Sorption and transport of acetylcholine in reconstituted biomembrane structures

Journal

of Molecular

Catalysis,

18 (1983)

11 - 22

11

SORPTION AND TRANSPORT OF ACETYLCHOLINE RECONSTITUTED BIOMEMBRANE STRUCTURES SACHIO

IN

HIROSE

Central

Research

Laboratories,

Mitsubishi

Petrochemical

Biochemical

Engineering,

Co., Ltd.,

Ami,

Inashiki,

Ibaraki

(Japan) WOLF

R. VIETH

Department NJ (U.S.A.)

of Chemical

and MASAOMI Department (Received

and

Rutgers

University,

Piscataway,

TAKAO

of Chemistry, February

Kagawa

University,

Takamatsu,

Kagawa

(Japan)

26, 1982)

Summary In this work, neurotransmitter transport was studied in structures similar to those found at the neuromuscular junction. Transport and sorption of acetylcholine in a collagenous model of the fibrous matrix of the synaptic cleft were investigated. Sorption level depended upon the degree of swelling of collagen. From diffusion experiments, mobile species concentrations were estimated and found to be roughly independent of the degree of swelling. In contrast, immobile concentrations were proportional to it. These phenomena are explained by two different penetrant binding affinity levels: one, a lower affinity for the mobile species which controls diffusion, and the other a higher affinity for the immobile species. In the range of low acetylcholine concentration, substantial accumulation of acetylcholine in collagen membranes was demonstrated and the effect was strongly amplified in reconstituted acetylcholine receptor-rich membrane vesicles from torpedo fish. Distribution coefficients ranged from 10 (collagen) to lo3 (receptor vesicles). It is thought that accumulated ligand binding can play an important role in the observed transport of penetrant across biological membranes generally, and in the strength of the neurotransmitter-receptor interaction, in particular.

Introduction Recently, it was found that adaptation of Dual Sorption Theory [l] to sugar transport in a biopolymeric membrane (i.e., collagen) is successful. Ludolph et al. [ 21 indicate that DST is applicable to sugar transport through collagen membrane and enzyme-bound collagen membranes. Soddu and 0304-5102/83/0000-0000/$03.00

@ Elsevier Sequoia/Printed

in The Netherlands

12

Vieth [3, 41 identified two types of sites in the medium, to which the substrate (sugar) has different affinities. That is, the substrate fraction bound to the high-affinity sites is considered to be immobilized. It was suggested that this model may be gainfully adapted to describe a number of the observed transport properties of other biological membranous structures. This suggestion leads to a more extensive investigation into the applicability of DST for biological membranes. An important representative biological membrane system is the structure comprising the synaptic cleft ending in the receptor-rich postsynaptic membrane. Binding of acetylcholine to the latter triggers the all-ornone opening of discrete channels or pores through which Na+ and K+ ions flow along their electrochemical gradients [ 51. In this biomembrane, binding phenomena in relation to the nerve mechanism and roles of several substrat.es in neurotransmission have been studied biologically in detail 16, 71. Heidmann and Changeux [ 51 and Couteaux [ 81 have investigated the mechanism of the response of an excitable membrane. Elliot et al. [9] and Lindstrom et al. [ 10, 111 have demonstrated successful purification and characterization of the structures of acetylcholine receptor from the electro-organs of torpedo fish and electric eel. As the first step in a series of works, acetylcholine iodide and collagen were selected as models of the penetrant and the fluid-filled collagenous matrix of the synaptic cleft [12], respectively. Our aim was to characterize the basic phenomena of the transport process across the synaptic cleft experimentally. Experimental data on transport and sorption under the conditions of varying acetylcholine iodide (AChI) ~once~ltrations were investigated. In addition, as the beginning of the second phase of work, accumulations of AChI at low bulk concentrations in collagen membranes and vesicles which comprise ACh receptor-rich membrane purified from torpedo fish by established general methods, were investigated.

Experimental Materials Microfibrillar collagen hydrochloride (collagen) was supplied by FMC Inc. (Princeton, NJ). Acetyl~holine iodide (AChI) was purchased from Kodak Co., Ltd. (Rochester, NY) and radioactive AChI (r4C(U)) (NEC-350, 4.8 mCi/mmol, 47.1 mg) and radioactive 2ZNa+ (0.2 mCi/ml), from New England Nuclear Co. (Boston, MA). Liquid scintillation cocktail (ReadySolv@ HP) was purchased from Beckman Instruments, Inc. (Fullerton, CA). Torpedo California electroplax organs (Pacific Biomarine Co., CA) obtained from freshly killed animals were frozen in liquid N2 and stored at -90 “C until use. Other reagents were commercially available analytical reagents or laboratory-grade chemicals. Glass distilled water was used in all procedures. The Beckman liquid scintillation counter (Model LS-133) was used for radioactive AChI measurements.

13

Preparation

of the collagen membrane

A suspension containing 0.5% (w/w) of collagen was ground for 4 periods of 30 s at high speed in a commercial Waring blender and then deaerated for 4 h with a vacuum pump. The solution obtained was cast on a Mylar@ sheet in a frame. After evaporation for a few days, dry collagen membrane was obtained.

Measurements

of membrane

properties

The thickness and surface area of a membrane specimen were measured in dry and wet states. For the latter, a specimen was swollen in a phosphate buffer (0.05 M, pH 7.0). The degree of swelling (DS) has been defined as the ratio between swollen and dry thicknesses. Sorption level was measured by the following methods. Equivalent pieces of collagen membrane (1 cm2 in area), from the same casting, were swollen in phsophate buffer (0.05 M, pH 7.0) for at least one day. Once the swollen thickness was measured by microgauge, the volume of the specimen could be calculated easily. Each specimen was placed into AChI solutions (10 ml) in the range from lop6 to 2.0 mol/l of AChI concentrations. Radioactive AChI (14C) (0.1 ml, stock solution of ca. 20 Ci/ml) was added to the solutions, in order that sorption experiments under the conditions of low AChI concentrations were easily possible. Sorption equilibration was reached within a few days, and the specimen was rinsed with 200 ml distilled water to remove excess radioactive AChI on the membrane surface. It was then desorbed in a phosphate buffer (0.05 M, pH 7.0, 5.0 m). Samples of 0.1 ml of sorption solution before immersion of the specimen with 0.4 ml of water, and of 0.5 ml of desorption solution after desorption equilibrium were pipetted into 10 ml of cocktail. The AChI concentrations in these samples were measured by a liquid scintillation counter (LSC). Sorption level was defined as the AChI moles per unit volume of collagen membrane obtained from the above AChI concentrations. Time-lag and diffusivity were measured using the same method and apparatus as in a previous paper [ 131, employing a diffusion cell consisting of two compartments. AChI solution recycled upstream while phosphate buffer (0.05 M, pH 7.0) passed over the downstream side. The total amount ( Qt) of penetrant (AChI) that permeates through the membrane in time (t) can be calculated as follows: Qt=

jJAdt=

i(V$i

0

0

+qx t

)dt

(1)

where J, flux of AChI, is the number of moles of AChI transported per unit membrane area and unit time, x is the downstream concentration of AChI in time t, A is the membrane area (10.17 cm2, 36 mm diameter), V is the volume of the diffusion cell (downstream, 18.0 ml) and q is the flow rate of the phosphate buffer (0.05 M, pH 7.0, 18 ml/min). The time-lag (0) was schematically obtained from the plots of Qt against t. The asymptotic time-lag, 8,,,,, was obtained from plots of 8 against upstream AChI

14

displaying a nonlinear transition region asymptotically concentration, tending to a steady value in higher AChI concentrations. The diffusivity (Da,, m) of the penetrant (AChI) can be determined as follows: D aSyIn= L216&iS,In

(2)

where L is swollen membrane thickness. For such a system, Dasym would be the intrinsic membrane diffusion coefficient. was then used to calculate the mobile This asymptotic value (D,,,,) concentrations. At a steady state the concentration (C,) of the mobile species varies linearly through the membrane. Thus, Ficks’s First Law can be written J=

dCh4=D

-Dasym -

dL

aSYm

cMU

-

L

cMD

i

where D,, and CMn are the upstream and downstream concentrations of the mobile species, respectively. In this experiment, the concentration of the mobile species in the downstream side (CM,) is much smaller than that in the upstream side (CM,). Therefore, eqn. (3) can be rewritten as: J=D

asumCMUIL

CMu is then expressed CMlJ

=

by:

(5)

JLID,,,,

J can be calculated

concentration by:

from the time-lag plot, using eqn. (1). Furthermore, the (C,) of the immobile species in the membrane is expressed

CI=cT-CMU

(6)

where CT is the total concentration obtained from sorption level which is defined as the number of AChI moles sorbed in the collagen membrane. Purification

and reconstitution

of vesicles from

torpedo

fish

This procedure followed the general approach of several laboratories in which acetylcholine receptor (AChR)-rich membrane vesicles were purified. The method used in these experiments is outlined as follows: After Torpedo California electroplax organs were dissected away, the organ (ca. 100 g) was minced into small pieces and an equal volume of cold buffer (10 mM sodium phosphate pH 7.8, 400 mM NaCl, 5 mM EDTA, 0.02% NaN,, 5 mM iodoacetamide) was added. After 2 min initial grind at high speed in a commercial Waring blender, the homogenate was reground in small portions for four periods of 30 s at 30 000 rev/min in a Virtis 60. Connective tissue and other large particles were pelleted by centrifugation of the homogenate at 5 000 rev/min for 10 min in a Sorvall GS-2 at 2 “C!. The supernatant was passed through two layers of cheesecloth and centrifuged at 16 000 rev/min for 1 h in a Sorvall GS-2 rotor at 2 “C. The pellet obtained was AChR-rich membrane vesicles to be further reconstituted

15

by soybean lipids. Vesicles were resuspended in reconstitution buffer (2% Na cholate (Sigma); 25 mg/ml of soybean L-cY-phosphatidylcholine (Sigma); 100 mM NaCl; 10 mM phosphate buffer, pH 7.5; 10 mM NaN,) at concentrations of 0.02 to 0.1 g of vesicles/ml. After blending on a Vortex mixer and stirring to provide a homogeneous suspension, this mixture was dialyzed overnight against at least 100 volumes of 100 mM NaCl, 10 mM Na phosovernight phate, pH 7.5, and 10 mM NaN,, followed by an additional dialysis against flux buffer (145 mM sucrose, 10 mM Na phosphate, pH 7.5, 5 mM NaN,). Carbamylcholine-induced

22Na’ and AChI uptakes of reconstituted

vesicles

[lOI The 22Na+ uptake was conducted at room temperature behind suitable lead shielding. In microfuge tubes, 22Na+ (5 ~1 of 0.2 mCi/ml) and water (5 ~1) or carbamylcholine (5 1.11of 1 X 10v4 M) were mixed. The assay was initiated by pipetting in 40 ~1 of vesicles in flux buffer with an Eppendorf pipetter. After mixing by five up-and-down strokes of the pipetter, the mixture was transferred to a column (0.5 X 8 cm) packed with Dowex 5OW-X8-100 (Sigma) treated with Trizma base (Sigma). And then, sucrose solution (3 ml of 175 mM) was carefully added to elute the vesicles. The tube containing the eluate of each column was placed in the y-counter and counted. The carbamylcholine-induced 22Na+ uptake was defined as the difference between the counts per minute in the water samples and those containing carbamylcholine. The AChI uptake of reconstituted vesicles was carried out by almost the same method as above. That is, in microfuge tubes, AChI in the range from low6 to 10-l M (450 pl), radioactive AChI (30 ~1 0.01 mCi/ml) and reconstituted vesicles (20 ~1) were mixed. The mixture was transferred to the column (0.5 X 8 cm) and sucrose solution (3 ml of 175 mM) was carefully added to elute the vesicles. The eluate (0.5 ml) was mixed into liquid scintillation cocktail (10 ml) and AChI concentrations in vesicles were calculated. Results Collagen membranes

In order to measure the effect of AChI concentration on the swelling of collagen, membrane samples were swollen in AChI solution at pH 7.0 (phosphate buffer, 0.05 M). In Fig. 1, the experimental data were plotted in the range to 2.0 mol/l of AChI, when using a collagen membrane of swelling degree 16.6. Swollen membrane thickness in the phosphate buffer as a control is 830 pm. At AChI concentrations of ca. 2.0 mol/l, there is an abrupt change in swelling. It was noticed that immersion of the collagen membranes at high AChI concentrations resulted in shrinkage due to interaction between AChI and collagen. This type of result was previously observed with sucrose and collagen by Soddu and Vieth [ 81.

16

f 0

b. 0

O.-l

0.2

Con~entfation

0.5

1.0

2.0

of ACh I 1mot /

5.0

I1

Fig. 1. Swelling of collagen membrane at pH 7.0; degree of swelling, 16.6.

The sorption of AChI in collagen membranes was studied over a wide range of concentrations and degrees of swelling. For these experiments, collagen membranes having various degrees of swelling were prepared and are summarized in Table 1. The results of sorption experiments in which these membranes were used are shown in Fig. 2. Large differences in the levels of sorption were found for different degrees of swelling, depending on AChI concentrations. For high degrees of swelling, the level of sorption proportionally increased with increasing AChI concentrations. For swelling degrees 9.9, 13.4 and 16.6, AChI was slightly accumulated in the collagen membrane compared with bulk AChI concentrations, For low degrees of swelling 6.3 and 3.1, which were obtained by a long-term aging of collagen and the reaction of glut~~dehyde, respectively, the slope reached a plateau at AChI concentrations higher than 1.5 mol/l. Highly cross-linked membranes did not accumulate AChI. For high degrees of swelling, the structure is rather open and all microdomains are accessible to the substrate. Excess AChI occupies affinity sites of collagen. In contrast, accessibility of microdomains is the limitation of highly cross-linked structures at low degrees of swelling. Nonetheless, a substantial fraction of the microdomains are still accessible to the substrate TABLE 1 Preparation for the various kinds of collagen membranes Degree of swelling

Swollen membrane thickness (Mm)

Aging conditions

16.6 13.4 9.9 6.3 3.1

830 1075 893 506 301

4 4 31 10 5

days at 25 days at 25 days at 25 days at 65 min in 3%

“C “C % “C and 90% relative humidity (v/v) of glutaraldehyde solution

17

0.5

Fig. 2. Sorption and (A) 3.1.

1.5

1.0

Concentration

of

AChl

2.0 I mol/

level at different

I1

degrees

of swelling:

(X)

16.6,

(0) 13.4,

(0)

9.9,

(A) 6.3

in a highly cross-linked structure, Therefore, saturation effects of sorption level were observed in higher AChI concentrations. These effects indicate that the number of accessible affinity sites from different types of collagen membranes is controlled by the microstructure of the biopolymeric medium in the sense of microdomains and networks. Time-lag experiments were performed using collagen membrane of degree of swelling 16.6. The time-lags (0) are shown in Fig. 3. The time-lag asymptotically decreased with increasing upstream AChI concentrations, following the approach to 8,,,,, 2.0 min. This type of behavior is predicted by the analysis of Bhatia and Vieth [ 141 and Gondo and Vieth

[I51 *

0

’ 0

1.0

0.5

Upstream

AChl

1.5

Concentration

2.0 (molll)

Fig. 3. Relation between time-lags membrane; degree of swelling 16.6.

and

upstream

AChI

concentrations

with

collagen

18

Furthermore, AChI concentrations of the mobile species (CM,) were calculated from the flux and the asymptotic diffusivity, using eqns. (l), (2) and (5), for degrees of swelling 6.3, 13.4 and 16.6. The AChI concentrations (C,) of the immobile species were also calculated as the difference between the value of the total sorption level of AChI in collagen membrane and the above-obtained AChI concentrations of the mobile species, using eqn. (6). The results are illustrated in Figs. 4, 5 and 6. The mobile species concentration increased with increasing upstream AChI concentration, while it was roughly independent of the degree of swelling. A sigmoidal curve for the immobile species was obtained for all degrees of swelling, characteristic of an allosteric transport effect [4]. It should be noticed that the slope of sorption level decreased with

0

0.5

Upstream

AChl

Fig. 4. Resolution collagen membrane

1.0

1.5

Concentration

,MMOBlLE

._ .i

p’

L

E

0

of AChI concentrations of both at 25 C; degree of swelling, 16.6.

E ,= s

I

[mol/ll

,.o-

mobile

and

immobile

species

in

Yd

/

@

s i P

0 Upstream

0.5 AChl

1.0

1.5

2.0

Concentration[mol/l)

Upstream

AChl

Concentration

(mol/l)

Fig. 5. Resolution collagen membrane

of AChI concentrations of both at 25 “C; degree of swelling, 13.4.

mobile

and

immobile

species

in

Fig. 6. Resolution collagen membrane

of AChI concentrations of both at 25 “C; degree of swelling, 6.3.

mobile

and

immobile

species

in

19

decreasing degrees of swelling. Therefore, the immobile species concentrations remarkably decreased with decreasing degrees of swelling. These results are also in agreement with the previous experimental findings which are obtained from collagen membranes and sugar as a penetrant [4]. Collagen, a biopolymeric medium, permits the coexistence of two penetrant species of different site affinities (higher and lower), where the mobile species controls the process of diffusion. Therefore, it is thought that the high affinity sites in which the immobile species could be occupied are consumed or buried under the conditions of low degrees of swelling, due to the presence of a highly cross-linked structure. Vesicles It is well-known that cation channel activity of AChR-rich membrane vesicles is dependent on many factors, for instance, purification steps from torpedo fish, conditions of reconstitution of vesicles, concentrations of inducing carbamylcholine, as well as general environment conditions such as pH, ionic strength and temperature [9 - 111. In particular, the balances of vesicle and soybean lipid concentrations in reconstitution buffer solution have great effects on the survival of cation channel activities. In these experiments, in order to make an appropriate recipe of reconstituted AChR-rich membrane vesicles, carbamylcholine ( lop4 M)induced 22Na+ uptakes under the conditions of different vesicle concentrations were studied, following dilution with flux buffer. Figure 7 shows that carbamylcholine-induced 22Na+ uptake increased linearly with the concentration of receptor present in the reconstituted vesicles for the different vesicle concentrations. The presence of relatively low amounts of initial reconstituted vesicles in reconstitution buffers effectively preserved cation 250

Lipids

t

Concentration

,I

0. 0

0.02

Vesicles

0.04

0.06

OP5

Concentration

Fig. 7. Effect of reconstituted 10e4 M carbamylcholine.

0.10

(g/g

1

vesicle concentration

on uptake of **Na+ in response to

20

channel

activity. From our previous experiments and those of Lindstrom no cation channel activity survived without addition of lipids into [51, reconstitution buffer. On the other hand, Na uptakes of reconstituted vesicles decrease proportionally with dilution of flux buffer. Therefore, it is thought that an optimum ratio between reconstituted vesicles and soybean lipids exists to obtain higher cation channel activity. From Anholt’s results on the relation between Na uptake and AChR concentrations, a value of AChR concentration, approximately 0.1 PM, was estimated under the conditions of 0.10 g of vesicles per g reconstitution buffer. In the range from 1OVj to 10-l mol/l of immersing AChI concentrations, sorption levels of AChI in collagen specimens having the degree of swelling 16.6, were measured. AChI uptakes of reconstituted vesicles (0.02 g/g) used in Fig. 7 were also measured. K value is defined as the ratio of sorption level (AChI uptake) and bulk AChI concentration and is shown in Fig. 8. It is of interest that the AChI concentration in collagen membrane was accumulated at least 10 times in low AChI concentration solution, compared to immersing AChI concentrations, while cu. 1000 times in vesicles. For the latter case, it is possible to estimate the value of an apparent Michaelis constant for the receptor. We obtain 2.0 X 10e4 mol/l, in good agreement with the estimated value of ACh concentration in the synaptic cleft during the process of neurotransmission [ 51. Solute transport across biological membranes can be divided into two classes, active and passive, In the former class, a species is transported against its trans-membrane electrochemical gradient; the free energy necessary for this movement comes from a chemical reaction directly coupled to the transport. However, in some instances, the existence of a very high accumulation internal to a membrane, owing to a large immobilized penetrant population, might be misconstrued as an uphill gradient. In the case of a unit volume of collagen membrane with a distribution coefficient of 10.0 for ACh, the steady-state accumulation is (K/2) or 5.0 when the penetrant

10

10

Concentration

10 of AChl

10 [ mol/

It? II

Fig. 8. Distribution coefficients US. AChI concentrations 16.6) and reconstituted AChR-rich vesicles.

for collagen

membrane

(DS =

21

is continuously removed from the downstream reaction (or as in a time-lag experiment). In this jumps uphill, going from the bulk solution to the gradient runs smoothly downhill from the bulk, the downstream side. These phenomena suggest that two affinity the ligand can play an important role in the solute membranes.

face, as by an enzymatic instance, the concentration membrane, but the activity through the membrane to states of the medium for transport across biological

Discussion Transport and sorption of acetylcholine iodide (AChI) across collagen membranes were studied, in order to adapt and extend the Dual Sorption Theory to biological systems, particularly those which are similar to the structure at the neuromuscular junction. In higher than 1.5 mol/l AChI concentrations, shrinkage of collagen membranes occurred due to strong interaction between AChI and the biopolymeric membranes (Fig. 1). It was also found that strong penetrant-site interactions have a close relation to the degree of swelling from sorption experiments (Fig. 2). Accessible microdomains with binding sites capable of strong interaction control the extent of sorption of the penetrant in the biopolymeric membrane. Resolutions of sorption level for the various kinds of swelling degrees of collagen membranes were analyzed through results of diffusion experiments (Figs. 4 - 6). That is, an effective ACh diffusion coefficient, Delf = 2.0 X lop6 cm*/s, of collagen membranes obtained by time-lag experiments was found which was also in good agreement with the value found when sucrose was transported through collagen membranes [ 21. Through sorption and diffusion experiments, two modes of sorption were, indeed, found to be present in this study. The mobile species concentration was roughly independent of the degree of swelling, while the immobile concentration was proportional to it. The apparent decrease of affinity of the ligand (the immobile species) for the high-affinity sites with decreasing degree of swelling could be due to the more compact microstructure of the medium. Our results provide support for the assumption that the affinity of the ligand for the mobile state is much lower than the affinity for the immobile state, and only one mode of sorption (low-affinity sites) controls the diffusion. Furthermore, AChI sorption of reconstituted acetylcholine receptor (AChR)-rich membrane vesicles and collagen membranes under the conditions of relatively lower AChI concentration similar to those encountered in neurotransport were investigated. That is, AChR-rich membrane vesicles purified from torpedo fish were selected as a close model of the active component of the biological system while collagen is known to be one of the framework components supporting it in neuro-systems. Penetrant-biopolymer interactions in such structures are of obvious interest. It is found that biological

22

structures of this type accumulate a large amount of the ligand, due to high affinity. Moreover, this phenomenon plays an important role in the transport of the ligand. A penetrant may apparently be moved uphill, against its concentration gradient, (but not, of course, against its thermodynamic activity gradient) in such biological systems. This result shows that the transport of mobile AChI in the biological membrane structure is consonant with accumulation and retention of immobilized AChI. Local equilibrium between the mobile and immobile species populations provides the pool of mobile species which controls the process of diffusion.

References Sci., 1 (1976) 177. 1 W. R. Vieth, J. M. Howell and J. H. Hsieh, J. Membrane W. R. Vieth, K. Venkatasurbramanian and A. Constantinides, J. Mol. 2 R. A. Ludolph, Catal., 5 (1979) 197. W. R. Vieth and A. Soddu, J. Mol. Cotal., 7 (1980) 415. A. Soddu and W. R. Vieth, J. Mol. Catal., 7 (1980) 491. T. Heidmann and J.-P. Changeux, Ann. Rev. Biochem., 47 (1978) 317. R. D. O’Brien (ed.), The Receptors, Plenum Press, New York, 1980. J. Cheymol (ed.), Neuromuscular Blocking and Stimulating Agents, Pergamon Press, Oxford, 1972. Experimental Cell Research, Suppl. 5, (1958) 323. 8 R. Couteaux, W. Wu, J. Miller, C. D. Straden, P. Hartig, H.-P. Moore, 9 J. Elliot, S. G. Blanchard, J. Rats and M. A. Raftery, Biochem. J., 185 (1980) 667. 10 J. Lindstrom, R. Anholt, B. Einarson, A. Engel, M. Osame and M. Montal, J. Biot. Chem., 255 (1980) 8340. 11 R. Anholt, J. Lindstrom and M. Mental, Eur. J. Biochem., 109 (1980) 481. 236 (1977) 106. 12 H. A. Lester, Sci. American, M. Hayashi and W. R. Vieth, J. Membrane Sci., submitted 13 S. Hirose, E. Yasukawa, for publication. Sci., 6 (1980) 351. 14 D. Bhatia and W. R. Vieth, J. Membrane 15 S. Gondo, W. R. Vieth and A. Soddu, Time-Lags for Penetrants in Protein Membranes, Paper presented to Japan Society of Chemical Engineers, Yokohama, (Sept., 1980).