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.

     

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.

     

Immunochemical and immunohistochemical determination of ubiquitin in molluscan (Mytilus edulis) smooth muscle

• 1.1. Immunochemical and immunohistochemical distribution of ubiquitin in the anterior byssus retractor muscle (ABRM) of Mytilus edulis was investigated.
• 2.2. In immunostaining, specific ubiquitin immunoreactivity was observed in the cross-sectioned ABRM, and was uniformly localized in this section.
• 3.3. The amount of free ubiquitin in the ABRM homogenate was 130 ± 4.6 ng/mg protein by western blot analysis, and ubiquitin conjugates were found at about 25, 29 and 200–230 kDa.
• 4.4. These findings were similar to those obtained in the skeletal muscle of rat.

     

Proteolysis of skeletal muscle in yellowfin tuna (Thunnus albacares): Evidence of calpain activation

• 1.1. White muscle of yellowfin tuna is subject to a form of deterioration known as “burnt tuna”.
• 2.2. TEM and SDS-PAGE were used to quantify cellular differences in deteriorated white muscle of yellowfin tuna.
• 3.3. Electron micrographs showed a significant loss of Z-disc integrity and an increase in intracellular edema in burnt tuna.
• 4.4. Electrophoresis established that a specific doublet of proteins, 42 kD and 46 kD was lost.
• 5.5. Proteolysis of isolated myofibrils incubated in calpain (EC 3.4.22.17) was greatest at pH 7.5 and was selective for intermediate molecular weight proteins.
• 6.6. This evidence suggests that burnt tuna is a specific and limited proteolysis of myofibrillar structural proteins characteristic of calpain proteolysis.

     

Hydro- and thermodynamic properties of bovine heart AMP-deaminase

• 1.1. Sedimentation velocity and sedimentation equilibrium studies of bovine heart AMP-deaminase were performed. Molecular weights of the native enzyme and subunit were determined as 161,000 and 43,000 dallons respectively.
• 2.2. The kinetic data indicate that in the presence of 100 mM KCl the enzyme may be active as a dimer.
• 3.3. The influence of temperature on the enzyme kinetics was investigated, from which activation energy (E_a and the heat of enzyme-substrate complex formation (ΔH_s) were calculated.
• 4.4. It is suggested that an equilibrium may exist between a dimeric and tetrameric form of AMP-deaminase in the heart.

     

DNase-I-like enzyme from the carp liver—Inhibition by muscle and endogenous actin

• 1.1. DNase-I-like activity occurs in the carp (Cyprinus carpio) liver cytosol (supernatant 105,000g).
• 2.2. The enzyme resembles DNase I from bovine pancreas in respect to the molecular mass (~31 kDa), pH (7.4) and ion requirements (Mg²⁺, Ca²⁺) and the ability to degrade native as well as denatured DNA.
• 3.3. As judged by comparison of DNase zymograms obtained after native- and SDS-PAGE, the enzyme occurs in the three molecular forms of similar molecular weight and different charges.
• 4.4. All these forms are inhibited by rabbit skeletal muscle actin as well as by endogenous actin isolated from the carp liver cytosol.
• 5.5. DNase from the carp liver cytosol does not interact with the antibodies directed against DNase I from bovine pancreas and against DNase I from the rat and bovine parotid glands.

     

Polymorphism and tissue specificity of scallop tropomyosin

• 1.1. Molecular polymorphism of tropomyosin from various muscle sources of the scallop, Patinopecten yessoensis, was investigated by electrophoretic and immunochemical methods.
• 2.2. Treatment of the muscle sources with trichloroacetic acid (TCA) prior to tropomyosin preparation was found useful to prevent proteolytic degradation of this protein.
• 3.3. Electrophoretic and immunochemical analysis revealed that at least six kinds of tropomyosin isoforms may exist in scallop muscle tissues.
• 4.4. The tropomyosin isoforms showed tissue-specific distribution in amounts and molecular species among the various muscle sources.

     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

     

Extraction and rapid identification of law molecular weight compounds from marine organisms

• 1.1. Different methods for extraction of low molecular weight compunds (lipids, terpenoids, steroids, glycosides, etc.) from Black Sea invertebrates and algae were examined.
• 2.2. The composition and quantity of the extracts were found to depend strongly on the type of the organisms and on the solvents used for extraction.
• 3.3. Thin-layer chromatography was found to be convenient for rapid identification of low molecular weight compounds in the extracts.
• 4.4. An approach has been developed which was suitable for field experimentrsand was successfully applied for the analysis of five algae and seven invertebrates from the Black Sea.

     

Purification and characterization of lipoprotein lipase from the white adipose,skeletal muscle,cardiac muscle,mammary gland and lung tissues of the rat

• 1.1. Lipoprotein lipase (LPL) was isolated from five rat tissues: white adipose, skeletal muscle, cardiac muscle, mammary gland and lung.
• 2.2. Specific activity of the preparations varied from 75 U/mg for skeletal muscle and 720 U/mg for adipose.
• 3.3. The preparations were further analysed using SDS-PAGE and a single component identified. The mol. wt of 61,000 Da of this component was consistent for all five of the tissue sources.
• 4.4. Significant differences in the values of the isoelectric points of the enzyme species were revealed. The values varied from 7.23 (SEM 0.022) for cardiac and lung to 7.51 (SEM 0.037) for mammary.
• 5.5. Two-dimensional electrophoresis, using isoelectric focusing in the first dimension and SDS-PAGE in the second revealed differences in the patterns of stained material derived from the five tissue sources.

     

The high intracellular ph buffering of hagfish (Eptatretus cirrhatus) dental plate retractor muscle cannot be attributed to the known histidine-related compounds present in other vertebrates

• 1.1. Intracellular pH buffering capacity of hagfish (Eplatretus cirrhatus) dental plate retractor muscles is among the highest reported for any vertebrate muscle.
• 2.2. Over 80% of the pH buffering capacity of hagfish retractor and myotome muscle is due to components other than proteins and phosphate.
• 3.3. The muscles have less than 0.5 μmol/g wet weight of l-histidine, and lack l-l-methyl histidine, l-3-methyl histidine and the histidine-containing dipeptides anserine, carnosine and ophidine.
• 4.4. Instead, they contain an unidentified low molecular weight acid-soluble compound to which the high pH buffering capacity can be attributed.

     

Purification and some properties of isocitrate dehydrogenase of a blowfly Aldrichina grahami

• 1.1. NADH-dependent isocitrate dehydrogenase has been purified 110-fold from the crude extract of the flight muscle mitochondria of Aldrichina grahami.
• 2.2. The purification procedure involved Triton X-100 treatment of isolated mitochondria, column chromatography on DEAE-cellulose, Affi-gel blue, and P-cellulose.
• 3.3. The purified enzyme was homogeneous by criteria of the polyacrylamide gel electrophoresis.
• 4.4. The enzyme of the blowfly contains more acidic amino acids and less hydrophobic amino acids than that of pig heart.
• 5.5. The molecular weight was determined to be 330,000 daltons. The subunit construction differs from ghat of mammalian isocitrate dehydrogenase.

     

Reduced muscle protein breakdown in septic rats following treatment with interleukin-1 receptor antagonist

• 1.1. The role ofinterleukin-1 (IL-1) in sepsis-induced muscle proteolysis was assessed by treating septic rats with recombinant IL-1 receptor antagonist (rIL-Ira).
• 2.2. In initial experiments, we tested the effectiveness of IL-Ira in preventing muscle proteolysis induced by administration of IL-1.
• 3.3. When normal rats were treated with rIL-α (three intraperitoneal doses of 100 μ g/kg body weight each over 16 hr), total and myofibrillar muscle protein breakdown rates, measured as release oftyrosine and 3-methylhistidine, respectively, by incubated extensor digitorum longus muscles, were significantly increased.
• 4.4. This metabolic response to IL-α was completely abolished by rIL-Ira, administered as three intraperitoneal doses of 3 mg/kg body weight each over 16hr.
• 5.5. In subsequent experiments, sepsis was induced in rats by cecal ligation and puncture (CLP); non-septic rats were sham-operated.
• 6.6. Treatment of septic rats over 16hr with a total dose of 25mg/kg body weight of rIL-Ira reduced, but did not normalize, the increased muscle protein breakdown rates seen during sepsis.
• 7.7. When the dose of rIL-Ira was more than doubled and given as a constant infusion at a rate of 4.2 mg/kg body weight/hr for 16 hr, the increased rate of muscle proteolysis in septic rats was normalized.
• 8.8. The present study offers the first direct evidence that IL-1 is involved in the regulation of muscle proteolysis during sepsis.

     

The isolation of skeletal muscle plasma membranes from rainbow trout (Salmo gairdneri)

• 1.1. Plasma membranes were isolated from caudal flank skeletal musculature of rainbow trout by discontinuous sucrose gradient centrifugation.
• 2.2. Na⁺−K⁺-ATPase was enriched 8-fold and 5′-nucleotidase activities 4-fold in a fraction isolated at the 8–25% sucrose interface.
• 3.3. A cholesterol: phospholipid ratio of 0.37 in the plasma membrane fraction was 85% greater than that observed in adjacent subcellular fractions.
• 4.4. Electron microscopy provided morphological confirmation of enrichment and integrity of skeletal muscle plasma membranes at the 8–25% sucrose interface.

     

The metabolic output of avian (Sturnus vulgaris,Calidris alpina) adipose tissue liver and skeletal muscle: Implications for BMR/body mass relationships

• 1.1. The oxygen uptake rate of avian adipose tissue, liver and skeletal muscle slices were measured.
• 2.2. The energy consumption of fat was less than one tenth that of liver and muscle.
• 3.3. Thus, interspecific allometric equations for the prediction of basal metabolic rate from body mass will not be accurate throughout the avian annual cycle unless changes in body composition are taken into account.

     

Comparative study on AMP deaminase in gill,muscle and blood of fish

• 1.1. High AMP deaminase activities were determined in the gill of one selachian, Scyliorhinus caniculus, and five teleosts, Anguilla anguilla, Cyprinus carpio, Salmo gairdneri, Perca fluviatilis and Esox lucius.
• 2.2. The highest activity was generally found in skeletal white muscle, except in A. anguilla and S. caniculus.
• 3.3. In s. caniculus a very high AMP deaminase activity was found in the blood where it was shown to be tightly regulated by inorganic phosphate.
• 4.4. Seasonal variations were observed for AMP deaminase activity in gill and white muscle, but also for blood Hb and protein concentration in the three tissues examined.

     

Isolation of dna and rna from Streptococcus sobrinus OMZ176 using CsTFA gradients

• 1.1. A simple procedure for isolation of high molecular weight genomic DNA, and RNA, from Streptococcus sobrinus OMZ176 is described.
• 2.2. Cell cultures were grown aerobically for 10 hr.
• 3.3. Spheroplast formation and lysis was achieved by mutanolysin/lysozyme-dependent digestion of the cell wall, followed by N-lauroylsarcosinate-mediated lysis.
• 4.4. Nucleic acids were isolated directly from cell-lysates using cesium-trifluoroacetate (CsTFA) densitygradient centrifugation.
• 5.5. Three different centrifugation regimes were tested: self-generated density gradients in a fixed angle rotor; self-generated density-gradients in a swinging-bucket rotor; pre-formed density-gradients in a swinging-bucket rotor.
• 6.6. Genomic DNA isolated by the CsTFA-procedure was found to have higher purity as compared to genomic DNA isolated in a conventional CsCl gradient.
• 7.7. Isolated DNA was shown to be of a quality suitable for applications in molecular biology.

     

Carbonic anhydrase III content in various equine muscles

• 1.1. In this study, carbonic anhydrase III (CA-III) content in 18 equine muscles was determined by enzyme immunoassay.
• 2.2. It was found to differ in several muscles.
• 3.3. That in external intercostal muscle, rectus abdominis muscle and splenius muscle from four horses was very high.
• 4.4. Although the masseter muscle had only type I fibers, CA-III content was similar to that in mixed-fiber type muscles such as the biceps femoris muscle.
• 5.5. It thus appear that equine type I fibers can be further subgrouped.

     

Variation in turtle myoglobins (subfamily emydinae: Testudines) examined by isoelectric focusing

• 1.1. Myoglobins from heart and skeletal muscle of turtles were analyzed by thin-layer isoelectric focusing.
• 2.2. Within the subfamily Emydinae, variation in the occurrence of two myoglobin electromorphs (pI 6.8 and 6.9) was detected.
• 3.3. Patterns of myoglobin polymorphism support dividing the Emydinae into two subfamilies and help resolve controversial theories on relationships of the genus Deirochelys.
• 4.4. Possible adaptive significance of the myoglobin variants (isoforms) remains to be determined.

     

Modification of glyceraldehyde-3-phosphate dehydrogenase,EC 1.2.1.12, isolated from rainbow trout during acclimation at 5 or 15°C—II. Biochemical characterization of G3PDH muscle isolates

• 1.1. G3PDH was isolated from the lateral muscle of rainbow trout (Salmo gairdneri) acclimated at 5°C (cold) and 15°C (warm).
• 2.2. No differences were found in muscle concentration, molecular weights, isoelectric focusing patterns, amino acid compositions or peptide maps between cold and warm isolates.
• 3.3. Cold and warm G3PDH contained mannose in variable concentration but no other prosthetic groups.