Role of substrate binding forces in exchange-only transport systems: I. Transition-state theory

Summary An analysis of transition-state models for exchange-only transport shows that substrate binding forces, carrier conformational changes, and coupled substrate flow are interrelated. For a system to catalyze exchange but not net transport, addition of the substrate must convert the carrier from an immobile to a mobile form. The reduction in the energy barrier to movement is necessarily paid for out of the intrinsic binding energy between the substrate and the transport site, and is dependent on the formation of two different types of complex: a loose complex initially and a tight complex in the transition state in carrier movement. Hence the site should at first be incompletely organized for optimal binding but, following a conformational change, complementary to the substrate structure in the transition state. The conformational change, which may involve the whole protein, would be induced by cooperative interactions between the substrate and several groups within the site, involving a chelate effect. The tightness of coupling, i.e., the ratio of exchange to net transport, is directly proportional to the increased binding energy in the transition state, a relationship which allows the virtual substrate dissociation constant in the transition state to be calculated from experimental rate and half-saturation constants. Because the transition state is present in minute amount, strong bonding here does not enhance the substrate's affinity, and specificity may, therefore, be expressed in maximum exchange rates alone. However, where substrates largely convert the carrier to a transport intermediate whose mobility is the same with all substrates, specificity is also expressed in affinity. Hence the expression of substrate specificity provides evidence on the translocation mechanism.     

Cardiac alpha-crystallin. I. Isolation and identification

A water soluble protein, a major component of the cytosolic fraction of rat heart cells, was purified using either reverse phase HPLC or antibodies affinity chromatography procedures and characterized. The protein has an apparent Mr of 24 k, as judged by SDS-gel electrophoresis. Under non-denaturing conditions, however, the protein occurs as a homomultimer (Mr between 400 and 650 k) of the monomeric 24 kDa species and could be selectively enriched by fractionation of the cytosolic fraction on 10 to 40% sucrose gradients. Polyclonal antibodies, raised against the denatured 24 kDa protein, were used to investigate its tissue distribution. Besides the heart, where it is very abundant, the 24 kDa protein is expressed also in other red muscles and in kidneys, but was not detectable in stomach, thymus, liver, and brain. The amino acid composition of the protein and the partial amino acid sequence of various proteolytic fragments was determined. A search for homologies of the primary structure of known proteins has shown that the 24 kDa protein is strikingly similar, if not identical to alpha-B-crystallin. In fact, the two proteins were found to be indistinguishable also by immunological criteria. This study demonstrates that the lens protein alpha B-crystallin is a major cytosolic component of heart cells.     

The equine endometrosis: new insights into the pathogenesis

This paper describes the histomorphological and immunohistochemical characterisation of phenotypic variations of endometrosis as well as potential etiological factors which may influence disease progression. In total, 779 endometrial biopsies were examined. These biopsies were taken in the breeding and non-breeding season (n=509), on defined days during the estrous cycle (n=70) and before and after experimentally induced bacterial endometritis (n=200). In addition to conventional histopathology, selected biopsies were investigated using alcianblue staining as well as immunohistochemical methods for the detection of steroid hormone receptors, Ki-67-antigen, vimentin, desmin, fibronectin, smooth-muscle-alpha-actin and laminin. The equine endometrosis can be divided into a destructive and a non-destructive form. Based on the morphology of the stromal cells involved, an active or inactive state can be distinguished in fibrotic foci. In all types of endometrosis, fibrotic stromal cells show a distinctly reduced expression of steroid hormone receptors in comparison to the intact stroma, indicating their dedifferentiation. However, the steroid hormone receptor expression of involved glandular epithelia seems to depend on the activity of the fibrosis. These results suggest an independency of all fibrotic foci from the hormonal control mechanism of the uterus. The characteristical features of destructive endometrosis are a large number of smooth-muscle-alpha-actin containing myofibroblasts, a pronounced epithelial vimentin expression, excessive extracellular matrix accumulation and a progressive alteration of the basal lamina. Furthermore, the frequently seen cystic glandular dilatation and mechanical destruction of the uterine glands may occur due to the contractibility of the myofibroblasts involved. As shown in this study, a simultaneous endometritis can cause a temporary activation of fibrotic stromal cells. However, cyclic and seasonal endocrine changes seem to have no effects on progression of the disease. It can be concluded that the various types of endometrosis represent different stages in the fibrotic process, possibly leading to the destruction of the glands and subsequently resulting in the development of a stromal fibrosis.     

Determination of heart rate and heart rate variability in the equine fetus by fetomaternal electrocardiography

Heart rate is an important parameter of fetal well-being. We have analyzed fetal heart rate (HR) and heart rate variability (HRV) by fetomaternal electrocardiography (ECG) in the horse (Equus caballus) from midpregnancy to foaling. It was the aim of the study to detect changes in the regulation of fetal cardiac activity over time and to establish normal values in undisturbed pregnancies. A total of 22 mares were available for the study. Fetomaternal electrocardiography was a reliable technique to detect cardiac signals in fetuses between Day 173 of gestation and foaling. Fetal HR decreased from 115 ± 4 beats/min (Days 170 to 240 of gestation) to 83 ± 3 beats/min (Day 320) to 79 ± 1 beats/min (1 d before foaling; P < 0.001). Mean beat to beat (RR) interval and standard deviation of the RR interval (SDRR) increased (P < 0.001). Gestational age thus affects RR interval and HR in the equine fetus. From Days 270 to 340 of gestation, SDRR increased from 11.4 ± 1.3 msec on Day 270 to 27.8 ± 3.6 msec on Day 340 (P < 0.05), and the root mean square of successive RR differences (RMSSD) tended to increase (P = 0.07), indicating maturation of the fetal autonomous nervous system. For the last 10 d before foaling, fetal HR and HRV remained constant and did not allow predicting the onset of parturition in the horse. Only during the last 30 min before the foal was born, in 4 of 5 fetuses, HR decreased and RR interval increased. Accelerations and decelerations in HR were detectable at all times, but neither their number nor duration changed over time.     

Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ: I. Pharmacological properties of the electroplaque

The membrane potential (average = ?52 mV) of a freely exposed electroplaque from a dissected prism of Torpedo marmorata electric organ is recorded with an intracellular glass microelectrode. The resting potential decreases with external potassium concentration. Acetylcholine (in the presence of O,O′-diethyl S-(β-diethylamino)ethyl phosphorothiolate), decamethonium, phenyltrimethyl-ammonium and carbamylcholine added to the bath cause a decrease of membrane potential, i.e. behave as agonists. Their effect is blocked in a competitive manner by d-tubocurarine, gallamine and hexamethonium, and in a non-competitive way by prilocaine; 1 μg Erabutoxin/ml completely abolishes the response to carbamylcholine. The apparent dissociation constants for seven cholinergic ligands are determined from the dose-response curves, and found to be closely related to those previously determined with Electrophorus electricus electroplaque with, however, a few differences. During these experiments it was noticed that potassium ions affect, in a differential manner, the response of T. marmorata electroplaque to carbamylcholine and decamethonium.     

Studies on the equine placenta. III. Ultrastructure of the uterine glands and the overlying trophoblast

Ultrastructural studies of the uterine glands at intervals during pregnancy in the mare show that secretory activity continues after formation of the placental exchange units. The nature of the glandular secretion appeared initially to be proteinaceous, but cellular debris was also present during the last third of gestation. These secretions were absorbed by the trophoblast overlying the mouths of the glands. The fate secretions and their significance for the fetus and placenta are unknown.