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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Korean Society for Biochemistry and Molecular Biology
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Volume & Issues
Volume 33, Issue 6 - Nov 2000
Volume 33, Issue 5 - Sep 2000
Volume 33, Issue 4 - Jul 2000
Volume 33, Issue 3 - May 2000
Volume 33, Issue 2 - Mar 2000
Volume 33, Issue 1 - Jan 2000
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Regulation of the Phagocyte Respiratory Burst Oxidase by Protein Interactions
Lambeth, J. David ;
BMB Reports , volume 33, issue 6, 2000, Pages 427~439
The activity of the phagocyte respiratory burst oxidase is regulated by complex and dynamic alterations in protein-protein interactions that result in the rapid assembly of an active multicomponent NADPH oxidase enzyme on the plasma membrane. While the enzymatic activity has been studied for the past 20 years, the past decade has seen remarkable progress in our understanding of the enzyme and its activation at the molecular level. This article describes the current state of knowledge, and proposes a model for the mechanism by which protein-protein interactions regulate enzyme activity in this system.
Expression of Recombinant HBV Pol Proteins in HepG2 Cells
Cho, Ginam ; Na, Seun-Gon ; Suh, Se-Won ; Jung, Gu-Hung ;
BMB Reports , volume 33, issue 6, 2000, Pages 440~447
In this study HepG2 cells were used to express and purify HBV pol proteins. In order to facilitate purification of HBV pol proteins, HBV pol and its deletion mutants were fused to MBP (Maltose Binding Protein). As a result we successfully expressed and partially purified both wild type and mutant recombinant HBV pol proteins by using an amylose resin and anti-MBP antibody. In the case of wild type, the anti-MBP antibody detected three bands. One was full-length and the others were generated by proteolysis of the terminal domain region. The expressed MBP/POL proteins were localized both in the cytoplasm and in the perinuclear region. The purified proteins had polymerase activity toward an exogenous homo-polymer template. The MBP/POL protein also had DNA synthesis activity in vivo, since the MBP/POL expression construct was able to complement a HBV polymerase mutant in trans.
Identification of Receptor-like Protein for Fructose-1,6-bisphosphatase on Yeast Vacuolar Membrane
Ko, Je-Sang ;
BMB Reports , volume 33, issue 6, 2000, Pages 448~453
In yeast the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is selectively targeted from the cytosol to the lysosome (vacuole) for degradation when glucose starved cells are replenished with glucose. The pathway for glucose induced FBPase degradation is unknown. To identify the receptor-mediated degradation pathway of FBPase, we investigated the presence of the FBPase receptor on the vacuolar membrane by cell fractionation experiments and binding assay using vid mutant (vacuolar import and degradation), which is defective in the glucose-induced degradation of FBPase. FBPase sedimented in the pellets from vid24-1 mutant after centrifugation at
for 15 min, suggesting that FBPase is associated with subcellular structures. Cell fractionation experiments revealed that FBPase is preferentially associated with the vacuole, but not with other organelles in vid24-1. FBPase enriched fractions that cofractionated with the vacuole were sensitive to proteinase K digestion, indicating that FBPase is peripherally associated with the vacuole. We developed an assay for the binding of FBPase to the vacuole. The assay revealed that FBPase bound to the vacuole with a Kd of
. The binding was saturable and specific. These results suggest that a receptor for FBPase degradation exists on the vacuolar membrane. It implies the existence of the receptor-mediated degradation pathway of FBPase by the lysosome.
Regulation of IgE and Type II IgE receptor expression by insulin-like growth factor-1: Role ofSTAT6 and
Koh, Hyun-Ja ; Park, Hyun-Hee ; Lee, Choong-Eun ;
BMB Reports , volume 33, issue 6, 2000, Pages 454~462
Interleukin-4(IL-4) is known to be a major cytokine regulating immunoglobulin E(IgE) response by the induction of IgE production and type II IgE receptor(IgER II: CD23) expression. Recently, however, the role of neuroendocrine factors has been implicated in modulating the IgE response. Among various neuroendocrine growth factors, we investigated the effects of the insulin-like growth factor-1(IGF-1) since IL-4 and IGF-1 share common intracellular signaling molecules, such as the insulin receptor substrate-1/2(IRS-1/2) to induce a specific cellular response. In the human peripheral blood mononuclear cell (PBMC) cultures, IGF-1 was capable of inducing a substantial level of IgE production in a dose-dependent manner. It also noticeably upregulated the IL-4-induced or IL-4 plus anti-CD40-induced IgE production. Similarly, the IGF-1-induced IgE production was enhanced by IL-4 or anti-CD40 in an additive manner, which became saturated at high concentrations of IGF-1. Although IGF-1 alone did not induce IgER II (CD23) expression, it augmented the IL-4-induced surface CD23 expression in a manner similar to the action of anti-CD40. These results imply that IGF-1 is likely to utilize common signaling pathways with IL-4 and anti-CD40 to induce IgE and IgER II expression. In support of this notion, we observed that IGF-1 enhanced the IL-4-induced signal transducers and activators of transcription 6(STAT6) activation and independently induced
activation. Both of these bind to the IgE(C) or IgER II (CD23) promoters. Together, our data suggest that IL-4 and IGF-1 work cooperatively to activate STAT6 and
. This leads to the subsequent binding of these transcription factors to the
and CD23 promoters to enhance the expression of IgE and IgER II. The observed differential ability of IGF-1 on the induction of IgE vs. IgER II is discussed based on the different structure of the two promoters.
Modulation of Interleukin Production in Anthrax Lethal Toxin-treated Macrophages by Melatonin and Dehydroepiandrosterone
Shin, Sung-Ho ; Hur, Gyeung-Haeng ; Yeon, Kyu-Baek ; Kim, Yun-Bae ; Park, Kyung-Jin ; Park, Young-Min ; Lee, Woo-Sung ; Cho, Bong-Huey ; Kim, Won-Yong ; Chung, Sang-In ; Choi, Chul-Soon ;
BMB Reports , volume 33, issue 6, 2000, Pages 463~468
Anthrax lethal toxin, which consists of two separate protein, protective antigen (83 KDa) and lethal factor (85 KDa) is responsible for major symptoms and death from systemic infection of Bacillus anthracis. High concentrations of this toxin are cytolytic to macrophages, whereas sublytic concentrations of lethal toxin induce these cells to produce interleukin
). It is proposed that melatonin and dehydroepiandrosterone (DHEA) may play an important role in modifying immune dysfunction. In this study, we investigated whether or not melatonin and DHEA could prevent
production that is induced by anthrax lethal toxin in mouse peritoneal macrophages. Treatment of melatonin or DHEA alone, as well as together, prevented the production of
caused by anthrax lethal toxin. We found that melatonin at a concentration of
production induced by anthrax lethal toxin. As expect, treatment of DHEA at a concentration
M also suppressed production of
by lethal toxin stimulated macrophages. The results of these studies suggest that melatonin and DHEA, immunomodulators, may have an important role in reducing the increase of cytokine production in anthrax lethal toxin-treated macrophages.
Excess Taurine Induced Placental Glutathione S-transferase Positive Foci Formation in Rat
Kweon, Sang-Hui ; Kim, Yoon ; Choi, Hay-Mie ; Kwon, Woo-Jung ; Chang, Kyung-Ja ;
BMB Reports , volume 33, issue 6, 2000, Pages 469~475
The purpose of this study was to examine the chemopreventive potential of taurine at various levels on the diethylnitrosamine (DEN)·induced hepatocarcinogenesis. Male Sprague-Dawley rats were fed on diets containing 0, 1, 2, 3% taurine or 5%
for taurine depletion. Then they were treated with DEN and 2/3 partial hepatectomy. The number of placental glutathione S-transferase positive (
) foci, as a preneoplastic marker in the 1 % taurine group was lower than the control diet group. However the difference was insignificant. Although taurine diets reduced the thiobarbituric acid reactive substance (TBARS) level, the number of
foci was increased in 3% taurine diet group. The 1 % taurine diet increased the glutathione (GSH) level and GST activity, however they unfortunately did not suppress the foci formation. In the 3% taurine group, the GSH level and GSH peroxidase (GPx) activity were significantly decreased. Excess taurine supplementation of the pharmaceutical dose worked against hepatic chemoprevention, which might result from modulation of GPx activity and GSH utility. On the contrary, taurine might work as an antioxidant against TBARS production as the 1 % taurine diet increased GSH level. The potency of the cancer preventive effect of taurine still remains and further studies should investigate the effect of taurine with less than 1 % levels on the prevention of hepatic cancer.
[L-Lysinamide-Carbamoyl] Cholesterol Cationic Lipid as a Biocompatible Vector for Efficient Gene Transfer
Choi, Joon-Sig ; Lee, Eun-Jung ; Jang, Hyung-Suk ; Park, Jong-Sang ;
BMB Reports , volume 33, issue 6, 2000, Pages 476~482
In this paper, we report a new cationic lipid composed of L-lysinamide and cholesterol as a potent gene delivery vector.
[L-Lysinamide-carbamoyl] cholesterol could self-assemble with plasmid DNA forming discrete lipoplexes. From atomic force microscopic images of the complexes, the size distribution was observed to range from 100 to 150 nm in diameter. The transfection efficiency of this amphiphile on different cell lines was evaluated as a micellar solution in the absence of the fusogenic helper lipid, dioleoyl phosphatidyletbanolamine (DOPE). Transfection experiments were performed as a function of charge ratio (lipid/DNA) and transfection time. Cytotoxicity and in vitro transfection efficiency of the amphiphile was demonstrated and compared with those of commercially available Lipofectin and polyethylenimine (PEI).
High Level Production of Glycoprotein H of HSV-1 (F) Using HcNPV Vector System
Kang, Hyun ; Cha, Soung-Chul ; Han, You-Jin ; Park, In-Ho ; Lee, Min-Jung ; Byun, Si-Myung ; Lee, Hyung-Hoan ;
BMB Reports , volume 33, issue 6, 2000, Pages 483~492
The Herpes simplex virus type 1 (HSV-1) strain F glycoprotein H (gH) gene in the pHLB-4 plasmid was recombinated into a baculovirus expression vector (lacZ-HcNPV) to construct a recombinant virus GH-HcNPV expressing gH. The sequences of gH and its expression were analyzed. The gH gene was located in the 6.41 kb BglII fragment. The open reading frame (ORF) of the gH gene was 2,517 bp and codes 838 amino acid residues. Insect cells infected with this recombinant virus synthesized a high level of the matured and gX-gH fusion protein with approximately 112 kDa. The fusion gH protein was localized on the membrane of the insect cells as seen by using immunofluorescence assay and accumulated in the cultured media by the SDS-PAGE and immunoprecipitation assays. The amino acid sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane sequence. Antibodies raised in mice to this recombinant protein recognized viral gH and neutralized the infectivity of HSV-1 in vitro. These results demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV; accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.
Identification of a Mature form and Characterization of Thermostability of a Serine-type Protease from Aquifex pyrophilus
Kim, Yun-Kyeong ; Choi, In-Geol ; Nam, Won-Woo ; Yu, Yeon-Gyu ;
BMB Reports , volume 33, issue 6, 2000, Pages 493~498
Aquifex pyrophilus, a hyperthermophilic bacterium, has a serine-type protease that is located at the cell wall fraction with a mature size of 43 kDa. Molecular cloning of the protease gene revealed that it has an ORF of 619 amino acids with homologous catalytic site of serine-type proteases [Choi, I.-G., Bang, W.-K., Kim, S.-H., Yu, G. Y., J. Biol. Chem. (1999), Vol. 274, pp. 881-888]. Constructs containing different regions of the protease gene, including a alanine-substituted mutant at the active site serine, were constructed, and the factors affecting the expression level of the cloned protease gene in E. coli were examined. The presence of the C-terminus hydrophobic region of the protease hindered over-expression in E. coli. Also, the proteolytic activity of the expressed protein appeared to toxic to E. coli. An inactive form that deleted both of the N-terminal signal sequence and the C-terminal polar residues was over-expressed in a soluble form, purified to homogeneity, and its thermostability examined. The purified protein showed three disulfide bonds and three free sulfhydryl group. The thermal denaturation temperature of the protein was measured around
using a differential scanning calorimeter and circular dichroism spectrometry. The disulfide bonds were hardly reduced in the presence of reducing agents, suggesting that these disulfide bonds were located inside of the protein surface.
Plasma Phospholipids, including Plasmalogens, after Consumption of Diets Enriched in Long-chain n-3 Fatty Acids
Yeo, Young-K. ; Kim, Jong-S. ; Lee, Jong-R. ; Lee, Ji-Y. ; Chung, Sang-W. ; Kim, Hyo-J. ; Horrocks, Lloyd A. ; Park, Young-S. ;
BMB Reports , volume 33, issue 6, 2000, Pages 499~505
The level of long-chain n-3 fatty acids in chicken and pork can be increased by changing the diet of the animals. Increased levels of these essential fatty acids improve cardiovascular health in humans. The purpose of this study was to study the effects of the consumption of pork and chicken enriched in docosahexaenoic acid (DHA) on plasma lipids. The consumption of these products decreased the levels of two cardiovascular risk factors, LDL-cholesterol and triacylglycerols, in the plasma of female college students. The effect on LDL-cholesterol differed from that of fish oil, which does not affect the level of LDL-cholesterol. The proportions of DHA in the triacylglycerols and the glycerophospholipids were increased markedly. The greatest changes in the glycerophospholipids were in the ether types of the ethanolamine glycerophospholipids. Dietary DHA appears to be incorporated preferentially into the plasma ethanolamine plasmalogens, which can act as antioxidants. This agrees with our hypothesis that DHA stimulated the transcription of the genes for peroxisomal enzymes that are required for plasmalogen synthesis.
Stable Isotope Labeled Cytochrome
from Desulfovibrio vulgaris on a Defined Medium as Sole Nitrogen Source
Kim, Andre ; Shim, Yoon-Bo ; Kang, Shin-Won ; Park, Jang-Su ;
BMB Reports , volume 33, issue 6, 2000, Pages 506~509
To obtain Cytochrome
labeled with a stable isotope, the conditions of cultivation and the composition of medium for DvMF were examined. The growth of DvMF was steady and reproducible under purging with
and under pH control. DvMF was able to go on a defined medium without natural products. The composition of the medium containing a small amount of
as sole nitrogen source was established. Then, uniformly
was obtained during the culture of DvMF in a defined medium with
; it was confirmed by
Amino Acid Composition Analysis of the 32 kDa Sperminogen
Yi Lee, S.H. ;
BMB Reports , volume 33, issue 6, 2000, Pages 510~513
Boar sperminogen was purified from the acid extracts of the washed epididymal spermatozoa by gel filtration through a Sephadex G-100 column, followed by preparative SDS-PAGE. The 32 kDa sperminogen band was sliced out from the preparative SDS-PAGE and 32 kDa sperminogen was eluted from the gel matrix. The purified 32 kDa sperminogen was subjected to amino acid composition analysis. The amino acid composition of the 32 kDa boar sperminogen showed significant differences from that of either boar proacrosin or
, which signifies that 32 kDa sperminogen might not be a breakdown product of proacrosin-acrosin system and that the 32 kDa sperminogen is a different protein from proacrosin-acrosin system.
Characterization of Haemophilus influenzae Peroxiredoxins
Hwang, Young-Sun ; Chae, Ho-Zoon ; Kim, Kang-Hwa ;
BMB Reports , volume 33, issue 6, 2000, Pages 514~518
Two open reading frames of Haemophilus influenzae, HI0572 and HI0751, showing homology to a yeast thioredoxin peroxidase II (TPx II) and an E. coli thiol peroxidase
, respectively, were cloned and expressed in E. coli, and then the proteins were subsequently purified and characterized. HI0751 protein showed the thioredoxin (Trx)-dependent peroxidase activity, whereas HI0572 protein showed glutathione-dependent peroxidase. The HI0572 is the first peroxiredoxin with glutathione peroxidase activity rather than thioredoxin peroxidase. Purified HI0572 and HI0751 proteins protected specifically the inactivation of glutamine synthetase by metal catalyzed oxidation (MCO) systems composed of
and mercaptans such as dithiothreitol,
and glutathione (GSH). Unlike the HI0751 protein, the HI0572 protein was more effective in protecting glutamine synthetase from inactivation by the
system. It seems that these unique properties of the HI0572 protein are due to the structure containing a glutaredoxin domain at it's C-terminal in addition to a peroxiredoxin domain.
Effects of the Heptasequence SPTSPTY of Rat Nuclear Factor 1-A on Interactions between the C-Terminal Regions of Mammalian Nuclear Factor 1 Proteins
Hwang, Jung-Su ; Kim, Ji-Young ;
BMB Reports , volume 33, issue 6, 2000, Pages 519~524
NF1 proteins are a family of DNA binding proteins which consist of two separate domains, N-terminal DNA binding domain and C-terminal transcription activation domain. The N-terminal 220 amino acids are highly conserved and are also known to mediate dimerization of NF1 proteins. The C-terminal regions of different type of NF1 proteins are heterogeneous and responsible for transcriptional activation. In this study, we tested the interaction between different domains of rat NF1-A protein by yeast two hybrid analysis and observed the interaction between C-terminal regions of NF1-A which do not contain the N-terminal dimerization domain. Our results showed that the C-terminal region of rat NF1-A between residues 231 and 509 strongly interacted not only with itself, but also with human NF1/CTF1 which is a different type of NF1. When the C-terminal region was divided into two fragments, one from residue 231 to 447 and the other from 448 to 509, the two fragments were able to interact with the C-terminal region of NF1-A significantly. This indicates that both fragments contain independent interaction domains. Analysis of the interactions with alanine substituted fragments showed that substitutions of the heptasequence, SPTSPTY of NF1-A, affected interaction between NF1 proteins. Our results strongly suggest that C-terminal regions may also be important for the formation of homo- and heterodimers in addition to the N-terminal dimerization domain. Also, the heptasequence motif may play some roles in dimer formation.
Involvement of Cytosolic Phospholipase
in Nerve Growth Factor-Mediated Neurite Outgrowth of PC12 Cells
Choi, Soon-Wook ; Yu, Eun-Ah ; Lee, Young-Seek ; Yoo, Young-Sook ;
BMB Reports , volume 33, issue 6, 2000, Pages 525~530
The nerve growth factor (NGF) induces neuronal differentiation and neurite outgrowth of PC12 cells, whereas epidermal growth factors (EGF) stimulate growth and proliferation of the cells. In spite of this difference, NGF-or EGF-treated PC12 cells share various properties in cellular-signaling pathways. These include the activation of the phosphoinositide (PI)-3 kinase, 70 kDa S6 kinase, and in the mitogen-activated protein (MAP) kinase pathway, following the binding of these growth factors to intrinsic receptor tyrosine kinases (RTKs). Therefore, many studies have been attempted to access the critical signaling events in determining the differentiation and proliferation of PC12 cells. In this study, we investigated the cytosolic phospholipase
) in neurite behavior in order to identify the differences of signaling pathways between the NGF-induced differentiation and the EGF-induced proliferation of PC12 cells. We have showed here that the
was translocated from cytosol to membrane only in NGF-treated cells. We also demonstrated that this translocation is associated with NGF-induced activation of phospholipase
, which elevates intracellular
concentration. These results reveal that the translocation of
may be a requisite event in the neuronal differentiation of PC12 cells. Various phospholipase inhibitors were used to confirm the importance of these enzymes in the differentiation of PC12 cells. Neomycin B, a PLC inhibitor, dramatically inhibited the neurite outgrowth, and two distinct
inhibitors, 4-bromophenacyl bromide (BPB) and arachidonyltrifluoro-methyl ketone (
) also suppressed the neurite outgrowth of the cells, as well Taken together, these data indicated that
is involved in NGF-induced neuronal differentiation and neurite outgrowth of PC12 cells.
The Carboxyl Terminal Amino Acid Residues Glutamine276-Threonine277 Are Important for Actin Affinity of the Unacetylated Smooth
Cho, Young-Joon ;
BMB Reports , volume 33, issue 6, 2000, Pages 531~536
Tropomyosin (TM) is an important actin binding protein involved in regulation of muscle contraction. Unacetylated striated tropomyosin failed to bind to actin whereas unacetylated smooth tropomyosin bound well to actin. It has been demonstrated that high actin affinity of unacetylated
was ascribed to the carboxyl terminal amino acid residues. In order to define the role of the carboxyl terminal residues of tropomyosin molecule on actin binding, two mutant tropomyosins were constructed. TM11 is identical to the striated tropomyosin except that the carboxyl terminal last three amino acids was replaced with
whereas in TM14
was substituted with smooth specific
. TM11 and TM14 were overproduced in Escherichia coli and analyzed for actin affinity. The apparent binding constants (Kapp) of unacetylated tropomyosins were
for striated, respectively. This result indicated that higher actin affinity of the unacetylated smooth tropomyosin was primarily attributed to the presence of QT residues in the smooth sequence. In case of the Ala-Ser (AS) dipeptide extension of the amino terminus of tropomyosin, Kapp were
for AS-striated. AS-TM11 showed considerably higher actin affinity than AS-TM14, implying that interaction of Ala-Ser of the amino terminus with the carboxyl terminal residues. Since Kapp of AS-TM11 was significantly lower than that of AS-sm9, the presence of QT might be required for restoration of high actin affinity of the smooth
. These results suggested that the carboxyl terminal amino acid residues Glutamine275-Threonine276 are important for actin affinity of the recombinant smooth
, particularly of unacetylated smooth
A Simple and Reliable Method for Preparation of Cross-Contamination-Free Plant Genomic DNA for PCR-Based Detection of Transgenes
Hwang, Seon-Kap ; Kim, Young-Mi ;
BMB Reports , volume 33, issue 6, 2000, Pages 537~540
A simplified but reliable method was developed for the polymerase chain reaction (PCR)-based detection of genetically modified (GM) plants. The modified CTAB (mCTAB) method enabled us to prepare a high quality of genomic DNA from several hundred plant leaf samples in one day. Using DNA samples prepared from seven dicots and two monocots, approximately 1.75-kb regions spanning 17 S to 25 S ribosomal RNA genes were successfully amplified in a 2X PCR pre-mix containing BLOTTO. Further fidelity assessment of the mCTAB method by PCR analysis with Roundup Ready soybean (RRS) and non-RRS plants showed that the DNA samples prepared alternately from each of two lines were evidently free of cross-contamination. These results demonstrate that the mCTAB method is highly recommended for the rapid detection of transgenes in large numbers of leaf samples from diverse transgenic plants.
Transbilayer Effects of Chlorpromazine.HCl on Rotational Mobility of Synaptosomal Plasma Membrane Vesicles Isolated from Bovine Brain
Ahn, Ki-Weon ; Choi, Chang-Hwa ; Kim, Inn-Se ; Chung, In-Kyo ; Cho, Goon-Jae ; Jang, Hye-Ock ; Yun, Il ;
BMB Reports , volume 33, issue 6, 2000, Pages 541~547
Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to evaluate the effects of chlorpromazine HCl on the range of the rotational mobility of bulk bilayer structure of the synaptosomal plasma membrane vesicles (SPMV) isolated from a bovine brain. In a dose-dependent manner, chlorpromazine HCl increased the anisotropy (r), limiting anisotropy (
) and order parameter (S) of DPH in the membranes. Cationic 1-[4-(trimethylammonio)-phenyl]-6-phenylhexa-1,3,5-hexatriene (TMA-DPH) and anionic 3-[p-(6-phenyl)-1,3,5-hexatrienyl]-phenylpropionic acid (PRO-DPH) were utilized to examine the range of transbilayer asymmetric rotational mobility of the neuronal membranes. The anisotropy (r) of TMA-DPH in the inner monolayer was 0.034 greater than the value of PRO-DPH in the outer monolayer of the membranes. Both cationic TMA-DPH and anionic PRO-DPH were also used to examine the transbilayer asymmetric effects of chlorpromazine HCl on the range of rotational mobility of the membranes. Chlorpromazine HCl have a decreasing effects on the rotational mobility of the bulk bilayer structures and have a greater decreasing effect on the mobility of the inner monolayer as compared to the outer monolayer of the membranes. It has been proven that chlorpromazine HCl exhibit a selective rather than nonselective fluidizing effect within the transbilayer domains of the SPMV.