Beta Decay of Molecular Tritium.

The beta decay of tritium in the form of molecular T_{2} is the basis of sensitive experiments to measure neutrino mass. The final-state electronic, vibrational, and rotational excitations modify the beta spectrum significantly and are obtained from theory. We report measurements of the branching ratios to specific ionization states for the isotopolog HT. Two earlier, concordant measurements gave branching ratios of HT to the bound HHe^{+} ion of 89.5% and 93.2%, in sharp disagreement with the theoretical prediction of 55%-57%, raising concerns about the theory's reliability in neutrino mass experiments. Our result, 56.5(6)%, is compatible with the theoretical expectation and disagrees strongly with the previous measurements.

Physiological and molecular responses to an acute bout of reduced-exertion high-intensity interval training (REHIT).

PURPOSE: We have previously shown that 6 weeks of reduced-exertion high-intensity interval training (REHIT) improves VO2max in sedentary men and women and insulin sensitivity in men. Here, we present two studies examining the acute physiological and molecular responses to REHIT. METHODS: In Study 1, five men and six women (age: 26 ± 7 year, BMI: 23 ± 3 kg m(-2), VO2max: 51 ± 11 ml kg(-1) min(-1)) performed a single 10-min REHIT cycling session (60 W and two 20-s 'all-out' sprints), with vastus lateralis biopsies taken before and 0, 30, and 180 min post-exercise for analysis of glycogen content, phosphorylation of AMPK, p38 MAPK and ACC, and gene expression of PGC1α and GLUT4. In Study 2, eight men (21 ± 2 year; 25 ± 4 kg·m(-2); 39 ± 10 ml kg(-1) min(-1)) performed three trials (REHIT, 30-min cycling at 50 % of VO2max, and a resting control condition) in a randomised cross-over design. Expired air, venous blood samples, and subjective measures of appetite and fatigue were collected before and 0, 15, 30, and 90 min post-exercise. RESULTS: Acutely, REHIT was associated with a decrease in muscle glycogen, increased ACC phosphorylation, and activation of PGC1α. When compared to aerobic exercise, changes in VO2, RER, plasma volume, and plasma lactate and ghrelin were significantly more pronounced with REHIT, whereas plasma glucose, NEFAs, PYY, and measures of appetite were unaffected. CONCLUSIONS: Collectively, these data demonstrate that REHIT is associated with a pronounced disturbance of physiological homeostasis and associated activation of signalling pathways, which together may help explain previously observed adaptations once considered exclusive to aerobic exercise.

Moving the insulin-regulated glucose transporter GLUT4 into and out of storage.

The glucose transporter isoform GLUT4 is unique among the glucose transporter family of proteins in that, in resting cells, it is sequestered very efficiently in a storage compartment. In insulin-sensitive cells, such as fat and muscle, insulin stimulation leads to release of GLUT4 from this reservoir and its translocation to the plasma membrane. This process is crucial for the control of blood and tissue glucose levels. Investigations of the composition and structure of the GLUT4 storage compartment, together with the targeting motifs that direct GLUT4 to this compartment, have been extensive but have been controversial. Recent findings have now provided a clearer consensus of opinion on the mechanisms involved in the formation of this storage compartment. However, another controversy has now emerged, which is unresolved. This concerns the issue of whether the insulin-regulated step occurs at the level of release of GLUT4 from the storage compartment or at the level at which released vesicles fuse with the plasma membrane.

Leucosulfakinin, a sulfated insect neuropeptide with homology to gastrin and cholecystokinin.

A sulfated, myotropic neuropeptide termed leucosulfakinin (Glu-Gln-Phe-Glu-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2) was isolated from head extracts of the cockroach Leucophaea maderae. The peptide exhibits sequence homology with the hormonally active portion of the vertebrate hormones human gastrin II and cholecystokinin, suggesting that these peptides are evolutionarily related. Six of the 11 amino acid residues (55 percent) are identical to those in gastrin II. In addition, the intestinal myotropic action of leucosulfakinin is analogous to that of gastrin.

Meaningful use's benefits and burdens for US family physicians.

Objective: The federal meaningful use (MU) program was aimed at improving adoption and use of electronic health records, but practicing physicians have criticized it. This study was aimed at quantifying the benefits (ie, usefulness) and burdens (ie, workload) of the MU program for practicing family physicians. Materials and Methods: An interdisciplinary national panel of experts (physicians and engineers) identified the work associated with MU criteria during patient encounters. They conducted a national survey to assess each criterion's level of patient benefit and compliance burden. Results: In 2015, 480 US family physicians responded to the survey. Their demographics were comparable to US norms. Eighteen of 31 MU criteria were perceived as useful for more than half of patient encounters, with 13 of those being useful for more than two-thirds. Thirteen criteria were useful for less than half of patient encounters. Four useful criteria were reported as having a high compliance burden. Discussion: There was high variability in physicians' perceived benefits and burdens of MU criteria. MU Stage 1 criteria, which are more related to basic/routine care, were perceived as beneficial by most physicians. Stage 2 criteria, which are more related to complex and population care, were perceived as less beneficial and more burdensome to comply with. Conclusion: MU was discontinued, but the merit-based incentive payment system within the Medicare Access and CHIP Reauthorization Act of 2015 adopted its criteria. For many physicians, MU created a significant practice burden without clear benefits to patient care. This study suggests that policymakers should not assess MU in aggregate, but as individual criteria for open discussion.

A new deadly Syn?

Insulin-mediated regulation of the exocytosis of vesicles containing the glucose transporter GLUT4 has similarities to regulated synaptic transmission. A recent study has now identified a key regulated component of the fusion step in the exocytosis of these GLUT4-containing vesicles.

The myth of standardized workflow in primary care.

OBJECTIVE: Primary care efficiency and quality are essential for the nation's health. The demands on primary care physicians (PCPs) are increasing as healthcare becomes more complex. A more complete understanding of PCP workflow variation is needed to guide future healthcare redesigns. METHODS: This analysis evaluates workflow variation in terms of the sequence of tasks performed during patient visits. Two patient visits from 10 PCPs from 10 different United States Midwestern primary care clinics were analyzed to determine physician workflow. Tasks and the progressive sequence of those tasks were observed, documented, and coded by task category using a PCP task list. Variations in the sequence and prevalence of tasks at each stage of the primary care visit were assessed considering the physician, the patient, the visit's progression, and the presence of an electronic health record (EHR) at the clinic. RESULTS: PCP workflow during patient visits varies significantly, even for an individual physician, with no single or even common workflow pattern being present. The prevalence of specific tasks shifts significantly as primary care visits progress to their conclusion but, notably, PCPs collect patient information throughout the visit. DISCUSSION: PCP workflows were unpredictable during face-to-face patient visits. Workflow emerges as the result of a "dance" between physician and patient as their separate agendas are addressed, a side effect of patient-centered practice. CONCLUSIONS: Future healthcare redesigns should support a wide variety of task sequences to deliver high-quality primary care. The development of tools such as electronic health records must be based on the realities of primary care visits if they are to successfully support a PCP's mental and physical work, resulting in effective, safe, and efficient primary care.

Infinite cis influx of cyclic AMP into human erythrocyte ghosts.

Infinite cis uptake of cyclic AMP into red blood cell ghosts has been measured. The Koiic is calculated from two different integrated rate equations that are applicable when the substrate concentration is unsufficient to cause volume changes. Values of 0.69 mM and 0.66 mM are obtained for the infinite cis Km at 30 degrees C using these procedures. These values are only slightly higher than that predicted from zero trans net flux experiments. Lowering the temperature reduces Koiic from 0.69 mM at 30 degrees C to 0.478 mM at 20 degrees C, 0.108 mM at 10 degrees C and 0.072 mM at 4 degrees C (Q10 = 2.4). The Q10 for activation of influx permeability of 10(-5) M cyclic AMP is 1.55.

An allosteric pore model for sugar transport in human erythrocytes.

Glucose transport in human erythrocytes is characterized by a marked asymmetry in the V and Km values for entry and for exit. In addition, they show a high Km and a high V for equilibrium exchange that low Km values for infinite cis and for infinite trans exit and entry. An allosteric pore model has been proposed to account for these characteristics. In this model, substrate-induced conformational changes destabilize the interfaces between protein subunits (the pore gates). Pores doubly occupied from inside destabilize the transport gates and result in high Km and high V transport parameters. This effect is less marked when pores are doubly occupied from outside and therefore transport asymmetry results.

Cyclic AMP transport in human erythrocyte ghosts.

10(-5) M cyclic AMP has high permeability in human erythrocyte ghosts (p = 0.061-10(-6) cm.s-1). Saturation of influx and efflux occurs. Koizt = 4.43 mM. Voizt = 259.6 micron.min-1-Kiozt = 0.475 micron. Viozt = 28.3 micron.min-1 at 30 degrees C. Equilibrium exchange entry of cyclic AMP has similar kinetics to zero trans influx, though the system does show counterflow. Cytochalasin B is an apparent competitive inhibitor of cyclic AMP exit. (Ki = 3.9.10(-7) M). Control experiments indicated that cyclic AMP remains intact during incubation with red blood cell ghosts and is contained within the intravesicular space during the transport experiments.

Photolabelling of the hexose transporter at external and internal sites: fragmentation patterns and evidence for a conformational change.

The human erythrocyte sugar transporter has been labelled at its internal site with cytochalasin B and at its outside site by the azidosalicoyl derivative of bis(D-mannose) (ASA-BMPA). The cleavage of the transporter by various proteinases has been studied. Chymotrypsin, subtilisin and V8 proteinase give parallel fragmentation patterns for the two labels down to fragments as small as 7 kDa. Thus the binding sites for the two labels can only be separated by a small span of protein. 2-Nitro-5-thiocyanobenzoic acid (NTCB) cleaves at cysteines to give a 15 kDa fragment from the two labels. N-Bromosuccinimide (a reagent which preferentially cleaves at tryptophan residues) has revealed differences in fragmentation of the transporter labelled with either cytochalasin B or with ASA-BMPA. A major cleavage site is proposed to occur at tryptophan 186 which leaves a C-terminal fragment containing both labels. A tryptophan cleavage at residue 388 divides the cytochalasin B site and the ASA-BMPA site. A further tryptophan cleavage gives a cytochalasin B labelled 3 kDa fragment probably from residues 388-412. This gives an assignment of the cytochalasin B site as the inside of the hydrophobic span H 10. Since the ASA-BMPA site is probably only 7 kDa from residue 388 and is on the same 15 kDa NTCB fragment as cytochalasin B we assign this to the outside of hydrophobic span H 9. Thermolysin only cleaves the transporter labelled with cytochalasin B and not with ASA-BMPA. A 18 kDa cytochalasin B labelled fragment is formed. This is indicative of a change in conformation of the transporter when an outside ligand is bound such that the inside of the hydrogen bonding transmembrane segments H 7 and H 8 (and containing the proposed thermolysin cleavage site) are withdrawn from the cytosolic surface. Thus it appears that the core of the transporter (including the external and internal sites plus the transmembrane channel) is located between segments H 7 and H 10.

D-galactose accumulation in rabbit ileum. Effects of theophylline on serosal permeability.

The effects of theophylline and dibutyryl cyclic AMP, on in vitro unidirectional galactose fluxes across the mucosal and serosal borders of rabbit ileum have been studied. 1. When Ringer [galactose] = 2mM, theophylline and dibutyryl cyclic AMP reduce both mucosal-serosal and serosal-mucosal galactose flux by approx. 50%. The K1 for theophylline inhibition of flux in both directions is 2 mM. 1 mM dibutyryl cyclic AMP elicits a maximal inhibitory response. Concurrent with the inhibition in transmural galactose fluxes, theophylline and dibutyryl cyclic AMP increase the tissue accumulation of [galactose] and the specific-activity ratio R of 3H : 14C-labelled galactose coming from the mucosal and serosal solutions respectively. It is deduced that theophylline and dibutyryl cyclic AMP are without effect on the mucosal unidirectional permeability to galactose but cause a symmetrical reduction in serosal entry and exit permeability. 2. Reduction in the asymmetry of the mucosal border to galactose by reducing Ringer [Na], raising Ringer [galctose] or adding ouabain reduces the theophylline-dependent increase in galactose accumulation. 3. Hypertonicity in the serosal solution increases the permeability of the serosal border to galactose and reduces tissue galactose accumulation. Serosal hypertonicity partially reverses the theophylline-depedent effects on galactose transport. Replacing Ringer chloride by sulphate abolishes the theophylline-dependent effects on galactose transport. 4. It is considered that the theophylline-dependent increase in galactose accumulation results from the reduction in serosal permeability. This is shown to be a quantitatively consistent inference. 5. Further support for the view that the asymmetric transport of galactose in rabbit ileum results from convective-diffusion is presented.

Galactose transport across the serosal border of rabbit ileum and its role in intracellular accumulation.

Unidirectional fluxes of D-galactose across the brush and serosal border of rabbit ileum were determined using the method described previously (Naftalin, R. J. and Curran, P.F. (1974) J. Membrane Biol. 16, 257-278). With ringer [Na] equals 75 meguiv., the Km for galactose influx across the brush-border is 5mM, with 0.1 mM ouabain present K-m equals 50 mM, the V (2.0 munol - CM-2-H-1) remains unaltered. The Michaelis parameters for galactose influx across the serosal border are K-m equals 59 plus or minus 9 mM and V equals 4.7 plus or minus 0.24 mumol-cm-2-h-1 and for efflux K-m equals 85 plus or minus 10 mM and V equals 6.8 plus or minus 0.7 mumol-CM-2-H-1. 2. 2-Deoxy-D-glucose and methyl beta-D-glucopyranoside inhibit galactose entry exclusively at the serosal and mucosal borders respectively, while 3-O-methyl-D-glucose inhibits galactose influx at both borders. 0.1 mM ouabain increases the K1 of 3-O-methylglucose for the serosal transport system (100 mM) is unaffected by ouabain. Inhibition of mucosal galactose transport by ouabain or by competition with other sugars results in a reciprocal increase in exit permeability and decrease in entry permeability. Inhibition of serosal galactose transport results in inhibition of both the entry and exit permeability, entry is more affected. 3. There is a small degree of permeability asymetry at the serosal border to galactose which is reduced by ouabain or removel of Na+ from the Ringer. Uptake of 14C-labelled galactose from the serosal solution into the tissue is also inhibited by addition of ouabain or Na+ removal. It is therefore considered that there is a weak active transport system for galactose at the serosal border. 4. Net transepithelial galactose flux is sufficiently high and serosal permeability to galactose sufficiently low to be consistent with the view that galactose is concentrated within the tissue fluid, after conviction (Naftalin, R.J. and Holman, G.D. (1974) Biochim. Biophys. Acta., 373, 453-470) across the mucosal border because it is reflected at the serosal boundary.

Symmetrical kinetic parameters for 3-O-methyl-D-glucose transport in adipocytes in the presence and in the absence of insulin.

3-O-Methyl-D-glucose transport in isolated adipocytes in the presence of insulin is symmetric in zero-trans experiments (transport into sugar-free solutions). K oi zt = 6.10 +/- 1. 65 mM, V oi zt = 1.20 +/- 0.19 mM/s; K io zt = 2.66 +/- 0.26 mM, V io zt = 1.19 +/- 0.07 mM/s. (The superscripts o and i and subscript zt refer to outside, inside and zero-trans conditions, respectively). In the absence of insulin K oi zt = 5.41 +/ 0.98 mM, V oi zt = 0.034 +/- 0.014 mM/s; K io zt = 4.09 +/- 1.05 mM, V io zt = 0.153 +/- 0.023 mM/s. For insulin pre-treated cells, infinite-cis experiments (transport into solutions of varying sugar concentrations) are also symmetric K oi ic = 6.51 +/- 0.83 mM, V oi ic = 0.09 mM/s; K io ic = 3.60 +/- 1.33 mM, V io ic = 1.76 +/- 0.63 mM/S (the subscript ic refers to the infinite-cis condition). In the absence of insulin, K oi ic = 9.03 +/- 3.28 mM, V oi ic = 0.066 +/- mM/s; K io ic = 4.54 +/- 1.32 mM, V io ic = 0.106 +/- 0.026 mM/s. The infinite-cis parameters are shown to be technically easier to measure than the zero-trans parameters. The uses of integrated rate equations for studying rapid transport are demonstrated. The results show that the adipocyte sugar transport system handles 3-O-methyl-D-glucose symmetrically, and that insulin does not change either the internal or the external affinity constants for this glucose analogue.

Hydrogen bonding requirements for the insulin-sensitive sugar transport system of rat adipocytes.

(1) The t 1/2 for 1.3 mM D-allose uptake and efflux in insulin-stimulated adipocytes is 1.7 +/- 0.1 min. In the absence of insulin mediated uptake of D-allose is virtually eliminated and the uptake rate (t 1/2 = 75.8 +/- 4.99 min) is near that calculated for nonmediated transport. The kinetic parameters for D-allose zero-trans uptake in insulin-treated cells are Koizt = 271.3 +/- 34.2 mM, Voizt = 1.15 +/- 0.12 mM . s-1. (2) A kinetic analysis of the single-gate transporter (carrier) model interacting with two substrates (or substrate plus inhibitor) is presented. The analysis shows that the heteroexchange rates for two substrates interacting with the transporter are not unique and can be calculated from the kinetic parameters for each sugar acting alone with the transporter. This means that the equations for substrate analogue inhibition of the transport of a low affinity substrate such as D-allose can be simplified. It is shown that for the single gate transporter the Ki for a substrate analogue inhibitor should equal the equilibrium exchange Km for this analogue. (3) Analogues substituted at C-1 show a fused pyranose ring is accepted by the transporter. 1-Deoxy-D-glucose is transported but has low affinity for the transporter. High affinity can be restored by replacing a fluorine in the beta-position at C-1. The Ki for D-glucose = 8.62 mM; the Ki for beta-fluoro-D-glucose = 6.87 mM. Replacing the ring oxygen also results in a marked reduction in affinity. The Ki for 5-thio-D-glucose = 42.1 mM. (4) A hydroxyl in the gluco configuration at C-2 is not required as 2-deoxy-D-galactose (Ki = 20.75 mM) has a slightly higher affinity than D-galactose (Ki = 24.49 mM). A hydroxyl in the manno configuration at C-2 interferes with transport as D-talose (Ki = 35.4 mM) has a lower affinity than D-galactose. (5) D-Allose (Km = 271.3 mM) and 3-deoxy-D-glucose (Ki = 40.31 mM) have low affinity but high affinity is restored by substituting a fluorine in the gluco configuration at C-3. The Ki for 3-fluoro-D-glucose = 7.97 mM. (6) Analogues modified at C-4 and C-6 do not show large losses in affinity. However, 6-deoxy-D-glucose (Ki = 11.08 mM) has lower affinity than D-glucose and 6-deoxy-D-galactose (Ki = 33.97 mM) has lower affinity than D-galactose. Fluorine solution at C-6 of D-galactose restores high affinity. The Ki for 6-fluoro-D-galactose = 6.67 mM. Removal of the C-5 hydroxymethyl group results in a large affinity loss. The Ki for D-xylose = 45.5 mM. The Ki for L-arabinose = 49.69 mM. (7) These results indicate that the important hydrogen bonding positions involved in sugar interaction with the insulin-stimulated adipocytes transporter are the ring oxygen, C-1 and C-3. There may be a weaker hydrogen bond to C-6. Sugar hydroxyls in non-gluco configurations may sterically hinder transport.

The effects of removal of sodium ions from the mucosal solution on sugar absorption by rabbit ileum.

Net absorption and accumulation of D-galactose, beta-methyl D-glucose and low concentrations of 3-0-methyl-D-glucose by sheets of rabbit ileum are observed even when Na+ in the mucosal solution is replaced by choline. This indicates that active sugar transport can occur in the direction opposite to the brush-border Na+ gradient.

Transport of 3-O-methyl D-glucose and beta-methyl D-glucoside by rabbit ileum.

The intestinal transport of three actively transported sugars has been studied in order to determine mechanistic features that, (a) can be attributed to stereo-specific affinity and (b) are common. The apparent affinity constants at the brush-border indicate that sugars are selected in the order, beta-methyl glucose greater than D-galactose greater than 3-O-methyl glucose, (the Km values are 1.23, 5.0 and 18.1 mM, respectively.) At low substrate concentrations the Kt values for Na+ activation of sugar entry across the brush-border are: 27, 25, and 140 mequiv. for beta-methyl glucose, galactose and 3-O-methyl glucose, respectively. These kinetic parameters suggest that Na+, water, sugar and membrane-binding groups are all factors which determine selective affinity. In spite of these differences in operational affinity, all three sugars show a reciprocal change in brush-border entry and exit permeability as Ringer (Na) or (sugar) is increased. Estimates of the changes in convective velocity and in the diffusive velocity when the sugar concentration in the Ringer is raised reveal that with all three sugars, the fractional reduction in convective velocity is approximately equal to the (reduction of diffusive velocity)2. This is consistent with the view that the sugars move via pores in the brush-border by convective diffusion. Theophylline reduces the serosal border permeability to beta-methyl glucose and to 3-O-methyl glucose relatively by the same extent and consequently, increase the intracellular accumulation of these sugars. The permeability of the serosal border to beta-methyl glucose entry is lower than permeability of the serosal border to beta-methyl glucose exit, which suggested that beta-methyl glucose may be convected out of the cell across the lateral serosal border.

Photolabeling of erythrocyte and adipocyte hexose transporters using a benzophenone derivative of bis(D-mannose).

The benzophenone derivative of 1,3-bis(D-mannos-4-yloxy)-2-propylamine (BB-BMPA) has been tested as an exofacial photoaffinity label for the sugar transport systems of human erythrocytes and rat adipocytes. The half-maximal inhibition constants for the reagent are 971 microM in erythrocytes and 536 microM in basal and 254 microM in insulin-treated adipocytes. The photolabelling of erythrocyte membranes is very specific for the 50 kDa transporter peptide and is completely displaced by D-glucose. The exofacial photoaffinity labelling of adipocytes also shows labelling of a 50 kDa transporter peptide, which is displaced by cytochalasin B, but extensive nonspecific labelling of a 75 kDa plasma membrane peptide occurs. The transporter is labelled in insulin-treated cells but not in basal cells which indicates that this in situ labelling technique selectively reveals only those transporters that visit and are active in the plasma membrane during the labelling period. This also indicates that in basal cells transporters do not turn over rapidly. Subcellular redistribution of transporters after the labelling period has been studied. Following incubation and washing at 37 degrees C in the presence of insulin, 30% of the transporters photolabelled at the plasma membrane are internalised and are found in the light microsome fraction of the cell. The proportion of transporter that is observed to be internalised is much greater than can be accounted for by a contamination of the light microsome fraction by plasma membrane. The labelled 50 kDa transporter peptide in the light microsomes is enriched when compared with the carry-over of the 75 kDa nonspecifically labelled plasma membrane peptide. Thus we have obtained direct evidence for transporter translocation.

Specificity and kinetics of hexose transport in Trypanosoma brucei.

Transport of 6-deoxy-D-glucose was studied in Trypanosoma brucei in order to characterise the kinetics of hexose transport in this organism using a nonphosphorylated sugar. Kinetic parameters for efflux and entry, measured using zero-trans and equilibrium exchange protocols, indicate that the transporter is probably kinetically symmetrical. Comparison of the kinetic constants of D-glucose metabolism with those for 6-deoxy-D-glucose transport shows that transport across the plasma membrane is likely to be the rate-limiting step of glucose utilisation. The transport rate is nevertheless very fast and 6-deoxy-D-glucose, at concentrations below Km, enters the cells with a half filling time of less than 2 s at 20 degrees C. Thus the high metabolic capacity of these organisms is matched by a high transport rate. The structural requirements for the trypanosome hexose transporter were explored by measuring inhibition constants (Ki) for a range of D-glucose analogues including fluoro and deoxy sugars as well as epimeric hexoses. The relative affinities shown by these analogues indicated H-bonds from the carrier to the C-3, C-4 and C-5 hydroxyl oxygens and from the C-1 and C-3 hydroxyl hydrogens to the binding site. Hydrophobic interactions are likely at the C-2 and C-6 regions of the glucose molecule. Spatial constraints appear to occur around C-4 indicating that the transport site at this position is not freely open to the external solution as is the case with the mammalian hexose transporter. However, the trypanosome transporter appears to accept D-fructose but the common mammalian (erythrocyte type) hexose transporter does not.

Binding of cytochalasin B to trypsin and thermolysin fragments of the human erythrocyte hexose transporter.

The cleavage of the human erythrocyte hexose transporter by the proteinases trypsin and thermolysin has been studied. When red cell membranes are treated with trypsin, washed and then photolabelled with cytochalasin B, a labelled peak at 18 kDa is obtained. This labelling of the cleaved transporter is D-glucose inhibitable. This probably indicates that the residual 36 kDa portion of the transporter is not required for binding of ligands. Extensive cleavage of the transporter with low concentrations of thermolysin only occurs when transporter is prelabelled with cytochalasin B. This indicates that covalently bound cytochalasin B can cause a conformational change which exposes the thermolysin cleavage site.