Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.     

Parallel bioassay of litorin and phyllolitorins on smooth muscle preparations

Phyllolitorin and Leu8-phyllolitorin, two nonapeptide amides from the skin of the South American frog Phyllomedusa sauvagei, are representatives of a novel bombesin subfamily, characterized by the occurrence in their molecule of a serine residue substituting the usual histidine residue at position 3 from the C-terminus. In parallel bioassay on ten different smooth muscle preparations and rat blood pressure, phyllolitorin and Leu8-phyllolitorin were virtually equiactive, but the two peptides appeared remarkably less potent that litorin in all test preparations, except the rat urinary bladder. The shape of contractions produced by the phyllolitorins and promptness of cessation of their action upon washing seem to indicate a looser binding of these peptides to their receptors and/or a more rapid inactivation, in comparison to litorin.     

The preparation of a sarcolemmal fraction from evacuated muscle slices

A novel procedure is described for preparing a plasma membrane fraction from skeletal muscle (i.e., sarcolemma). The procedure entails evacuating the myoplasm from muscle slices as a preliminary step to homogenization and fractionation. The evacuated muscle slices are composed of a stroma-containing sarcolemma, which is then homogenized and fractionated, utilizing a sequence of differential and discontinuous sucrose density gradient centrifugations. On the basis of electron microscopy, selective enzyme markers and α-bungarotoxin binding in innervated and denervated muscles, the fraction most enriched with sarcolemma is recovered from the 0.5/0.7 M interface of a discontinuous sucrose gradient.     

The structure and distribution of satellite cells of cardiac muscles in decapod crustaceans

Summary The structure and distribution of satellite cells of cardiac muscles were examined in twenty-one species of animals chosen from each tribe within the order Decapoda (Arthropoda, Crustacea). The satellite cells were found in all animals observed. Most of them are morphologically identical with those described in different striated muscles of other species, but some cells have unusual features. The decapod satellite cell occasionally lies right over the region corresponding to the intercalated disc between the apposed cardiac muscle cells. The cell sends cytoplasmic processes into the adjacent muscle cells, enabling the plasma membrane to make close contact with the cleft opening of the intercalated disc, and with the myofibril at the level of the Z-line. Another characteristic feature is the presence of paired cells. Such cells are clearly separated from each other over most of the contact area by the respective plasma membranes, which are smooth in appearance and devoid of specialized regions. The significance of the presence of satellite cells in decapod cardiac muscle and its possible role are discussed and compared with those described for other species.     

Arrangement of smooth muscle cells and intramuscular septa in the taenia coli

Summary Bands of electron-dense material beneath the cell membrane of smooth muscle cells of the guinea-pig taenia coli provide attachment to thin myofilaments and to intermediate (10 nm) filaments; about 50% of the cell membrane is occupied by dense bands in muscle cells transversely sectioned at the level of their nucleus, and between 50 and 100% in smaller cell profiles nearer the cell's ends. In addition to the known cell-to-cell junctions (intermediate contacts), more complex apparatuses anchor muscle cells together, either end-to-end or end-to-side or side-to-side. They consist of elaborate folds, invaginations and protrusions accompanied by large amounts of basal lamina material. In the end-to-end anchoring apparatuses numerous finger-like and laminar processes from the two cells interdigitate. Other muscle cells have a star-shaped profile in the last few microns of their length, or show longitudinal invaginations occupied by a thickened basal lamina and occasionally by collagen fibrils. The septa of connective tissue extend only for a few hundred microns along the length of the taenia. In taeniae fixed in condition of mild stretch the muscle cells form an angle of about 5° with the septa. In muscles fixed during isotonic contraction the angle increases to about 20–22°, and in longitudinal sections the muscle cells appear arranged in a herring-bone pattern. The collagen concentration in the taenia coli is 4–6 times greater that in skeletal and cardiac muscles. These various structures are discussed in terms of their possible role in the mechanism of force transmission.I thank Mr. S.J. Sarsfield and Miss E.M. Franke for expert technical assistance, and Dr. Adam Yamey for much help in the experiments on collagen content. This work is supported by grants from the Medical Research Council     

Activation of two types of fibres in ferret,Mustela putorius furo,cremaster muscle

Summary Some contractile, histochemical, morphological and electrophysiological properties of ferret, Mustela putorius furo, cremaster muscle have been estimated. Histochemical fibre typing revealed the presence of two types of fibres (type I 66.2%, type II 33.8%). Morphometry performed on ATPase-stained transverse sections showed that type I was composed of a large amount (40%) of small(<400 m²) cells. In mammalian Ringer two groups of fibres could be recognized on the basis of the values of resting potential (-69.7 mV and-59.1 mV) and intracellular sodium activity (8.3 mmol·l^-1 and 14.1 mmol·l^-1, respectively). In experiments on fibre bundles, the elevation of extracellular potassium concentration to 15–200 mmol·l^-1 produced contractures that consisted of a well-defined transient or phasic tension followed by a sustained or tonic tension. Properties of activation and inactivation of the tension analysed in small bundles of cut fibres (lengths 0.5–1.0 cm) were of fast- and slow-twitch type for phasic and tonic phase, respectively. In contrast to the phasic component of K contractures, the tonic phase was abolished by Ca²⁺ withdrawal and inhibited by Ni²⁺, Cd²⁺, Co²⁺, Gd³⁺ and gallopamil (D600). In Ca²⁺-free medium the sustained tension was restored by adding Sr²⁺. It is concluded that in ferret cremaster muscle the presence of slow-twitch fibres would give rise to the tonic component of the K contracture in which an extracellular source of activator Ca²⁺ is involved. The ability of these fibres to contract with a maintained tension for prolonged periods of time might participate in the temperature regulation of the testes.Abbreviations a _i ^Na intracellular sodium activity - ATPase myosin adenosine triphosphatase - D600 gallopamil - E _m membrane potential - E _r resting potential - EDL muscle, extensor digitorum longus muscle - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - e.c. excitation-contraction - SDHase succinate dehydrogenase - NADHase nicotinamide adenine, dinucleotide hydrogen-diaphorase - SOL muscle, soleus muscle - T time constant of relaxation - TEACI tetraethylammonium chloride - [Ca]_o, [K]_o, [Na]_o extracellular calcium, potassium, sodium concentration     

Nucleotide sequence of Rattus norvegiens mitochondrial DNA that includes the genes for tRNAile, tRNAgln and tRNAf-met

The nucleotide sequence of a segment of mtDNA from Rattus norvegiens (rat) which contains the genes for tRNA^ile, tRNA^gl and tRNA^f-met has been determined. A detailed comparison has been made between this sequence and the corresponding sequences of mouse, human and bovine mtDNAs with regard to the primary and secondary structure of the tRNA genes, the regions connecting the tRNA genes, and the regions flanking the tRNA genes which code for the carboxyl terminus of URF-1 and the amino terminus of URF-2. No differences were found in the nucleotide sequences of the genes for tRNA^ile, tRNA^gln and tRNA^f-met in mtDNAs from three different female lines of rats (SASCO-1, SASCO-2 and Wild-UT) that differ by substitutions of 0.8% to 1.8% of their total nucleotides.     

The membrane systems in different fibre types of the triceps surae muscle of cat

Summary The volume and surface area of mitochondria and sarcoplasmic reticulum in fast and slow twitch fibres of the cat triceps surae muscle were determined from thin sections. The width of the Z-line and the array of glycogen granules identified fast and slow twitch fibres.The relative volume occupied by mitochondria was largest in slow twitch gastrocnemius fibres. Fast twitch fibres showed the greatest scatter of mitochondrial content. This corresponds with the fact that motor units of the fast twitch type differ most with respect to resistance to fatigue.The relative volume of the sarcoplasmic reticulum was twice as large in fast as in slow twitch fibres. The volume fraction occupied by longitudinal tubules of the reticulum was the same in fast and slow twitch gastrocnemius fibres but was only half as large in the slow twitch soleus fibres. This difference may be related to post-tetanic potentiation: this property is present in all gastrocnemius fibres but is absent in soleus fibres.The specific tetanic force is 3 to 5 times smaller in slow twitch gastrocnemius than in slow twitch soleus fibres or fast gastrocnemius fibres. There was, however, no detectable morphological difference that might be related to this difference in force.Freeze fractures demonstrated directly that, in soleus fibres, terminal cisternae and longitudinal tubules of the reticulum were scarce as compared to gastrocnemius fibres. The plasma membranes of some gastrocnemius fibres displayed square arrays of 60-nm particles; these arrays were absent in other gastrocnemius fibres and in all soleus fibres. They probably characterize plasma membranes of fast twitch fibres.This study was supported by grants from the Danish Medical Research Council. I wish to thank Mrs. M. Bjærg for valuable technical help