increased synthesis of lactate dehydrogenase M-sub-units in human skeletal muscles during foetal development. 5. The weekly changes in the total lactate dehydrogenase activity and in lactate dehydrogenase isoenzymes are lower in muscles involved in support than in those involved in periodic contractility. 6. These findings, together with the literature available, are consistent with the morphological fact that foetal development of skeletal muscles mostly concerns the white muscle fibres and not the red muscle fibres.     

Isolation and characterization of heat-modifiable proteins from the outer membrane of Porphyromonas asaccharolytica and Acinetobacter baumannii

Active porins were isolated and purified from the outer membranes of the gram-negative anaerobic rod Porphyromonas asaccharolytica and the aerobic coccobacillus Acinetobacter baumannii. The porins from both bacteria appear to be monomers when isolated and purified. Both porins exhibited decreased mobility on SDS-PAGE after boiling for 10 min in the sample buffer. After heating, their molecular weight is estimated at 43 kDa while without heating they run as proteins with a molecular weight of approximately 37 kDa. Due to their characteristic heat-modifiability, these proteins were named HMP (heat-modifiable protein)-P. asaccharolytica and HMP-A. baumannii. Amino acid analysis revealed both porins to be hydrophilic proteins. These proteins have been shown to be active in transporting sugars when incorporated into liposomes. The permeability of both porins for L-arabinose was less than that produced by the porin of Escherichia coli B. Permeability to high molecular weight disaccharides was lower than for small monosaccharides. Western blot analysis did not reveal any antigenic cross reaction between HMP-A. baumannii and the HMP-P. asaccharolytica. The results obtained in this study confirm that although these heat-modifiable proteins are pore forming proteins and have similar activity they differ in their antigenicity.     

Miniaturized one-chip electrochemical sensing device integrated with a dialysis membrane and double thin-layer flow channels for measuring blood samples

We have developed a microfluidic device consisting of a gold film working electrode modified with lactate oxidase and Os-poly(vinylpyridine) mediator containing horseradish peroxide, and reference and counter electrodes in a microflow detection channel separated by a microdialysis membrane from another microflow channel used for sample injection. The dialysis membrane is cellulose with a molecular weight cut off of 10 kDa. We achieved control over a wide recovery rate range of 3-94% because the device is capable of controlling both flow rates in the dual thin-layer channels. We were able to measure the lactate concentration in blood samples within a few minutes without any pretreatment because biomolecules are simultaneously separated by molecular weight and detected in the device. We achieved quantitative and reproducible measurements of the lactate concentration in blood samples, and obtained a relative standard deviation of 1.5% (n = 8). With our device, the lactate concentration in dog whole blood was measured with high stability without any pretreatment.     

Impact of polymorphism of the regulatory subunit of the ��-calpain (CAPN1S) on the proteolysis process and meat tenderness of young cattle

The objective of this study was to estimate the impact of the polymorphism of ??-calpain (CAPN1S) gene on protein changes of the cattle muscle tissue and its tenderness during 10-day cold storage. The analysis was performed on the longest dorsal and lumbar muscles collected from 76 bulls 6 to 12 months of age. Polymorphism identification of the above-mentioned gene was conducted using the PCR-RFLP technique. Its effect on the course of the proteolysis process was assessed by monitoring changes in proportions of tissue proteins during 10-day process of meat ageing. Special attention was focused on changes in native titin (T1) share and products of its degradation (proteins of molecular weight (m.w.) of 2400 and 200 kDa), ??-actinin and protein of 37 kDa as well as myosin heavy chains (MHC). In the case of the last proteins, their polymorphism was evaluated as well. Meat tenderness was estimated measuring the value of shear force and sensorially. The highest tenderness was ascertained for the heterozygote. Its improvement was associated with a significant decrease in proportions of proteins of molecular weight of approximately 37 kDa accompanied by an increase of those with 200 kDa molecular weight. Muscles derived from cattle of CT genotype were characterised by the highest proportions of type 2a MHC isoform. Value differences between proportions determined for the heterozygote and CC and TT homozygotes of the CAPN1S gene were statistically significant. Therefore, it can be presumed that the process of meat tenderisation was especially connected with MHC polymorphism.     

Metabolic characteristics of experimental free vascularized canine gracilis muscle transfers

A canine gracilis model was used to study muscle energy metabolism and enzyme activities after free vascularized muscle transfer. Fifteen male mongrel dogs underwent orthotopic, free transfer of the left gracilis with microneurovascular anastomosis. After a minimum of 10 months' recovery, muscle biopsy specimens were obtained from the transfers and the contralateral controls and analyzed for relative fiber type areas and maximum activities of phosphorylase, hexokinase, phosphofructokinase, glycerol-3-phosphate dehydrogenase (GPDH), pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, 3-hydroxyacyl coenzyme A dehydrogenase (HAD), and creatine phosphokinase. Biopsy specimens obtained before and after a 10 minute, 20-Hz contraction were analyzed for glucose, glycogen, glycolytic intermediates, phosphocreatine, total creatine, and adenine nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, and inosine). There was no significant transfer versus control difference in type I relative fiber area (45 +/- 4 percent versus 44 +/- 3 percent). Total creatine was significantly reduced in the transferred muscles relative to control (83.1 +/- 3.0 mmol/kg versus 100.6 +/- 5.1 mmol/kg dry weight). Maximal activities of phosphorylase, pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, HAD, and creatine phosphokinase were diminished in transfers relative to controls, although hexokinase activity was significantly higher in the freely transferred gracilis muscles. During the 20-Hz contraction, muscle transfers produced less force initially, although the force/time integral over the 10-minute stimulation was similar in transfers (277 +/- 25 N/g/second) and controls (272 +/- 24 N/g/second). The contraction was associated with significant glvcogen use and lactate accumulation in both transfers and controls, although this was less pronounced for the transfers. Glycolytic flux appeared muted in the transfers relative to controls. Significant, similar high-energy phosphagen reductions and inosine monophosphate accumulation were noted during the contraction in both groups. Contractile activity is associated with the expected pattern of muscle metabolite changes following free vascularized transfer, indicating the components of cellular energy metabolism are not qualitatively altered after microneurovascular muscle transfer. In contrast, quantitative differences suggest that free vascularized muscle transfer can be associated with a muscle enzyme profile consistent with deconditioning and the presence of denervated muscles fibers in the absence of fiber type profile changes.     

Chaperonin TRiC assists the refolding of sperm-specific glyceraldehyde-3-phosphate dehydrogenase

The cytosolic chaperonin TRiC was isolated from ovine testes using ultracentrifugation and heparin-Sepharose chromatography. The molecular mass of the obtained preparation was shown to exceed 900 kDa (by Blue Native PAGE). SDS–PAGE yielded a set of bands in the range of 50–60 kDa. Electron microscopy examination revealed ring-shaped complexes with the outer diameter of 15 nm and the inner diameter of approximately 6 nm. The results suggest that the purified chaperonin is an oligomeric complex composed of two 8-membered rings.The chaperonin TRiC was shown to assist an ATP-dependent refolding of recombinant forms of sperm-specific glyceraldehyde-3-phosphate dehydrogenase, an enzyme that is expressed only in precursor cells of the sperms in the seminiferous tubules of the testes. In contrast, TRiC did not influence the refolding of muscle isoform of glyceraldehyde-3-phosphate dehydrogenase and assisted the refolding of muscle lactate dehydrogenase by an ATP-independent mechanism. The obtained results suggest that TRiC is likely to be involved in the refolding of sperm-specific proteins.     

Patterns of amino acid enzyme in domestic fowl breast and leg muscle during development

Activities of alanine and aspartate transaminases, glutamine synthetase, adenylate deaminase, glutamate and xanthine dehydrogenases and lactate dehydrogenase were measured in leg and breast muscles of developing chicks from day 10 in ovo to day 5 of free life, and compared with measurements for adult hens. Xanthine dehydrogenase activity was low in both muscles with adult levels attained on day 15 in ovo. Glutamine synthetase for chicks was maintained higher during development than for adults in both muscles. Minor differences were observed between both muscles in all enzymes tested up to day 18. With low embryonic values and important rises before hatching, the differences were initiated in the posthatching period. Important differences were observed between adult levels of activity. Leg muscle revealed higher enzyme values except for lactate dehydrogenase and indistinguishable levels for adenylate deaminase and xanthine dehydrogenase in both muscles. Alanine, instead of glutamine, is postulated as the main nitrogen transport between muscle and liver in the domestic fowl.     

Effect of denervation on enzyme levels in flight muscles of the adult migratory locust

The metathoracic dorso-longitudinal muscles of adult males of Locusta migratoria were denervated. Twenty days after denervation wet weight, protein content and the specific activities of enzymes, representative of aerobic glycolytic and β-oxidative pathways, and citric acid and glycerophosphate cycles are reduced in a statistically significant way. In contrast, the specific activity of lactate dehydrogenase is increased. It is demonstrated that locusts, in which the metathoracic dorso-longitudinal muscles are denervated on one side only, constitute an experimental system with proper experimental and control muscles.