The Effects of Physical States of Phospholipids on $Ca^{2+}$-ATPase Activity of Biological Membranes

하종식,Hah, Jong-Sik The Korean Physiological Society 1988 대한생리학회지 Vol.22 No.2

세포막을 구성하고 있는 지질의 물리학적 성상이 단백질의 세포막 속으로의 삽입과정 및 단백질의 기능에 미치는 영향을 관찰하기 위하여 골격근의 근세망(SR)으로부터 $Ca^{2+}-ATPase$ 단백질을 분리한 후 이를 세포막의 주 구성성분인 포스파티딜콜린(PC)과 포스파티딜에타노라민(PE)의 혼합지질과 재조합(reconstitution)시켰다. 이와같이 인공적으로 재조합된 구조물에서 $Ca^{2+}-ATPase$의 기능을 측정하기 위하여 칼슘지시색소인 아르세나죠III(AIII)를 이용한 분광방법과 방사선동위원소를 이용한 여과법으로 칼슘흡수율을 측정하였고 또한 ATP 가수분해 능력을 측정하였다. 실험결과 칼슘의 흡수율은 포스파티딜코린의 함량이 많은 혼합지질과 재조합시킬 때에 증가하였고, ATP 가수분해 능력은 포스파티딜함량이 25%까지는 포스타피딜코린의 양에 비례하여 증가하였으나 50%이상에서는 약간 감소하는 경향을 보였다. 한편 지질세포막속으로 단백질이 삽입되는 양은 포스파티딜 함량이 25%일 때 최고의 값을 보였으며 함량이 그 이하 또는 이상일 때는 감소하였다. 이상의 실험결과로보아 단백질의 기능은 세포막이 "bilayer" 구조를 갖출때에 증가하고 세포막속으로 단백질이 삽입되는 양은 세포막이 "non-bilayer" 구조를 형성할 때에 증가함을 알 수 있다. The $Ca^{2+}-ATPase$ of sarcoplasmic reticulum (SR) was solubilized and reconstituted into a mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of varying ratios in order to assess the effect of physical states of phospholipids on the incorporation and functions $Ca^{2+}-ATPase$. On the basis of the spectral data of Ca-arsenazo III, the $Ca^{2+}$ uptake of SR was increased linearly as the PC content increased in the reconstituted vesicles. The ATP hydrolysis activity also increased as PC content increased up to 25% and then decreased slightly as the PC content further increased. On the other hand the incorporation of $Ca^{2+}-ATPase$ into the reconstituted vesicls occured maximally at 25% PC and 75% PE mixture which is known to have a non-bilayer structure in reconstitution system. From the above results it is clear that preexisting defects in the lipid bilayer promote protein incorporation into the bilayer during reconstitution and lamellar structure of the bilayer facilitates the $Ca^{2+}-ATPase$ function.

인공적혈구의 제조 및 이용

하종식,조응행,김구자,Hah, Jong-Sik,Cho, Eng-Haeng,Kim, Ku-Ja 대한생리학회 1990 대한생리학회지 Vol.24 No.1

Hemoglobin was purified from the outdated human red blood cells. Phospholipids were purified from egg yolk and stored in chloroform. The artificial red blood cells (hemosome) were prepared by encapsulation of hemoglobin with phospholipid mutilayer using rotary vacuum evaporator. The shape and size of hemosomes were measured by phase contrast microscope and image analyzer. The function of hemosomes was tested by measuring oxygen dissociation curve using blood gas analyzer. In order to test whether hemosomes are useful as blood substitute they were infused into rats of which one third of total blood were drawn. The results obtained are summarized at followings. 1) Hemosomes were spherical shape and their mean diameter was 0.7 um. 2) Oxygen dissociation curve of hemosomes showed the same figure as that of normal red blood cells. 3) All rats given 1/3 transfusion with hemosomes survived until sacrificed whereas three of four rats given 1/3 transfusion with saline died within 1 hour and the rest of them died within 24 hours.

인슐린의 포도당 이동 촉진 기전에 관한 연구 -세포내부 미세구조와 Cytochalasin B 결합단백질의 분포-

하종식,Hah, Jong-Sik 대한생리학회 1990 대한생리학회지 Vol.24 No.2

What makes glucose transport function sensitive to insulin in one cell type such as adipocyte, and insensitive in another such as liver cells is unresolved question at this time. Recently it is known that insulin stimulates glucose transport in adipocytes largely by redistributing transporter from the storage pool that is included in a low density microsomal fraction to plasma membrane. Therefore, insulin sensitivity may depend upon the relative distribution of gluscose transporters between the plasma membrane and in an intracellular storage compartment. In hepatocytes, the subcellular distribution of glucose transporter is less well documented. It is thus possible that the apparent insensitivity of the hepatocyte system could be either due to lack of the constitutively maintained, intracellular storage pool of glucose transporter or lack of insulin-mediated transporter translocation mechanism in this cell. In this study, I examined if any intracellular glucose transporter pool exists in hepatocytes and this pool is affected by insulin. The results obtained summarized as followings: 1) Distribution of subcellular fractions of hepatocyte showed that there are $24.9{\pm}1.3%$ of plasma membrane, $36.9{\pm}1.7%$ of nucleus-mitochondria enriched fraction, $23.5{\pm}1.2%$ of lysosomal fraction, $9.6{\pm}1.0%$ of high density microsomal fraction and $4.9{\pm}0.5%$ of low density microsomal fraction. 2) In adipocyte, there were $29.9{\pm}2.6%$ of plasma membrane, $19.4{\pm}1.9%$ of nucleus-mitochondria enriched fraction, $26.7{\pm}1.8%$ of high density microsomal fraction and $23.9{\pm}2.1%$ of low density microsomal fraction. 3) Surface labelling of sodium borohydride revealed that plasma membrane contaminated to lysosomal fraction by $26.8{\pm}2.8%$, high density microsomal fraction by $8.3{\pm}1.3%$ and low density microsomal fraction by $1.7{\pm}0.4%$ respectively. 4) Cytochalasin B bound to all of subcellular fractions with a Kd of $1.0{\times}10^{-6}M$. 5) Photolabelling of cytochalasin B to subcellular fractions occurred on 45 K dalton protein band, a putative glucose transporter and D-glucose inhibited the photolabelling. 6) Insulin didn't affect on the distribution of subcellular fractions and translocation of intracellular glucose transporters of hepatocytes. 7) HEGT reconstituted into hepatocytes was largely associated with plasma membrane and very little was found in low density microsomal fraction which equals to the native glucose transporter distribution. Insulin didn't affect on the distribution of exogeneous glucose transporter in hepatocytes. From the above results it is concluded that insulin insensitivity of hepatocyte may due to lack of intracellular storage pool of glucose transporter and thus intracellular storage pool of glucose transporter is an essential feature of the insulin action.

Target Size of $(Na^++K^+)$-ATPase and $Na^+,\;K^+)$Pump of Human Erythrocytes

하종식,Hah, Jong-Sik,Jung, Chan Y. The Korean Physiological Society 1985 대한생리학회지 Vol.19 No.1

$(Na^++K^+)-ATPase$은 ${\alpha}$ 와 ${\beta}$의 두 subunits로 구성되어 있으며, 분자량이 약 300,000 daltons 정도되는 것으로 보아 ${\alpha}_2{\beta}_2$의 형태로 존재할 것으로 알려져 왔다 한편, 사람 적혈구막에 있는 $Na^+,\;K^+\;Pump$는 glycolytic enzymes과 complex를 이루고 있으리라는 보고도 있다. 우리는 이 실험에서 in situ상태의 사람 적혈구막$(Na^++K^+)-ATPase$의 분자량을 측정하기 위하여, 소위 말하는 ‘Target theory’를 radiation에 의한 ouabain sensitive한 $\Na^+$이동과, intact한 cells과 ghosts에서의 ATP가수분해능력의 inactivation data에 적용하였다. Intact한 cells은 cryoprotective agent의 존재하에서, ghosts는 직접적으로 액화질소의 용기속에 담고 온도를 $-45^{\circ}C$에서 $-50^{\circ}C$로 유지시키면서 1.5 MeV의 electron beam으로 조사한 후에 Pump의 기능내지 효소의 활성도를 측정하여 radiation에 따르는 inactivation의 정도를 측정하였다. 이득 활성도는 radiation의 양에 따라 simple exponential function으로 inactivation되었으며, 이로부터 radiation sensitive volume(target size)를 계산하였다. Target size는 intact한 cells을 사용하였을 경 우$(Na^++K^+)-ATPase$나 $Na^+,\;K^+\;Pump$ 모두 600,000 daltons으로 계산되었으며, 이 값은 만약 cells을 strophanthidin으로 먼저 처치하고 측정하면 약 325,000 daltons으로 감소하였다. Ghosts를 사용했을 경우에도$(Na^++K^+)-ATPase$의 target size는 역시 약 325,000 daltons이었다. 이상의 결과로 미루어 보아 intact한 cells에서는 $(Na^++K^+)-ATPase/Na^+,\;K^+\;Pump$가 $(\alpha\beta)_2$의 dimer 상태로 존재하거나 혹은 $(\alpha\beta)_2$의 monomer에 glycolytic enzymes과 같은 다른 enzymes이 붙어 functional한 구조를 이루고 있는 것이 아닌가 사료된다. 또한 실헐성적은 이러한 dimeric association 혹은 heterocomplex association은 ghost를 만드는 과정에서나 strophanthidin의 처치로 부서질 수 있음을 암시하고 있다. Previous biochemical studies indicate that $(Na^++K^+)-ATPase$ is composed of two subunits, ${\alpha}$ and ${\beta}$, in a form of ${\alpha}_2{\beta}_2$ with a molecular weight of approximately 300,000 daltons. There is also suggestive evidence that the $Na^+$, $K^+$ pump in human erythrocytes occurs in a complex with some glycolytic enzymes. We assessed here in situ assembly size of the $(Na^++K^+)-ATPase$ of human erythrocytes by applying classical target theory to radiation inactivation data of the ouabain-sensitive sodium flux and ATP hydrolysis of intact cells and ghosts. Cells(in the presence of cryoprotective agent) and ghosts were irradiated at $-45^{\circ}C$ to $-50^{\circ}C$ with an increasing dose of a 1.5 MeV electron beam, and after thawing, the pump and/or enzyme activities were assayed. Each activity measured was decreased as a simple exponential function of radiation dose, from which a radiation sensitive volume (target size) was calculated. When intact cells were used, the target size of both $(Na^++K^+)-ATPase$ and $Na^+$, $K^+$ pump was found to be approximately 600,000 daltons. This target size of the ATPase was reduced to approximately 325,000 daltons if the cells were pretreated with strophanthidin. When ghosts were used, the target size of the ATPase was again approximately 325,000 daltons. Our target size measurement suggests that, in intact cells, the $(Na^++K^+)-ATPase/Na^+,K^+$ pump exists either as a dimer of $(\alpha\beta)_2$ which is a functional unit or as a monomer of $(\alpha\beta)_2$ but in tight complex with other enzyme or enzymes. The results also suggest that this dimeric or heterocomplex association is dissociated during ghost preparation and strophanthidin treatment.

인체 적혈구막 Band 4.5 단백질의 기능적인 분자구조

하종식,Hah, Jong-Sik 대한생리학회 1986 대한생리학회지 Vol.20 No.2

The functional molecular weight of band 4.5 polypeptide was measured by applying the classical target theory to radiation inactivation data of the cytochalasin B binding. Band 4.5 polypeptides purified from human erythrocyte membranes were irradiated at -45 to $-50^{\circ}C$ with an increasing dose of 1.5 MeV electron beam, and after thawing, cytochalasin B binding activities were assayed. Each activity measured was reduced as a simple exponential function of radiation dose. $D_{37}$, dose appeared to be 6.7 mega rads, from which the target size (radiation sensitive mass) of band 4.5 polypeptide was calculated to be 95,500 daltons. This result with other informations available in literature suggests that band 4.5 polypeptide may exist as a dimer in human erythrocytes.

인삼(Panax Ginseng)주정추출액이 기관지 평활근의 수축력에 미치는 영향

하종식(Hah, Jong-Sik),이명호(Lee, Myoung-Ho),강두희(Kang, Doo-Hee) 대한생리학회 1977 대한생리학회지 Vol.11 No.2

It has been reported that administration of Ginseng powder to the Guinea pig reduces anaphylactic shook induced by horse serum (Lee, 1939). However, Lee et al. (1960) and Paik et al. (1976) have demonstrated that Ginseng increases capillary permeabilites and histamine release from the mast cell. These facts suggest that Ginseng acts directly on the bronchial muscle causing it to dilate. Recently, a number of investigators(Kidakawa & Iwasiro 1963; Takagi et al. 1973) have reported that Ginseng reverses acetylcholine- or histamine- induced contraction in the isolated Guinea pig ileum. We, therefore, undertook the present study to examine if Ginseng relaxes the spasm of bronchial muscle induced by acetylcholine or histamine. We have also attempted to identify the mechanism of the Ginseng effect. Male Guinea Pig was sacrificed by a blow on the head, The trachea was removed and sectioned with scissors into about 12 rings. After the C shaped ring of cartilage was sectioned the one end of ring was tied to the bottom of the incubation bath and the other end was connected to a force transducer (FTO 3C) to record tension on a Polygraph. When the antispasmodic action of Ginseng effect was first examined in the normal trachea which was not treated by the drug. And then the Ginseng effect was tested in the muscle treated by histamine hydrochloride, acetylcholine hydrochloride or barium chloride. The results indicate that Ginseng alcohol extract relaxes the contraction of isolated tracheal muscle induced by histamine (1μg/ml ~ 10μg/ml), acetylcholine (1μg/ml ~ 5μg/ml) and barium chloride (1.5 mg/ml). The mechanism of this action is in Pa.1 due to nonspecific antimuscarinic and antihistaminic effect and in part by predominant action in the adrenergic β-receptor although the α-receptor is also involved. We, therefore, conclude that Ginseng can be act as a bronchodilator.

사람 적혈구막의 (Na⁺+K⁺)-ATPase/Na⁺, K⁺ Pump의 Target Size

하종식(Hah, Jong-Sik),Jung, Chan Y. 대한생리학회 1985 대한생리학회지 Vol.19 No.1

(Na⁺+K⁺)-ATPase은 α 와 β의 두 subunits로 구성되어 있으며, 분자량이 약 300,000 daltons 정도되는 것으로 보아 α₂β₂의 형태로 존재할 것으로 알려져 왔다 한편, 사람 적혈구막에 있는 Na⁺, K⁺ Pump는 glycolytic enzymes과 complex를 이루고 있으리라는 보고도 있다. 우리는 이 실험에서 in situ상태의 사람 적혈구막(Na⁺+K⁺)-ATPase의 분자량을 측정하기 위하여, 소위 말하는 ‘Target theory’를 radiation에 의한 ouabain sensitive한 Na⁺이동과, intact한 cells과 ghosts에서의 ATP가수분해능력의 inactivation data에 적용하였다. Intact한 cells은 cryoprotective agent의 존재하에서, ghosts는 직접적으로 액화질소의 용기속에 담고 온도를 -45℃에서 -50℃로 유지시키면서 1.5 MeV의 electron beam으로 조사한 후에 Pump의 기능내지 효소의 활성도를 측정하여 radiation에 따르는 inactivation의 정도를 측정하였다. 이득 활성도는 radiation의 양에 따라 simple exponential function으로 inactivation되었으며, 이로부터 radiation sensitive volume(target size)를 계산하였다. Target size는 intact한 cells을 사용하였을 경 우(Na⁺+K⁺)-ATPase나 Na⁺, K⁺ Pump 모두 600,000 daltons으로 계산되었으며, 이 값은 만약 cells을 strophanthidin으로 먼저 처치하고 측정하면 약 325,000 daltons으로 감소하였다. Ghosts를 사용했을 경우에도(Na⁺+K⁺)-ATPase의 target size는 역시 약 325,000 daltons이었다. 이상의 결과로 미루어 보아 intact한 cells에서는 (Na⁺+K⁺)-ATPase/Na⁺, K⁺ Pump가 (αβ)₂의 dimer 상태로 존재하거나 혹은 (αβ)₂의 monomer에 glycolytic enzymes과 같은 다른 enzymes이 붙어 functional한 구조를 이루고 있는 것이 아닌가 사료된다. 또한 실헐성적은 이러한 dimeric association 혹은 heterocomplex association은 ghost를 만드는 과정에서나 strophanthidin의 처치로 부서질 수 있음을 암시하고 있다. Previous biochemical studies indicate that (Na⁺+K⁺) is composed of two subunits, α and β, in a form of α₂β₂ with a molecular weight of approximately 300,000 daltons. There is also suggestive evidence that the Na⁺, K⁺ pump in human erythrocytes occurs in a complex with some glycolytic enzymes. We assessed here in situ assembly size of the (Na⁺+K⁺) of human erythrocytes by applying classical target theory to radiation inactivation data of the ouabain-sensitive sodium flux and ATP hydrolysis of intact cells and ghosts. Cells(in the presence of cryoprotective agent) and ghosts were irradiated at -45℃ to -50℃ with an increasing dose of a 1.5 MeV electron beam, and after thawing, the pump and/or enzyme activities were assayed. Each activity measured was decreased as a simple exponential function of radiation dose, from which a radiation sensitive volume (target size) was calculated. When intact cells were used, the target size of both (Na⁺+K⁺) and Na⁺, K⁺ pump was found to be approximately 600,000 daltons. This target size of the ATPase was reduced to approximately 325,000 daltons if the cells were pretreated with strophanthidin. When ghosts were used, the target size of the ATPase was again approximately 325,000 daltons. Our target size measurement suggests that, in intact cells, the (Na⁺+K⁺)-ATPase/Na⁺,K⁺ pump exists either as a dimer of (αβ)₂ which is a functional unit or as a monomer of (αβ)₂ but in tight complex with other enzyme or enzymes. The results also suggest that this dimeric or heterocomplex association is dissociated during ghost preparation and strophanthidin treatment.

적혈구막 Glucose Transporter 의 메칠화 부위와 재조합 리포좀에서 glucose Uptake

전길자,하종식 ( Gil Ja Jhon,Jong Sik Hah ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.2

For the purpose of showing the physiological role of methyl esterification by protein carboxyl methyltransferase as a post-translational modification, the glucose transporter of human erythrocyte membrane was partially purified using octyl glucoside. The activity of human transporter was increased by 34% as a result of the methylation of transporter. The increase of methylatability and decrease (47%) of the amount of glucose transporter in erythrocyte of diabetic rats could be explained from the above result. It was also found that a small amount of glucose transporter (about 1/20 of human erythrocytes) was existed in rat erythrocytes. The physiological significance of this methylation is not clear, but our results support that protein carboxyl methyltransferase functions in increasing the activity of damaged erythrocyte glucose transporter.

수종의 알레르기 관련 약물이 흰쥐의 복강내 비만세포에서 Hyaluronidase 및 히스타민 유리에 미치는 영향

유신애,김구자,하종식,Yoo, Shin-Ae,Kim, Ku-Ja,Hah, Jong-Sik 대한생리학회 1988 대한생리학회지 Vol.22 No.2

Type I allergic reaction and it's related clinical manifestations are known to occur by the effects of various chemical mediators. These chemical mediators are released from circulating basophils and tissue mast cells, which become 'sensitized' through the binding of antigens and antibodies of the IgE type to their cell surface receptors. Efforts to elucidate the mechanism of the release of these mediators, especially that of histamine, have been persued for years. The mechanism is not yet clarified at the present time. Recent reports of hyaluronidase, an enzyme known to be involved in the tissue inflammatory process, as possible participant in type I allergic reaction, initiated this study. Relationships between the hyaluronidase activity and histamine release from the sensitized rat peritoneal mast cells were investigated. Also anti-allergic agents, tranilast and disodium cromoglycate, along with known histamine releasers, morphine and compound 48/80, were used to observe the inhibitory and stimulatory effects of these substances on the hyaluronidase activity as well as histamine release from the rat mast cells. The results obtained are summarized as follows: 1) Hyaluronidase activity and histamine release from sensitiaed rat peritoneal mast cells started to increase on the 4th day of postsensitization. Hyaluronidase activity reached it's peak value on the 7th day of postsensitization and that of histamine release on the 14th day of postsensitization. 2) Hyaluronidase activity and histamine release from sensitized rat peritoneal mast cells, pre-treated with tranilast revealed significant decrease in comparison with those of non-treated cells. 3) Hyaluronidase activity and histamine release from sensitized rat peritoneal mast cells, pre-treated with tranilast, followed by morphine injection, revealed significant increase in comparison with those of tranilast treated cells. 4) In vitro study of hyaluronidase activity and histamine release from un-sensitized rat peritoneal mast cells, using morphine and compound 48/80 as activators, revealed significant increase compared to those of non-activator used cells. 5) In vitro study of hyaluronidase activity and histamine release from un-sensitized rat peritoneal mast cells, pre-treated with tranilast and disodium cromoglycate, using confound 48/80 and morphine as activators revealed significant decrease in comparison with those of tranilast and disodium cromoglycate treated cells. From above results, participation of enzyme hyaluronidase in the process of histamine release from sensitized rat pertioneal mast cells, could be suggested. It was also quite evident that the clinically used anti-allergic agents, tranilast and disodium cromoglycate, have significant inhibitory function on the hyaluronidase activity and histamine release from sensitized rat peritoneal mast cells, while morphine significantly increased the hyaluronidase activity and histamine release from sensitized rat peritoneal mast cells.

장거리 (마라톤)선수에서의 전 경기중 심박동수의 변화

김인교,이중우,하종식,유연희,최정옥,김기호,Kim, In-Kyo,Lee, Jung-Woo,Hah, Jong-Sik,Ryu, Yun-Hee,Choi, Jung-Ok,Kim, Ki-Ho 대한생리학회 1979 대한생리학회지 Vol.13 No.1

To evaluate the present status of physical fittness of Korean long distance runners, body fat, pulmonary functions, maximal oxygen intake and oxygen debt were measured in 5 elite marathoners (A group), 6 college student runners (B group) and 3 middle school student runners (C group). After laboratory tests, full course marathon running was performed in 2 elite marathoners during which their heart rates were monitored continuously. The results are summerized as follows: 1) Total body fat in all three groups are in the range of 13-15% of their body weight. 2) In all three groups, average values of various pulmonary functions were within the normal limits, but those of tidal volume were higher and respiratory rate were lower in comparison to normal values. These phenomena may represent respiratory adaptations against training. The average resting oxygen consumptions in A,B and C were $322{\pm}23$, $278{\pm}14$ and $287{\pm}16$m1/min, respectively. 3) In all three groups, resting blood pressures were in the normal range, but the resting heart rate was slightly lower in groups A $(56{\pm}3\;beats/min)$ and B $(64{\pm}2\;beats/min)$ and higher in group C $(82{\pm}9\;beats/min)$ in comparison to normal values. These changes in cardiovascular functions in marathoners may also represent adaptive phenomena. 4) During treadmill running the minute ventilation and oxygen consumption of the runners increased lineally with work load in all three groups. When the oxygen consumption was related to heart rate, it appeared to be a exponential function of the heart rate in all three groups. 5) The average maximal heart rates during maximal work were $196{\pm}3$, $191{\pm}3$ and $196{\pm}5\;beats/min$ for groups A,B and C, respectively. Maximal oxygen intakes were $84.2{\pm}3.3\;ml/min/kg$ in group A, $65.2{\pm}1.1\;ml/min/kg$ in group B and $58.7{\pm}0.4\;ml/min/kg$ in group C. 6) In all three groups, oxygen debts and the rates of recovery of heart rate after treadmill running were lower than those of long ditsance runners reported previously. 7) The 40 km running time in 2 elite marathoners was recorded to be $2^{\circ}42'25'$, and their mean speed was 243 m/min (ranged 218 to 274 m/min). The heart rate appeared to increase lineally with running speed, and the total energy expenditure during 40 km running was approximately 1360.2 Calories. From these it can be speculated that if their heart rates were maintained at 166 beats/min during the full course of marathon running, their records would be arround $2^{\circ}15'$. Based on these results, we may suspect that a successful long distance running is, in part, dependent on the economical utilization of one's aerobic capacity.