The mitochondrial acetoacetyl-CoA thiolase (T2) deficiency in Japanese patients: urinary organic acid and blood acylcarnitine profiles under stable conditions have subtle abnormalities in T2-deficient patients with some residual T2 activity.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism affecting isoleucine and ketone bodies in the catabolic process. Mutation analysis and expression analysis of mutant cDNAs have facilitated the division of T2-deficient patients into two groups: those with null mutations in either allele (group 1) and those with mutation(s) retaining some residual T2 activity in at least one of two mutant alleles (group II). Among 5 Japanese T2-deficient patients, GK01 belonged to group I and the other patients (GK19, GK19B, GK30 and GK31) to group II. As we have suggested previously, the severity of ketoacidotic episodes in the group II patients was similar to that in the group I patient. However, the urinary organic acid and blood spot acylcarnitine profiles under stable conditions differed between the two groups. The group I patient had typical profiles for the T2 deficiency. In contrast, in all four patients in group II, tiglylglycine was not or was only faintly detected and the 2-methyl-3-hydroxybutyrate levels were less than the cutoff value. Their tiglylcarnitine levels were within the normal range and 2-methyl-3-hydroxy-, butyrylcarnitine was detected just around the cutoff value in our newborn screening pilot test. Hence, these analyses under stable conditions are not reliable for diagnosing the T2 deficiency in the group II patients. The T2 deficiency (group II) can be misdiagnosed as normal if these analyses are performed under nonepisodic conditions and possibly during the newborn screening for inborn errors of metabolism.

[Usefulness of a simple expiratory gas monitor during sedation under spinal anesthesia].

We introduce a simple expiratory gas monitor during sedation under spinal anesthesia. A small extension tube for infusion used as a gas sampling line is placed in the nasal vestibule. It is necessary to make it sure that the point of the tube should not contact with the mucous membrane of the nose. Our method needs no special equipments such as Capnoxygen or Nazorcap, but a cheap extension tube available in any operating room. Therefore this is a simple method. Expiratory gas monitor can detect apnea early, airway obstruction and stenosis and predict PaCO2 during sedation. The change of fractional concentration of oxygen in inspired gas predicts the change of tidal volume. Increase in the former reflects a decrease in the latter under the administration of oxygen. It is possible to evaluate whether sedation became steady with analysis of respiratory pattern. However, nasal discharge may interrupt monitoring expiratory gas. Our simple method to monitor expiratory gas is useful during sedation under regional anesthesia.

[Effects of infusion methods of propofol on quality of sedation and ease of sedation control during gynecological laparotomy under spinal anesthesia].

Twenty patients were prospectively and randomly studied to investigate effects of infusion methods of propofol on quality of sedation and ease of sedation control during gynecological laparotomy under spinal anesthesia. After establishment of spinal anesthesia, patients were randomly assigned to one of the following two groups, i.e. conventional continuous infusion group (Cont group) and target-controlled infusion group (TCI group). In the Cont group, propofol was started at a rate of 6 mg.kg-1.hr-1 until response to command disappeared. In the TCI group, the initial target concentration of propofol was set at 1.2 micrograms.ml-1 until response to command disappeared. Thereafter infusion rate or target concentration was adjusted to maintain Mackenzie's score at 3 or 4. Predicted concentration of propofol was 1.2 +/- 0.01 micrograms.ml-1 at induction of sedation and 1.2 +/- 0.11 micrograms.ml-1 during stable sedation in the TCI group. Satisfaction VAS, anxiety VAS, discomfort VAS, sedation score and times of changing infusion condition were similar in both groups. Total dose of propofol was significantly less in the TCI group. In conclusion, quality of sedation and ease of control of sedation were comparable in both groups and continuous infusion method is simple.

Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis.

A major hallmark of apoptosis is normotonic shrinkage of cells. Here, we studied the relation between apoptotic cell shrinkage and apoptotic cell death. Induction of the apoptotic volume decrease (AVD) under normotonic conditions was found to be coupled to facilitation of the regulatory volume decrease (RVD), which is known to be attained by parallel operation of Cl(-) and K(+) channels, under hypotonic conditions. Both the AVD induction and the RVD facilitation were found to precede cytochrome c release, caspase-3 activation, DNA laddering, and ultrastructural alterations in three cell types after apoptotic insults with two distinct apoptosis inducers. Also, the AVD was not prevented by a broad-spectrum caspase inhibitor. When the AVD induction and the RVD facilitation were prevented by blocking volume-regulatory Cl(-) or K(+) channels, these cells did not show succeeding apoptotic biochemical and morphological events and were rescued from death. Thus, it is concluded that the AVD, which is caused by disordered cell volume regulation, is an early prerequisite to apoptotic events leading to cell death.

[Anesthesia for MIDCAB (minimally invasive direct coronary bypass) in a patient with Tangier disease: a case report].

Tangier disease is a rare, autosomally-inherited disorder of lipoprotein metabolism characterized by absence or marked deficiency of normal high density lipoprotein (HDL) cholesterol in plasma resulting in the accumulation of cholesteryl esters in various organs. The patient was a 55-yr-old male diagnosed as Tangier disease 16 years before. He had angina on exercise and his coronary angiogram revealed triple vessel disease including left main trunk (LMT) lesion. Stenosis of the right coronary artery was treated by percutaneous transluminal coronary angioplasty (PTCA). He was scheduled for a MIDCAB for further PTCA to be performed to relieve the stenosis of LMT. Preoperative laboratory data and physical examination showed total cholesterol 36 mg.dl-1, HDL-cholesterol 2 mg.dl-1, apoprotein A-I not-detected, pancytopenia, hyperplastic orange tonsils, splenomegaly and hepatomegaly. Clonidine 0.225 mg was orally given as a preanesthetic medication. Anesthesia was induced with fentanyl and midazolam and maintained with propofol, sevoflurane and supplemental fentanyl. Nitroglycerin and diltiazem were infused continuously. ST segment was elevated transiently during the clamping of the left anterior descending branch. Hemodynamic parameters were stable during the operation. He was extubated 2 hours after the end of the operation. No significant changes were found in postoperative EKG, total cholesterol, HDL-cholesterol and triglyceride. Perioperative course was uneventful.

Swelling-activated, cystic fibrosis transmembrane conductance regulator-augmented ATP release and Cl- conductances in murine C127 cells.

1. A hypotonic challenge, but not cAMP stimulation, was found to induce release of ATP measured by the luciferin-luciferase assay from both the murine mammary carcinoma cell line C127i and C127 cells stably transfected with the cDNA for human cystic fibrosis transmembrane conductance regulator (CFTR) protein (C127/CFTR). CFTR expression augmented swelling-induced ATP release by 10-20 times under hypotonic conditions (< or = 80 % osmolality). 2. Glibenclamide failed to suppress swelling-induced ATP release from C127/CFTR cells. In contrast, whole-cell patch-clamp recordings showed that both the cAMP-activated ohmic Cl- currents and volume-sensitive outwardly rectifying (VSOR) Cl- currents were prominently suppressed by glibenclamide. 3. Gd3+ markedly blocked swelling-induced ATP release but failed to suppress both cAMP- and swelling-activated Cl- currents in the CFTR-expressing cells. Even after pretreatment and during treatment with Gd3+, VSOR Cl- currents were activated normally. 4. The continuous presence of an ATP-hydrolysing enzyme, apyrase, in the bathing solution did not prevent activation of VSOR Cl- currents in C127/CFTR cells. 5. The rate of regulatory volume decrease (RVD) in C127/CFTR cells was much faster than that in C127i cells. When apyrase was added to the bathing solution, the RVD rate was retarded in C127/CFTR cells. 6. On balance, the following conclusions can be deduced. First, swelling-induced ATP release is augmented by expression of CFTR but is not mediated by the CFTR Cl- channel. Second, swelling-induced ATP release is not mediated by the VSOR Cl- channel. Third, the released ATP facilitated the RVD process but is not involved in the activation of VSOR Cl- channels in C127/CFTR cells.

Midazolam premedication reduces propofol requirements for sedation during regional anesthesia.

PURPOSE: Propofol is often used for sedation during spinal anesthesia. We investigated the effects of midazolam premedication on the propofol requirements and incidence of complications during sedation. METHODS: In a prospective randomized, controlled, and single-blinded study, 50 patients undergoing elective gynecological surgery were randomly divided into control and midazolam groups. Patients in the midazolam group received 2 mg midazolam im 30 min before arrival at the operation room. After spinal anesthesia was instituted with intrathecal injection of hyperbaric tetracaine, we provided sedation using continuous infusion of propofol. The level of sedation was controlled at a level between "eyes closed but rousable to command" and "eyes closed but rousable to mild physical stimulation" by adjusting the infusion rate. During sedation, the propofol requirements and complications were recorded and patients were asked, two hours after the end of operation, whether they remembered intraoperative events. RESULTS: In the midazolam group, the loading dose, steady state infusion rate, and overall infusion rate of propofol were 0.74 mg x kg(-1), 2.86 mg x kg(-1) x hr(-1), and 3.32 mg x kg(-1) x hr(-1), respectively, which were about 17% lower than those in the control group (P<0.05). Moreover, midazolam premedication reduced the incidence of intraoperative memory (P < 0.05), but had no effects on other complications. CONCLUSION: Midazolam premedication reduced propofol requirements and the incidence of intraoperative memory during sedation. These effects on sedation using propofol during spinal anesthesia are considered beneficial for patients.

Swelling-induced, CFTR-independent ATP release from a human epithelial cell line: lack of correlation with volume-sensitive cl(-) channels.

To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl(-) channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl(-) currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56-80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl(-) channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 microM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl(-) channel blocker, 4-acetamido-4'-isothiocyanostilbene at 100 microM. Glibenclamide (500 microM) and arachidonic acid (100 microM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl(-) channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd(3+), a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl(-) currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach approximately 13 microM by a biosensor technique using P2X(2) receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl(-) channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 microM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl(-) channel; and third, the ATP release is not a prerequisite to activation of the Cl(-) channel.

Brachial plexus birth injuries: anaesthesia for surgical nerve reconstruction and preoperative myelography and computed tomographic myelography.

Surgical nerve reconstruction for brachial plexus birth injuries and preoperative myelography and computed tomographic (CT) myelography require special anaesthetic considerations. Anaesthesia and medical records were retrospectively reviewed for the infants who underwent myelography, CT myelography (n=37) and microsurgical nerve reconstruction (n=34) at our institution from January 1993 to August 1996. Anaesthetic considerations include long duration of operation, perioperative respiratory complications and plaster application which makes reintubation difficult. Myelography for diagnosis requires a specific positioning of the patient with the head fixed in a midline and prone position.

Rapid gating and anion permeability of an intracellular aquaporin.

Aquaporin (AQP) water-channel proteins are freely permeated by water but not by ions or charged solutes. Although mammalian aquaporins were believed to be located in plasma membranes, rat AQP6 is restricted to intracellular vesicles in renal epithelia. Here we show that AQP6 is functionally distinct from other known aquaporins. When expressed in Xenopus laevis oocytes, AQP6 exhibits low basal water permeability; however, when treated with the known water channel inhibitor, Hg2+, the water permeability of AQP6 oocytes rapidly rises up to tenfold and is accompanied by ion conductance. AQP6 colocalizes with H+-ATPase in intracellular vesicles of acid-secreting alpha-intercalated cells in renal collecting duct. At pH less than 5.5, anion conductance is rapidly and reversibly activated in AQP6 oocytes. Site-directed mutation of lysine to glutamate at position 72 in the cytoplasmic mouth of the pore changes the cation/anion selectivity, but leaves low pH activation intact. Our results demonstrate unusual biophysical properties of an aquaporin, and indicate that anion-channel function may now be explored in a protein with known structure.

Tyrosine kinase-independent extracellular action of genistein on the CFTR Cl- channel in guinea pig ventricular myocytes and CFTR-transfected mouse fibroblasts.

The effects of genistein, a protein tyrosine kinase inhibitor, on the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel were studied in guinea pig ventricular myocytes and in NIH3T3 mouse fibroblasts stably transfected with CFTR cDNA by the whole-cell patch-clamp technique. Genistein did not activate whole-cell Cl- currents when applied to the intracellular (pipette) solution. In contrast, when applied to the extracellular solution, genistein alone promptly activated the Cl- current in a fully reversible manner. Also, extracellular genistein reversibly potentiated the forskolin-activated Cl- current. However, both basal and forskolin-activated Cl- currents were not affected by other protein tyrosine kinase inhibitors, including herbimycin A, lavendustin A, tyrphostin 21, tyrphostin 47, and tyrphostin 51. A nonspecific inhibitor of protein phosphatases, orthovanadate, had no effect on the genistein-induced activation of CFTR. Pretreatment with a protein kinase inhibitor, either H-89 or H-7, or with an adenylate cyclase inhibitor, SQ 22536, also had no effect on the genistein-induced response. Thus, it is concluded that genistein alone activates CFTR by a protein tyrosine kinase-independent and protein phosphatase-independent mechanism from the extracellular side, but not from the intracellular side.

Cell surface measurements of ATP release from single pancreatic beta cells using a novel biosensor technique.

To examine the possibility that ATP modulates insulin secretion by an autocrine mechanism, we measured the local concentration of released ATP at the surface of a single pancreatic beta cell by a new biosensor technique, using PC12 cells expressing ligand-gated cation channels, P2X2 receptors. Upon application of glucose or glibenclamide, a series of current spikes, whose amplitude equates to an ATP concentration of over 25 microM, were recorded from a PC12 cell using the whole-cell patch-clamp technique, when placed near a rat pancreatic beta cell at 37 degrees C. The current response was inhibited by cooling (below 30 degrees C) or by applying an ATP-hydrolysing enzyme (apyrase) or a P2 receptor blocker (suramin). Thus, it is concluded that pancreatic beta cells secrete ATP in response to glucose stimulation, thereby increasing the ATP concentration close to the cell surface sufficiently high enough to enhance insulin secretion from the pancreatic beta cells.

Activation of cAMP-dependent C1- currents in guinea-pig paneth cells without relevant evidence for CFTR expression.

1. To determine whether Paneth cells exhibit functional expression of cAMP-activated Cl- currents and molecular expression of the cystic fibrosis transmembrane conductance regulator (CFTR), we applied whole-cell patch clamp and single-cell mRNA analysis by reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification to single Paneth cells in crypts isolated from the guinea-pig small intestine. 2. Prominent activation of Cl- currents was consistently observed after stimulation with dibutyryl cAMP and forskolin or with vasoactive intestinal polypeptide (VIP). The cAMP-activated Cl- current was inhibited by removal of intracellular ATP or administration of an inhibitor of protein kinase A. 3. Many of the biophysical and pharmacological properties of the currents were phenotypically similar to those of the CFTR Cl- channel, such as the ohmic current-voltage relationship, the anion selectivity with a Type III sequence (Br- > Cl- > I- >> F- >= gluconate-), I--induced blockage, insensitivity to a stilbene-derivative Cl- channel blocker, and sensitivity to a carboxylate analogue Cl- channel blocker. The sensitivity of the current to glibenclamide was, however, much weaker than that reported for the CFTR Cl- channel current. In contrast to the time independence of CFTR currents, the inward component of the Paneth cell Cl- currents exhibited inactivation kinetics. 4. Expression of CFTR mRNA could not be detected by RT-PCR analysis in almost all single Paneth cells, although its expression was consistently detected at the whole-crypt level. The presence of a small number of CFTR-expressing epithelial cells, which were scattered both in villi and crypts but not at the crypt base where Paneth cells were located, was demonstrated by immunocytochemistry. 5. Taken together, it appears that guinea-pig Paneth cells functionally express cAMP-activated Cl- conductance without relevant evidence for molecular expression of CFTR. Functional expression of VIP receptors in the Paneth cells was also demonstrated.

Presteady-state currents of the rabbit Na+/glucose cotransporter (SGLT1).

The rabbit Na+/glucose cotransporter (SGLT1) exhibits a presteady-state current after step changes in membrane voltage in the absence of sugar. These currents reflect voltage-dependent processes involved in cotransport, and provide insight on the partial reactions of the transport cycle. SGLT1 presteady-state currents were studied as a function of external Na+, membrane voltage Vm, phlorizin and temperature. Step changes in membrane voltage-from the holding Vh to test values, elicited transient currents that rose rapidly to a peak (at 3-4 msec), before decaying to the steady state, with time constants tau approximately 4-20 msec, and were blocked by phlorizin (Ki approximately 30 microm). The total charge Q was equal for the application of the voltage pulse and the subsequent removal, and was a function of Vm. The Q-V curves obeyed the Boltzmann relation: the maximal charge Qmax was 4-120 nC; V0.5, the voltage for 50% Qmax was -5 to +30 mV; and z, the apparent valence of the moveable charge, was 1. Qmax and z were independent of Vh (between 0 and -100 mV) and temperature (20-30 degrees C), while increasing temperature shifted V0.5 towards more negative values. Decreasing [Na+]o decreased Qmax, and shifted V0.5 to more negative voltages 9by -100 mV per 10-fold decrease in [Na+]o). The time constant tau was voltage dependent: the tau-V relations were bell-shaped, with maximal taumax 8-20 msec. Decreasing [Na+]o decreased taumax, and shifted the tau-V curves towards more negative voltages. Increasing temperature also shifted the tau-V curves, but did not affect taumax. The maximum temperature coefficient Q10 for tau was 3-4, and corresponds to an activation energy of 25 kcal/mole. Simulations of a 6-state ordered kinetic model for rabbit Na+/glucose cotransport indicate that charge-movements are due to Na+-binding/dissociation and a conformational change of the empty transporter. The model predicts that (i) transient currents rise to a peak before decay to steady-state; (ii) the tau-V relations are bell-shaped, and shift towards more negative voltages as [Na+]o is reduced; (iii) taumax is decreased with decreasing [Na+]o; and (iv) the Q-V relations are shifted towards negative voltages as [Na+]o is reduced. In general, the kinetic properties of the presteady-state currents are qualitatively predicted by the model.

Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus oocytes.

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na+/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (Vm), the external myo-inositol concentration and the external Na+ concentration, yielding the kinetic parameters: KMI0.5, KNa0.5, and the Hill coefficient n. At 100 mM NaCl, KMI0.5 was about 50 microM and was independent of Vm. At 0.5 mM myo-inositol, KNa0.5 ranged from 76 mM at Vm = -50 mV to 40 mM at Vm = -150 mV. n was voltage independent with a value of 1.9 +/- 0.2, suggesting that two Na+ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent KI of 64 microM at Vm = -50 mV and 130 microM at Vm = -150 mV. To examine sugar specificity, sugar-induced steady-state currents (at Vm = -150 mV) were recorded for a series of sugars, each at an external concentration of 50 mM. The substrate selectivity series was myo-inositol, scylloinositol > L-fucose > L-xylose > L-glucose, D-glucose, alpha-methyl-D-glucopyranoside > D-galactose, D-fucose, 3-O-methyl-D-glucose, 2-deoxy-D-glucose > D-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at Vm = -150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: D-glucose, alpha-methyl-D-glucopyranoside, D-galactose, D-fucose, 3-O-methyl-D-glucose > D-xylose, L-xylose, 2-deoxy-D-glucose > myo-inositol, L-glucose, L-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na(+)-dependent uptake of 3H-D-glucose and 3H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mM Na+ that was abolished by phlorizin (0.5 mM). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in Vm. The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model.

Transport of cycasin by the intestinal Na+/glucose cotransporter.

The medicinal and food use of seed from the cycad plant (Cycas spp.), which contains the neurotoxin cycasin, is a proposed etiological factor for amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC), a prototypical neurodegenerative disease found in the western Pacific. Cycasin, the beta-D-glucoside of methylazoxymethanol might enter neurons and other cells via a glucose transporter. Since the intestinal brush-border Na+/glucose cotransporter plays a major role in the absorption of monosaccharides, the following studies were conducted to determine if cycasin, the beta-D-glucoside of methylazoxymethanol, is a substrate for the transporter. We measured the ability of cycasin to (i) inhibit Na+/glucose uptake into rabbit intestinal brush-border membrane vesicles, and (ii) to generate current by the cloned Na+/glucose cotransporter (SGLT1) expressed in Xenopus laevis oocytes. The results show that cycasin inhibits Na(+)-dependent sugar transport in the vesicles, and cycasin generates phlorizin-sensitive currents in oocytes. We conclude that cycasin is a substrate for the intestinal brush-border Na+/glucose cotransporter, albeit with a lower affinity than D-glucose. This suggests that cycasin may be absorbed from the gut lumen by the cotransporter, and as a result either cycasin or the aglycone is presented to the blood-brain barrier for uptake into the brain.

Properties of volume-sensitive Cl- channels in a human epithelial cell line.

A regulatory volume decrease is accomplished by parallel activation of Ca(2+)-dependent K+ channels and Ca(2+)-independent Cl- channels in cultured human intestinal epithelial cells (Intestine 407). The anion selectivity of whole-cell currents recorded in osmotically swollen cells falls into the Eisenman type I sequence corresponding to a low-field anion channel. The volume-sensitive Cl- channel has an intermediate unitary conductance. Both the whole-cell and single-channel Cl- currents exhibit unique voltage-dependency. The Cl- current can be maintained in the activated state in the physiological voltage range. However, at very large depolarizations (over +50 mV), the current is quickly inactivated. The Cl- current shows moderate outward rectification. The whole-cell Cl- current is sensitive to Cl- channel blockers such as SITS and NPPB as well as to cis unsaturated fatty acids such as arachidonic acid and oleic acid. The whole-cell current is totally independent of Ca2+ and cyclic AMP, but inhibited by increases in cytosolic free Mg2+ ions. Removal of intracellular ATP, but not Mg2+, abolishes the Cl- current. The ATP role can be substituted for non-hydrolyzable ATP analogs. Therefore, it is likely that intracellular ATP maintains the channel activity through non-hydrolytic binding.