Michael Laskowski

Biochemical and ultrastructural changes in skeletal muscle induced by a creatine antagonist

To evaluate the essentiality of creatine and phosphocreatine for the maintenance of the ultrastructure of skeletal muscle, chicks were fed a creatine antagonist, beta-guanidinobutyric acid (beta-GBA), as 2% of a Chow diet. Chicks fed beta-GBA exhibited growth retardation and weakness, and they accumulated large amounts of a monosubstituted guanidino compound, presumably beta-GBA, in their skeletal muscles. After 2 wk, there was a 74% decrease in the uptake of [14C]-1-creatine into pectoralis muscles of chicks fed beta-GBA. After 2 wk there as a significant decrease in phosphocreatine concentrations in pectoralis muscles from 20.1 +/- 2.8 mumoles per g wet weight (mean +/- S.D.) for 8 control chicks to 16.5 +/- 2.5 for 7 chicks fed beta-GBA. Selected fibers of the pectoralis and gastrocnemius muscles of chicks fed beta-GBA exhibited ultrastructural abnormalities including loss of thick and thin filaments, disruption of the Z band, dilated mitochondria, and dilated and displaced sarcoplasmic reticulum. The pectoralis muscles of chicks given 6% creatine in addition to 2% beta-GBA in the diet accumulated little beta-GBA, maintained normal phosphocreatine concentrations, and exhibited no significant ultrastructural abnormalities. These findings are the first experimental evidence that high concentrations of phosphocreatine are essential for the maintenance of the ultrastructural integrity of skeletal muscle.

[10] Study of protein-protein and of protein-ligand interactions by potentiometric methods☆

Publisher Summary This chapter focuses on study of protein-protein and of protein-ligand interactions by potentiometric methods. This application of potentiometric technique is described in this chapter. Protein–protein or protein–ligand association reactions are frequently pH dependent. It is equivalent to state that hydrogen ions are released or bound as these reactions take place or that the titration curves of the products differ from the titration curves of the reactants. The chapter discusses the methods of direct measurement of the difference titration curves for reactions of the form A + B → C , where A is a protein and B may be either a protein or a ligand that interacts with A to form a 1:1 complex. The chapter describes the method of measurement of large K assoc that bears a close relationship to the methods of measurement of very small K m s, where inconveniently small substrate concentrations would have to be employed in direct measurement and therefore competitive inhibitors are added.

Microsomal opiate receptors differ from synaptic membrane receptors in proteolytic sensitivity.

We have found that opiate receptors in smooth microsomal fractions differ from synaptic membrane-associated receptors in proteolytic sensitivity. With 3 proteases of different substrate specificities (trypsin, chymotrypsin and S. griseus protease) smooth microsomal opiate receptors from rat brain were consistently less sensitive to limited proteolysis than were synaptic membrane receptors. Thiamine pyrophosphatase, a luminal Golgi membrane marker enzyme, exhibited a similar resistance to S. griseus protease in microsomal preparations, while microsomal Na+/K+-ATPase (ouabain-sensitive) was readily destroyed by trypsin. We also discovered that smooth microsomal opiate receptors co-migrate with both Golgi membrane and endoplasmic reticulum marker proteins on equilibrium density gradients under isopycnic conditions. Electron microscopic examination of the Golgi-enriched fraction showed the typical cisternae frequently associated with isolated Golgi membranes. Synaptic junctions, presynaptic membranes, myelin and mitochondria were conspicuously absent from this fraction. Since the microsomes isolated in vitro showed similar topography to those in vivo, the binding sites for opiates could be localized on the luminal surface membranes of the microsomal fractions. The exquisite sensitivity of synaptic membrane opiate receptors to proteolysis suggests that these receptors are found on the extracellular surface of the synaptic junction.

The Ultrastructure of the Sinu-Atrial Node of the Bat

The sinu-atrial node (SAN) of the bat, Pipistrellus subflavus, is capable of generating a wide range of spontaneous activity varying from 20 bpm when hibernating to bursts of 800 bpm during active flight. Electrophysiological studies have shown an absence of arrhythmias even below 4 degrees C body temperature. In order to determine whether these physiological capabilities are based upon unique ultrastructural features of the bat SAN, the present study was conducted. We found that the structure of the SAN of the bat is typically mammalian. Diameters of all three cell types in the SAN (nodal, transitional, and atrial) are smaller than those observed in any other mammalian species. A morphometric analysis of cell junctions reveals that nodal-nodal and transitional-transitional cell contacts are primarily undifferentiated with few nexuses. Atrial-atrial cell contacts have a dominance of fasciae adherentes-type junctions with a small area left undifferentiated. Nexuses are much more prevalent in atrial-atrial cell contacts.