Sequence of critical events involved in fusion of phospholipid vesicles induced by clathrin.

Membrane fusion induced by clathrin is accompanied by several events such as conformational change, membrane binding and association of clathrin, and membrane aggregation (Maezawa et al. (1989) Biochemistry 28, 1422-1428; Maezawa and Yoshimura (1990) Biochem. Biophys. Res. Commun. 173, 134-140). To clarify the sequence of these events, we examined their time-courses by reducing the pH of the medium from 7.4 to a given pH in the range of 3.5-5.0 at 25 degrees C or 10 degrees C. Large unilamellar vesicles composed of phosphatidylserine and phosphatidylcholine were used in most experiments. The half-time for conformational change of clathrin was less than those for membrane binding and association of clathrin. The half-times and the initial rates of membrane binding and association of clathrin were similar order of magnitude, although the pH-profiles of the initial rates of the two events were somewhat different. Membrane aggregation started after membrane binding of clathrin. A lag phase was observed in the time-course of membrane fusion, whereas there was no lag phase in membrane binding and association of clathrin and membrane aggregation. Moreover, the lag time before fusion was independent of the clathrin concentration, although the initial rates of these three events were dependent on it, suggesting that the three reactions are not responsible for the lag phase before fusion, and that there is some other event(s) in the lag time. On the other hand, there was a threshould-pH in the pH profile of the lag-time and the threshold-pH coincided with the critical pH at which the final associated state of clathrin was apparently reversed in the presence and absence of liposomes, suggesting that the event(s) in the lag phase may be related to this final associated state of clathrin molecules on the liposome membranes. These results indicate that clathrin-induced fusion of liposomes is initiated through the following sequential events: conformational change of clathrin, membrane binding and association of clathrin, which occur simultaneously but independently, membrane aggregation, an event(s) in the lag phase, and actual fusion.

Determination of polypeptide chain molecular weights of human and bovine band 3 protein from erythrocyte membranes by low-angle laser light scattering combined with high-performance gel chromatography in the presence of sodium dodecyl sulfate.

Polypeptide chain molecular weights of human and bovine band 3 proteins which are glycoproteins of the erythrocyte membrane were determined as 101,000 +/- 2000 for the former and 107,000 +/- 2000 for the latter by using the low-angle laser light scattering technique combined with a high-performance gel chromatography column, an ultraviolet spectrophotometer and a differential refractometer in the presence of sodium dodecyl sulfate. The advantage of this method is that, unlike the sedimentation equilibrium technique, neither information on the binding to proteins of all ligands present nor the partial specific volume is required to evaluate the polypeptide chain molecular weight of proteins in a multicomponent system.

Molecular weights of alpha beta-protomeric and oligomeric units of soluble (Na+, K+)-ATPase determined by low-angle laser light scattering after high-performance gel chromatography.

The (Na+, K+)-ATPase of canine renal outer medulla was solubilized with a nonionic surfactant, octaethylene glycol n-dodecyl ether (C12E8), in the presence of 0.2 M sodium ion. The solubilized ATPase retained 74% of the enzymatic activity expressed before solubilization. Molecular species of the solubilized ATPase were analyzed by high-performance chromatography through a TSK-GEL G3000SW column in the presence of 1 mg/ml C12E8 at 23 degrees C. The eluate was monitored by one or two monitors chosen from the following: an ultraviolet absorption monitor, a precision differential refractometer and a low-angle laser light scattering photometer. The three kinds of elution pattern thus obtained can best be interpreted by assuming the presence of at least four kinds of protein component with molecular weights 1 740 000 +/- 230 000, 836 000 +/- 82 000, 286 000 +/- 30 000 and 123 000 +/- 8 000, respectively. Among them, those with the last two molecular weight were the major components. The amounts of the first three components were found to increase with time during the incubation before application to the column at the expense of that of the last one. The amounts of the last two were 18 and 73%, respectively, when measured immediately after the solubilization. A stoichiometric composition of 1:1 molar ratio for the alpha and beta polypeptide chains was obtained for the two major components as well as for the intact ATPase by high-performance gel chromatography in the presence of sodium dodecyl sulfate using the same column as above. The (Na+, K+)-ATPase was, thus, indicated to be solubilized with C12E8 to give the alpha beta-protomer and its dimer as the main components.

Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant.

An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry. It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane. The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.

Determination of subunit molecular weights of canine renal (Na+,K+)-ATPase by low-angle laser light scattering coupled with high performance gel chromatography in the presence of sodium dodecyl sulfate.

Subunit molecular weights of the (Na+,K+)-ATPase of canine renal outer medulla were estimated in the presence of sodium dodecyl sulfate (SDS) by the measuring system consisted of components connected in the following sequence: a TSK-gel G3000 SW column, a UV spectrophotometer, a low-angle laser light scattering photometer, and a differential refractometer. Polypeptide molecular weights of the alpha- and beta-subunits were determined to be 117,900 and 39,400, respectively. The measurement required the extinction coefficient at 280 nm of the sample polypeptide in addition to the outputs from the three detectors. The extinction coefficients at 280 nm of the alpha- and beta-subunits were determined to be 0.931 and 1.41 ml X mg-1 X cm-1, respectively by the quantitative amino acid analysis. The above procedure seems to be most appropriate to determine uniquely the composition of subunits molecular weights of an oligomeric membrane protein.

Exposure of hydrophobic domains of clathrin in its membrane fusion-inducible pH region.

Clathrin induces fusion of phospholipid membranes containing phosphatidylserine when the pH of the medium is reduced below about 6. The hydrophobicity of clathrin in the membrane fusion-inducible pH region was examined. In the presence of clathrin, the fluorescence maximum of 1-anilinonaphthalene-8-sulfonate was shifted to shorter wavelengths and its fluorescence intensity increased at pH values below about 6. Steep increase of the fluorescence intensity of the fluorescent probe, N-(1-anilinonaphthyl-4)maleimide covatently bound to clathrin was observed in a similar pH region. The efficiency of resonance energy transfer from tryptophan to anilinonaphthyl residues in the clathrin molecule showed similar pH dependency. When Triton X-114 solutions containing clathrin were subjected to phase separation by raising the temperature from 0 degree to 30 degrees C at different pH values, clathrin remained in the aqueous phase above pH 6, whereas the protein was partitioned between the aqueous and detergent phases at pH 5-6, and was present only in the detergent phase below pH 5. The effective hydrophobicity of clathrin determined by the fluorescence method using cis-parinaric acid also increased at pH values below 6. Moreover, clathrin was aggregated by lowering the pH below 6. These results show that exposure of hydrophobic domains of clathrin through conformational change occurs in its membrane fusion-inducible pH region, suggesting the crucial role of protein hydrophobicity in the initiation of membrane fusion.

Fusion of phospholipid vesicles induced by clathrin: modulation of fused liposome size.

The effects of lipid composition, temperature, and ionic strength on critical events in the membrane fusion reaction induced by clathrin, such as membrane binding and self-association of clathrin, membrane aggregation, and actual fusion, and the size of fused liposomes, were examined using large unilamellar vesicles (LUV) containing acidic phospholipids. When membrane fusion and aggregation of LUV with different lipid compositions were initiated at 25 degrees C in 0.1 M NaCl by lowering the pH of the medium from 7.40 to 4.75 in the presence of clathrin, two types of reaction processes were observed: fusion and aggregation of LUV containing phosphatidylserine and phosphatidic acid occurred slowly with a long fusion lag-phase and reached a high level, whereas the two reactions of LUV containing phosphatidylglycerol and phosphatidylinositol were induced faster with a much shorter fusion lag-time and leveled off in a shorter time. Similar differences in the fusion reactions were observed in media at different temperatures and ionic strengths with any type of LUV: slow and extensive aggregation and fusion occurred at low temperature and/or high ionic strength, but faster, less extensive reactions occurred at high temperature and/or low ionic strength, indicating that the two types of reaction pattern are due to the dependencies of the aggregation and fusion reactions on the temperature and ionic strength, but not on the lipid composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Pain provocation at lumbar discography as analyzed by computed tomography/discography.

Pain provocation was analyzed in 1477 intervertebral discs in 523 patients subjected to lumbar computed tomography/discography. The relation between pain provocation and the degree of general degeneration and anular disruption assessed according to the Dallas Discogram Description as indices of intradiscal deterioration was investigated. Pain provocation was also evaluated after categorizing the discs by the clinical diagnosis. Pain provocation showed little relation to intradiscal deterioration, whereas a strong relation was found between it and herniated nucleus pulposus. in herniated nucleus pulposus, discs with extraligamentous extrusion or sequestration, large protrusions, maximum protrusion site at the nerve root portion, and herniation routes passing through the central portion of the disc showed a high pain provocation ratio. Pain provocation ratios of discs associated with spinal canal stenosis were extremely low.

Traumatic Korsakoff syndrome.

A case of Korsakoff's syndrome occurring after a fall is reported. The patient was comatose on admission. Cerebral contusions were seen in the frontal lobes bilaterally, left temporal lobe, right caudate nucleus and cerebellar vermis. Cerebral blood flow (CBF) study showed diffuse hyperaemia. Although his conscious state improved gradually, he showed disorientation, antegrate and retrograde amnesia and confabulation 2.5 months later and was diagnosed as having Korsakoff's syndrome. Magnetic resonance imaging (MRI) and CBF studies during this period showed damage in the midbrain and general hypoperfusion. He recovered gradually, confabulation disappeared but a mild memory disturbance remained; hypoperfusion on CBF studies also improved up to the normal range. Among the limbic diencephalic structures, the frontal lobes were thought to be involved most prominently in this case of Korsakoff syndrome.

Coagulation and fibrinolysis study after local thrombolysis of a cerebral artery with urokinase.

Coagulation and fibrinolysis factors were studied in six patients after local thrombolysis with urokinase (720,000 IU). Transient abnormalities, such as prolonged prothrombin time, decreased plasminogen and alpha 2-antiplasmin activities, decreased fibrinogen, and increased fibrin degradation products were seen on the day after thrombolysis, but tended to return to the normal range on the 4th day except for one patient who suffered from disseminated intravascular coagulation. Antithrombin III activity did not change so much. Therefore, the dosage of urokinase should be as low as possible to prevent fluctuations in the coagulation and fibrinolysis system.

Effect of cisternal papaverine on cerebral blood flow in patients with subarachnoid hemorrhage--three case reports.

Three patients presenting with subarachnoid hemorrhage due to aneurysmal rupture underwent cerebral blood flow (CBF) measurements before and after cisternal injection of papaverine hydrochloride. One patient showed prominent increases in CBF in the frontal lobe and basal ganglia after injection of papaverine, but paradoxical decreases in the parietal lobe and corona radiata. The other two patients had poor CBF response. Dense clotting in the basal cisterns may have prevented diffusion of the agent so that only the proximal trunks of the internal carotid, anterior cerebral, and middle cerebral arteries were dilated in the former case. The dilation of proximal trunks of cerebral arteries might steal blood flow from the parietal lobe and corona radiata, where the intraparenchymal arteries were maximally dilated and cerebrovascular reserve capacity was poor.

Determination of the regions of the clathrin molecule inducing membrane fusion.

Clathrin induces fusion of liposome membranes containing phosphatidylserine at acidic pH [Maezawa, S., Yoshimura, T., Hong, K., Düzgünes, N., & Papahadjopoulos, D. (1989) Biochemistry 28, 1422-1428]. The regions of the clathrin molecule inducing membrane fusion were determined by examining the fusion abilities of clathrin fragments obtained by limited proteolysis of clathrin cages with thermolysin. Membrane fusion was assessed by resonance energy transfer assay in terms of the dilution of fluorescent phospholipids in liposome membranes. Proteolysis of clathrin decreased the fusion rate and the amount of protein but did not affect the specific fusion rate (i.e., the fusion rate per unit of protein), indicating that clathrin fragments retain the ability to induce fusion. Of the two proteolytic fragments of the clathrin heavy chain, the terminal domain and the residual proximal part, which were separated by ultracentrifugation or gel chromatography, only the proximal part showed fusion activity. Light chains seemed to have no role in membrane fusion, since they are susceptible to proteolytic digestion. The terminal domain induced reversible liposome membrane aggregation, which was also induced by the residual proximal part of the heavy chain and the whole molecule of clathrin. These results suggest that the terminal domain and the proximal portion of clathrin have critical roles in the steps of close apposition and fusion of membranes, respectively.

Assembly of clathrin molecules on liposome membranes: a possible event necessary for induction of membrane fusion.

Below pH6, clathrin induces fusion of liposomes containing phosphatidylserine (PS) [Maezawa et al. (1989) Biochemistry 28, 1422-1428]. Under similar conditions clathrin forms self-aggregates, suggesting that the associated form of clathrin may be involved in the fusion process. For examination of this possibility, the extent of fluorescence energy transfer from N-(p-(2-benzimidazolyl)phenyl)maleimide (BIPM)-labeled clathrin to N-(7-dimethyl-amino-4-methyl-3-coumarinyl)maleimide (DACM)-labeled clathrin in the presence of liposomes and the number of binding sites for clathrin in one liposome were examined in the pH region inducing membrane fusion. A high degree of transfer was observed, and the area on the membrane surface occupied by a clathrin molecule was estimated to be much less than that expected from its size, indicating that clathrin binds to the liposome membrane as an associated form, which may be essential for induction of membrane fusion.

Skeletal structure of clathrin triskelion in solution: experimental and theoretical approaches.

The physicochemical properties of the clathrin triskelion were determined by dynamic and static light-scattering and sedimentation analyses in Tris and triethanolamine (TEA) buffers of about pH 8, in which the clathrin triskelion has been found to be in different conformational states by electron microscopy [Heuser, J., & Kirchhausen, T. (1985) J. Ultrastruct. Res. 92, 1-27]. Dynamic light-scattering measurements provided diffusion coefficients (D0(20,w)) of 1.22 x 10(-7) and 1.23 x 10(-7) cm2/s, and ultracentrifugal analysis gave sedimentation coefficients (S0(20,w)) of 8.39 and 8.32 S in Tris and TEA buffer, respectively. The average Stokes radius of the protein was determined to be 175 A from its diffusion and sedimentation coefficients and its molecular weight. Static light-scattering analysis provided molecular weights of 6.58 x 10(5) and 6.41 x 10(5) and radii of gyration of 311 and 301 A in the respective buffers. These results indicate that the clathrin triskelion has a similar conformation in the two buffers. For clarification of the skeletal structure of the clathrin triskelion in solution, the physicochemical parameters were calculated by using two models in which the clathrin arms are bent at various angles in a plane, on the basis of the Bloomfield approximation and a formula derived to estimate the radius of gyration of proteins consisting of various structural units. Values for the Stokes radius, diffusion and sedimentation coefficients, and radius of gyration in the ranges of 178-170 A, (1.20-1.26) x 10(-7) cm2/s, 8.26-8.66 S, and 316-266 A, respectively, were obtained with these models with the arms bent in the range of 0-60 degrees.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between the level of urinary D-glucaric acid and the degree of activation of masked compound (FT-207) in cancer patients.

Phenobarbital stimulates the induction of liver microsomal drug-metabolizing enzyme, namely, cytochrome P-450, which enhances the rate of conversion of FT-207 to 5-FU, the active substance. When FT-207 is administered in combination with phenobarbital to cancer patients, the fluctuation in level of the drug-metabolizing enzyme, cytochrome P-450, should be taken into consideration. Therefore, it was investigated whether the urinary level of D-glucaric acid could be of value as an indicator for the evaluation of the activation of masked compounds, such as FT-207. The level of D-glucaric acid in urine was lower in cancer patients than in normal controls. The correlation between the level of urinary D-glucaric acid and that of 5-FU, which is an active metabolite of FT-207, in blood was statistically significant. The level of D-glucaric acid in urine was of use as an indicator for the evaluation of the activation of masked compounds, such as FT-207, in cancer chemotherapy.

Effects of inducer of liver drug-metabolizing enzyme on blood level of active metabolites of cyclophosphamide in rats and in cancer patients.

It was already reported that a masked compound, cyclophosphamide (Endoxan, EX) undergoes the first step metabolism by a drug-metabolizing enzyme in liver microsomes, cytochrome P-450. By pretreatment with phenobarbital as an inducer of P-450, the maximum blood level of active metabolites of EX (normustard-like substances) in normal rats was 2.3 times higher than that in non-treated rats, in conformity with the increase in amount of liver P-450 and in alkylating activity of EX. In YS (Yoshida sarcoma)-bearing rats, the value of liver P-450 went down day by day to 1/2 on the 4th day after inoculation, but it remained normal when animals were pretreated with phenobarbital. In parallel with this, the blood level of normustard-like substances after EX administration was normal or showed a tendency toward increase. In 11 clinical cases pretreated with phenobarbital, the blood level of normustard-like substances 1 to 3 hr after EX administration, at the time when it reaches the peak was 1.5 times higher on an average than that in cases without pretreatment.

Combined cancer chemotherapy with cyclophoshamide and an inducer of microsomal drug-metabolizing enzymes (cytochrome P-450) in tumor-bearing rats.

The fifty percent alive period in Yoshida sarcoma-bearing and AH66F-bearing rats was prolonged by an administration in a single dose of 100 mg cyclophosphamide/kg body weight in a group pretreated with phenobarbital as compared with that pretreated with saline. However, there was little difference in fifty percent alive period between AH109A (insensitive to cyclophosphamide)-bearing rats and controls even when they were pretreated with phenobarbital. In chemotherapy with anti-cancer drugs, inductive or inhibitory effects of the drugs on microsomal drug-metabolizing enzymes should be taken into consideration.