(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:20)
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1027
`
`

`

`Biochemistry: Buda et al.
`
`Proc. Natl. Acad. Sci. USA 91 (1994)
`
`8235
`
`ture-up shifted fish. Cell viability was shown by trypan blue
`exclusion to be 95%.
`Brain cell suspensions obtained from warm- and cold—
`acclimated fish were incubated at the alternative temperature
`for 80 min, subsequently restored to their acclimatization
`temperature, and then kept at that temperature for another 80
`min. During this time, fluorescence anisotropy measure-
`ments were carried out on loo-pl aliquots of cell suspension
`at intervals of 10 min throughout the duration of incubation.
`Steady-State Fluorescence Anisotropy Measurements. Cells
`were exposed to the opposite (experimental) temperature
`extremes for various times and then were labeled with 5 pl of
`1 mM fluorescent dye 3-[p-(6-phenyl-1,3,5-hexatrienyl)phe-
`nyl]propionic acid (DPH-PA) (Molecular Probes) in situ and
`steady-state fluorescence anisotropy measurements were
`carried out at the incubation temperature by using a com-
`puter-controlled thermostatable spectrophotofluorimeter
`(Hitachi MPF-ZA) according to Dey and Farkas (5). It has
`been found that DPH-PA labels the outer leaflet of plasma
`membrane (5, 6, 12). To correct the fluorescence intensity
`and anisotropy for light scattering, measurements were also
`made on an unlabeled sample under the same conditions as
`for the labeled samples. These measurements were based on
`Kuhry et al. (13). The error of these determinations was
`<0.005 unit.
`To study the effect of temperature on membrane structural
`order in the spectrophotofluorimeter cell itself, a different
`approach was followed. Concentrated cell suspensions from
`warm- and cold-acclimated fish were labeled in HBSS at 25°C
`and 5°C, respectively. They were then kept at their respective
`temperature for 20 min to ensure the labeling of the cells and
`equilibration of the fluorescent dye in the membrane. Next,
`they were transferred into the precooled (5°C) or preheated
`(25°C) chamber of the spectrophotofluorimeter in a concen-
`tration to give 0.05 OD unit, and the changes in the anisotropy
`were recorded at intervals of 2 min until the anisotropy
`assumed a constant value.
`Electron Spin Resonance (ESR) Spectroscopy. A cell sus-
`pension containing 1 mg of phospholipid was mixed with 10
`pg (1 mg/ml in ethanol) of l-palmitoyl-Z-[14-(4,4-dimethyl-
`N-oxyl)stearoyl]-sn-glycero-3-phosphoglycerol (14-PGSL)
`in HBSS at room temperature for 15 min. Samples were
`contained in l-mm (i.d.) capillary tubing. Phospholipid (5
`uM) in chloroform was mixed with 15 nmol of 14-PGSL in
`ethanol at room temperature for 5 min. For the labeling of
`phospholipids, the method of Dey et al. (6) was followed. The
`
`samples were transferred into the capillary and accommo-
`dated in a standard quartz ESR tube. Spectra were recorded
`between 5°C and 25°C with a computerized ECS-106 (Bruker,
`Billerica, MA) ESR spectrometer equipped with air-flow
`temperature regulation. The rate of motion of the spin label
`14-PGSL was quantitated with the efiecfive rotational cor-
`relation time (1:) according to Kivelson (14). Effective order
`parameters were calculated by the formula of lost et al. (15).
`Lipid Extraction and Analysis. Total lipids were extracted
`from the brains or the washed cells according to Folch et al.
`(16). Phospholipids from the total lipids were separated by
`silicic acid column chromatography, using chloroform to
`remove the neutral lipids and methanol to elute the polar
`lipids. Phospholipid subclasses were separated by one-
`dirnensional TLC according to Fine and Sprecher (17). Phos-
`phorus content was determined according to Rouser et al.
`(18). Methyl esters were separated on 10% FFAP on 80- to
`100-mesh Supelcoport (Supelco) in a 2-m column (2-mm i.d.).
`A Hitachi model 263-80 gas chromatograph connected to a
`Hitachi model 263-80 data processor was used. Peaks were
`identified by comparison with authentic standards (6).
`Molecular species composition was. determined according
`to Takamura et al. (19). The dinitrobenzoyl derivatives of
`diacylglycerols, obtained by phospholipase C hydrolysis
`(from Bacillus cereus for phosphatidylethanolamines and
`from Clostridium perfringens for phosphatidylcholines) (Sig-
`ma) of the phospholipids were separated by HPLC (Waters
`model 440) on a Nucleosil C13 column [5-p.m particle size; 4
`mm (i.d.) X 250 mm] using acetonitrile/Z-propanol (80:20,
`vol/vol) of HPLC grade (Carlo Erba, Milan) isocratically as
`the mobile phase (flow rate, 1.0 ml/min) and monitoring the
`eluent at 254 nm. The peaks were recorded and calculated by
`using a data processor (Hitachi model 263-80). Peaks were
`identified by 1,2-diacylglycerol derivatives of authentic stan-
`dards (6) and their relative elution times (20).
`
`RESULTS
`
`The carp brain total phospholipids proved to be rather rich in
`docosahexaenoic acid. Ethanolamine phosphoglycerides
`were richer in docosahexaenoic acid than choline phospho-
`glycerides, but the difference between cold-acclimated and
`warm-acclimated animals was not significant (Table 1). The
`ratio of saturated to unsaturated fatty acids varied with the
`temperature, being lower in the cold-adapted fish (Table 1).
`High levels of docosahexaenoic acid have been reported from
`
`Table 1. Fatty acid composition of total phospholipids, phosphatidylcholines (PC), and phosphatidylethanolamines in
`carp brain in relation to acclimation temperature
`
`Total
`
`wt % (mean 1 SD)
`PC
`
`PE
`
`25°C
`5°C
`25°C
`25°C
`5°C
`Fatty acid
`8.5 1 4.2
`8.1 1 3.6
`19.4 1 7.7
`13.8 1 3.2
`13.4 1 4.2
`16:0
`9.4 1 0.8
`7.6 1 4.6
`6.8 1 3.0
`8.5 1 1.5
`7.9 1 3.1
`16:1
`9.9 1 5.5
`5.3 1 1.3
`15.4 1 12.0
`12.5 1 1.3
`7.5 1 1.9
`18:0
`16.5 1 1.0
`16.8 1 5.4
`18.6 1 8.1
`17.2 1 1.4
`20.5 1 3.1
`18:1(n - 9)
`0.1 1 0.1
`1.4 1 1.3
`0.5 1 0.5
`0.6 1 0.5
`1.1 1 0.6
`18:2(n ~ 6)
`1.5 1 0.1
`2.6 1 0.4
`1.4 1 0.3
`1.0 1 0.6
`2.4 1 0.2
`18:3(n — 3)
`8.5 1 0.8
`12.9 1 1.8
`8.1 1 6.5
`8.6 1 0.6
`10.6 1 2.0
`20:4(n — 6)
`0.6 1 0.6
`0.7 1 0.6
`0.2 1 0.1
`0.2 1 0.1
`0.3 1 0.1
`20:5(n — 3)
`0.6 1 0.4
`0.5 1 0.3
`0.3 1 0.2
`0.5 1 0.1
`0.4 1 0.2
`22:4(n — 6)
`1.2 1 0.2
`1.3 1 0.5
`3.8 1 2.1
`3.3 1 0.5
`4.7 1 1.2
`24:1(n - 9)
`0.4 1 0.2
`1.3 1 0.5
`0.7 1 0.5
`0.3 1 0.1
`0.8 1 0.3
`22:5(n _ 3)
`31.7 1 2.0
`34.1 1 4.0
`18.9 1 7.1
`30.5 1 0.7
`25.5 1 5.2
`22:6(n — 3)
`11.5
`7.1
`5.9
`3.0
`4.9
`Others
`
`
`
`(0.35)(0.26)(sat/unsat)* (0.22) (0.33) (0.53) (0.15)
`
`
`n = 8 for 5°C fish and n = 6 for 25°C fish.
`*Saturated/unsaturated ratio.
`000002
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:21)
`
`17.1 1 6.9
`7.5 1 2.7
`11.2 1 6.5
`20.7 1 5.4
`0.8 1 0.3
`2.5 1 0.5
`9.7 1 0.5
`0.5 1 0.3
`0.3 1 0.2
`4.7 1 2.6
`0.8 1 0.6
`21.8 1 5.4
`
`
`
`

`

`8236
`
`Biochemistry: Buda et at.
`
`Proc. Natl. Acad. Sci. USA 9] (1994)
`
`Table 3. Molecular species composition of brain
`phosphatidylcholines in fish seasonally or evolutionarily
`adapted to contrasting temperatures
`% total
`
`At 5°C
`
`At 25°C
`
`Acyl
`groups
`22:6/22:6
`20:5/20z5
`20:4/20z4
`18:1/2025
`16:1/16:1
`18:1/22:6
`16:0/2226
`18:1/20:4
`16:0/20:4
`18:0/20:5
`18:0/2226
`18:0/1822
`18:0/20:4
`18:1/18:1
`16:0/18:1
`16:0/16:0
`18:0/18:1
`16:0/18:0
`18:0/1820
`Others
`
`C. carpio
`(n = 8)
`1.9 1 0.9
`0.2 1 0.1
`1.6 1 0.4
`0.3 1 0.2
`0.2 1 0.1
`3.5 1 1.3
`11.2 1 5.1
`1.7 1 0.6
`3.2 1 0.9
`1.0 1 0.2
`12.6 1 2.7
`2.7 1 2.0
`12.7 1 8.5
`3.4 1 0.1
`21.5 1 12.8
`3.4 1 1.1
`3.5 1 2.8
`8.5 1 4.7
`1.4 1 0.5
`5.2
`
`See legend to Table 2.
`
`A. cernua
`(n = 5)
`1.9 1 0.8
`0.4 1 0.1
`1.8 1 0.8
`1.2 1 0.2
`0.8 1 0.3
`5.6 1 0.6
`24.4 1 6.7
`1.0 1 0.5
`2.9 1 0.7
`1.8 1 0.5
`10.0 1 3.1
`Trace
`2.5 1 1.1
`1.6 1 0.8
`24.6 1 14.6
`1.7 1 1.2
`0.8 1 0.6
`9.4 1 3.2
`0.2 1 0.1
`11.3
`
`C. carpio
`(n = 6)
`1.7 1 0.7
`0.2 1 0.1
`1.6 1 0.4
`0.5 1 0.2
`Trace
`1.3 1 0.3
`8.4 1 5.1
`2.6 1 2.0
`2.6 1 0.7
`0.4 1 0.3
`23.5 1 15.0
`2.3 1 2.0
`13.5 1 2.4
`2.0 1 1.2
`20.4 1 14.5
`2.8 1 1.7
`3.7 1 2.7
`8.7 1 4.9
`1.6 1 1.4
`2.2
`
`C. calla
`(n = 7)
`0.6 1 0.4
`0.2 1 0.1
`0.5 1 0.1
`0.4 1 0.1
`0.1 1 0.08
`3.0 1 1.2
`27.3 1 10.2
`2.3 1 2.1
`1.2 1 0.3
`0.7 1 0.2
`19.5 1 3.1
`Trace
`2.5 1 1.5
`1.8 1 1.4
`30.5 1 18.7
`4.1 1 2.1
`0.8 1 0.5
`9.4 1 3.3
`Trace
`3.0
`
`indicating a slightly less
`warm-acclimated carp (Fig. 1),
`ordered environment of the spin probes in the former. The
`homeoviscous efficacy, calculated according to Wodtke and
`Cossins (4), was estimated to be around 10%. When cold- or
`warm-acclimated fish were shifted to the opposite extreme of
`the temperature in steps of 0.5°C/hr, neither the fatty acid
`composition (data not shown) nor the effective rotation
`correlation time (Fig. l) of the isolated phospholipids was
`adapted to the new temperature. In contrast, vesicles from
`cold-acclimated temperature-upshifted animals become less
`ordered in the lower temperature regions and vice versa. In
`a separate set of experiments, the isolated brain cells were
`labeled with the spin probe to study the responses given by
`the intact membranes. To avoid thermal shock to the cells,
`both the preparation and the labeling procedure were done at
`the acclimation temperature; moreover, the ESR tempera-
`4.00
`’r
`l
`l
`"T‘
`r
`
`other fish neural elements (9, 21, 22), but, as in our case, an
`effect of environmental temperature on docosahexaenoic
`acid has not been observed. Chang and Roots (21, 22) found
`that a reduction in the ratio of saturated to unsaturated fatty
`acids in the mitochondria and microsomes accompanied cold
`adaptation in goldfish (Carassius auraus) brains, but carp
`nerve phospholipids (9) and garfish (Lepisosteus osseus)
`axon phospholipids (22) did not show a similar response.
`There were characteristic differences with acclimation
`temperature in molecular species composition between phos-
`phatidylcholines and phosphatidylethanolamines of carps.
`Similar differences were seen also with fish evolutionarily
`adapted to the temperature (Tables 2 and 3). The 18:0/22z6,
`1810/2014, and 1620/18zl species were predominant in the
`phosphatidylcholines, with additionally 16:0/22:6 in the phos-
`phatidylethanolamines. However, 16:0/ 18:1 was only a mi-
`nor component in the phosphatidylethanolamine fraction.
`The level of 1820/22:6 tended to be higher in the phosphati-
`dylcholines and phosphatidylethanolamines in the brains of
`warm-acclimated fish than those in cold-acclimated fish. In
`agreement with findings in the present study, the level of
`18:0/22:6 has been found to be low in the brains of marine and
`freshwater fish adapted to low environmental temperature
`(20, 23). A characteristic difference between the two phos-
`pholipids is observed in the level of 18:1/22:6, which was
`significantly higher in the phosphatidylethanolamines than in
`the phosphatidylcholines in both cold- and warm-adapted
`animals. Moreover, cold-acclimated fish accumulated 2—3
`times more of this species, along with 18:1/20:4 and 18:1/
`18:1, than the warm-acclimated fish, and its level also varied
`slightly with an upshift of the temperature (from 14.1 1 1.8
`to 10.7 1 0.7 wt % in the case of 18:1/22z6). Elevated levels
`of 18:1/22:6 phosphatidylethanolamine in the livers of fresh-
`water and marine fish adapted to low temperature have
`already been reported (23-25). The rotational correlation
`time of 14-PGSL embedded in phospholipid vesicles pre-
`pared from brains of cold-acclimated carp was less than for
`
`Table 2. Molecular species composition of brain
`phosphatidylethanolamines in fish seasonally or
`evolutionarily adapted to contrasting temperatures
`% total
`
`At 5°C
`
`At 25°C
`
`Acyl
`groups
`22:6/22:6
`20:5/20:5
`20:4/20:4
`18:1/20:5
`16:1/16:1
`18:1/22:6
`16:0/22:6
`18:1/20:4
`16:0/20:4
`1820/2025
`l8:0/22:6
`1820/2024
`18:1/18:1
`l6:0/18:1
`16:0/16:0
`1820/2224
`18:0/18zl
`1620/18:0
`18:0/18:0
`Others
`
`C. carpio
`(n = 8)
`2.6 1 1.2
`0.2 1 0.1
`6.0 1 1.0
`0.5 1 0.2
`0.3 1 0.2
`14.1 1 1.8
`12.9 1 3.9
`7.2 1 2.5
`2.3 1 1.5
`1.6 1 0.5
`24.8 1 4.4
`9.8 1 1.0
`6.6 1 1.4
`4.0 1 1.7
`0.3 1 0.2
`0.4 1 0.2
`2.0 1 1.0
`1.6 1 1.3
`0.4 1 0.2
`8.9
`
`A. cernua
`(n = 5)
`0.5 1 0.3
`0.3 1 0.2
`2.0 1 0.9
`2.0 1 0.1
`0.8 1 0.4
`14.4 1 1.6
`26.1 1 2.5
`3.4 1 1.2
`1.6 1 1.4
`4.0 1 1.8
`32.4 1 2.5
`4.8 1 2.5
`2.1 1 1.4
`2.2 1 0.8
`0.2 1 0.1
`0.1 1 0.08
`0.3 1 0.1
`1.0 1 0.9
`0.3 1 0.2
`1.5
`
`C. carpio
`(n = 6)
`2.7 1 2.8
`0.4 1 0.3
`2.1 1 0.7
`0.2 1 0.1
`0.2 1 0.1
`5.1 1 2.4
`13.8 1 2.1
`2.1 1 1.0
`2.5 1 0.5
`0.9 1 0.3
`39.1 1 8.6
`8.5 1 2.0
`3.7 1 1.2
`5.5 1 0.4
`0.6 1 0.4
`0.4 1 0.2
`2.7 1 1.5
`4.0 1 3.3
`0.7 1 0.4
`4.8
`
`C. catla
`(n = 7)
`1.3 1 0.7
`0.7 1 0.5
`0.8 1 0.3
`0.5 1 0.2
`Trace
`2.5 1 1.5
`27.3 1 2.1
`2.3 1 1.5
`1.2 1 0.9
`0.7 1 0.2
`49.1 1 5.5
`4.3 1 2.1
`0.4 1 0.1
`3.6 1 0.3
`0.4 1 0.3
`Trace
`Trace
`3.8 1 2.5
`0.3 1 0.2
`0.8
`
`Cyrinus carpio was collected in summer (25°C) and winter (5°C) in
`Hungary, Acerina cernua in Finland (Vaasa, 5°C), and Calla calla in
`India (25°C).
`000003
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:22)
`
`T
`
`AT..
`
`L
`‘;~
`1‘
`~ .
`K}
`
`
`
`
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`H
`
`|
`13
`
`r
`18
`
`.1.
`23
`
`r
`23
`
`33
`
`3.40
`
`2.80
`
`w
`I:
`.
`E 2.20
`
`1.60
`
`1.00
`
`3
`
`Temp., °C
`
`FIG. 1. Temperature dependence of the rotational correlation
`time (11) of 14-PGSL embedded in phospholipid vesicles prepared
`from brains of cold (5°C) and warm (25°C) temperature-acclimated
`carps or shifted to the other temperature extreme (means 1 SD of
`five experiments). 0, Warm-acclimated; A, shift down; v, cold-
`acclimated; D, shift up.
`
`

`

`Biochemistry: Buda et al.
`
`Proc. Natl. Acad. Sci. USA 91 (1994)
`
`8237
`
`4.00
`
`3.40
`
`2.80
`
`2.20
`
`1.60
`
`7R,ns
`
`
`
`FIG. 2. Temperature dependence of the rotational correlation
`time (111) of 14—PGSL embedded in membranes of brain cells of carps
`adapted to summer or winter temperature or shifted to the other
`temperature extreme (means 1 SD of five experiments). 0, Wann-
`acclimated; A, shift down; A, cold—acclimated; 0, shift up.
`
`ture scannings were run in the heating cycle for the cold-
`adapted cells, and in the cooling cycle for the warm-adapted
`cells. This experimental approach, in sharp contrast to the
`isolated phospholipids (Fig. 1), demonstrated an ~80% com-
`pensation of the membrane structural order for the temper-
`ature (Fig. 2). There were no significant differences in the
`effective rotation correlation times of warm-acclimated and
`cold-acclimated temperature-upshifted cells and vice versa
`(Fig. 2).
`This experiment suggested that adjustment of the mem-
`brane physical state of the brain cells to the new temperature
`was a rapid and reversible process. To confirm this, brain
`cells prepared from warm-adapted fish were cooled under in
`vitro conditions to 10°C and the time course of the fluores-
`cence anisotropy of DPH-PA embedded in the plasma mem-
`brane was followed, after the removal of aliquots of the cells
`from the incubation medium. The anisotropy parameter
`started to decrease shortly after the cells were exposed to the
`reduced temperature, and it assumed a constant low value
`after 15—20 min (Fig. 3). When the same cell population was
`rewarmed, the anisotropy increased and reached the original
`value within a few minutes. Similar results were obtained
`when cells from warm- or cold-adapted fish brains were
`exposed to cold or warm temperature, respectively, in the
`cell of the spectrophotofluorimeter. In a typical experiment
`in which cells from cold-adapted fish were dropped into the
`spectrophotofluorimeter cell set to 25°C, the fluorescence
`anisotropy started to increase within a few minutes (Fig. 3
`0.32
`
`Inset). We interpret these results as a manifestation of an
`active adaptation of the physical properties of the intact brain
`cell membrane to the new temperature. Without this adap-
`tation, opposite results would have been obtained—Le, a
`decrease in the anisotropy parameter in temperature-
`upshifted cells, and vice versa, as demonstrated in model
`experiments using phospholipid vesicles (data not shown).
`
`DISCUSSION
`
`Fatty acid composition in mammalian brain cell is fairly
`stable: only long-term dietary effects can cause some changes
`in it (26—28), especially in the phosphatidylethanolamine
`fraction (27, 29). Long-chain polyunsaturated fatty acids
`present in high concentration in mammalian nerve cells
`originate in the liver and are transported to the brain by the
`circulatory system (30). The same is also undoubtedly true
`for fish, but the effect of the environmental temperature must
`also be taken into consideration. Ethanolamine phosphoglyc-
`erides gave the most sensitive response to changes of the
`environmental temperature. Despite the expected slowing
`down of metabolic processes with decreasing temperature, a
`turnover of molecular species composition of the phosphati-
`dylethanolamines took place in the brains of thermally ac-
`climated carp. The changes were characterized by accumu-
`lation of 1-monounsaturated/2-polyunsaturated and dioleoyl
`species with a fall in the environmental temperature (Table
`2). It seems conceivable that these changes are a direct effect
`of temperature on the lipid metabolism of carp brain and do
`not stem from the diet, since these fish stop feeding at or
`below 10°C. Previous studies demonstrated increases in the
`formation (31) and level (32) of long-chain polyunsaturated
`fatty acids in carp liver acclimated or exposed to cold, and it
`is tempting to speculate that a proportion of these fatty acids
`might have been transported to the brain during the accli-
`mation to reduced temperatures. Fish brain cells have been
`shown to take up and selectively incorporate unsaturated
`fatty acids from the incubation medium in vitro (33, 34). The
`level of 18: 1/22:6 phosphatidylethanolamine in the brain cells
`of carp acclimated to reduced temperatures resembles that
`which has been found in the brains of boreal and subtropical
`fish species (Table 2) as well as in brains of cod and rainbow
`trout adapted to low (5—7°C) temperatures (20, 23). It requires
`further investigations to decide whether the accumulation of
`this species in response to cooling is a result of a selective
`deacylation/reacylation reaction, an intensive desaturation
`of the existing 18:0/22:6 species, or its transfer from the liver.
`In another connection, it has been shown that carp liver cells
`preferentially ester-ify 18:1 at the sn-1 position of phosphati-
`dylethanolamine (32) under in vitro conditions in the cold
`and, moreover, that high levels of this species characterize
`the phosphatidylethanolamines in the livers of marine and
`freshwater fish evolutionarily adapted to low temperatures
`(24).
`Parallel to changes in the acyl composition of the brain
`phosphatidylethanolamines, there was also a high level (70—
`80%) of compensation of membrane fluidity for temperature
`changes, as measured by spin label and fluorescence polar-
`ization techniques. Although the individual membranes were
`not separated in this study, based on the data obtained for the
`brains of rat (28) and Channa punctatus (8) and for the nerve
`of carp (9), we propose that the fatty acid composition of carp
`brain total phospholipids is close to that of the plasma
`membranes. Thus,
`it can be speculated that both labels
`(14-PGSL and DPH-PA) indicate the ordering state of these
`structures. However, it still remains to be elucidated whether
`the observed changes in acyl-group composition of phos-
`phatidylethanolamines are directly related to the observed
`high degree of compensation of membrane physical state for
`temperature change. Some physical properties of the pre-
`
`T
`
`30
`
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`
`0
`
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`30.27
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`0
`
`1
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`10
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`20
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`40
`Time, min
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`80
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`in vitro on fluorescence
`FIG. 3. Effect of temperature shift
`anisotropy of DPH-PA embedded in membranes of brain cells
`prepared from warm-adapted fish (means 1- SD of five experiments).
`(Inset) Change in fluorescence anisotropy of DPI-I—PA in brain cell
`membranes of a cold-adapted carp in response to exposure to the
`other temperature extreme (25°C).
`000004
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:23)
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`

`

`8238
`
`Biochemistry: Buda et al.
`
`Proc. Natl. Acad. Sci. USA 91 (I994)
`
`dominant phosphatidylcholine species (1620/ 18:1, 1620/22:6,
`and 18:0/22:6) are rather close (35, 36) and the same might be
`true also for the phosphatidylethanolamines. Indeed, the
`compensation was only about 10% when total phospholipids
`were assayed (Fig. 1).
`A principal difference between the extracted total phos-
`pholipids and the intact membranes is the presence of the
`proteins in the latter. One of the functional roles of l-mono-
`unsaturated/Z-polyunsaturated phosphatidylethanolamines
`might be the prevention of the contraction of the bilayer due
`to reduced thermal motion and thereby the maintenance of a
`higher degree of disorder in cold. We speculate that certain
`nonlipid membrane components, such as membrane pro-
`teins, are responsible for the observed high degree of com-
`pensation of membrane ordering state for the temperature.
`Due to their specific molecular architecture and their effects
`on packing properties of the bilayer these phosphatidyleth-
`anolamines might contribute to these proteins assuming the
`appropriate configuration in cold. Phosphatidylcholines such
`as 16:0/18:1 or 16:0/22:6 may not share this property of
`phosphatidylethanolamines and serve merely as a matrix of
`the membranes.
`
`We thank Dr. A. B. Das for collecting brains of Carla catla, Dr.
`T. Wiik for collecting brains ofAcerina cernua, and Dr. D. R. Tocher
`for help in the separation of brain cells. Thanks go also to the Tisza
`Fish Farm, Szeged, for supplying fish for the experiments and to D.
`Marsh, Gottingen, Germany, for 14-PGSL. This work has been
`supported by the Hungarian National Scientific Research Founda-
`tion under Contracts T5163 and 903/90.
`
`1. Sinensky, H. (1974) Proc. Natl. Acad. Sci. USA 71, 522—525.
`2. Cossins, A. R. & Prosser, C. L. (1982)Biochim. Biophys. Acta
`687, 303—309.
`3. Lee, J. A. C. & Cossins, A. R. (1990) Biochim. Biophys. Acta
`1026, 195—203.
`4. Wodtke, E. & Cossins, A. R. (1991) Biochim. Biophys. Acta
`1064, 343-350.
`5. Dey,1. & Farkas, T. (1992) Fish Physiol. Biochem. 10, 347-355.
`6. Dey, 1., Szegletes, T., Buda, Cs., Nemcsok, J. & Farkas, T.
`(1993) Lipids 28, 743—746.
`7. Cossins, A. R. & Prosser, C. L. (1978) Proc. Natl. Acad. Sci.
`USA 75, 2040—2043.
`8. Roy, R., Ghosh, D. & Das, A. B. (1992) J. Therm. Biol. 17,
`209—215.
`9. Harper, A. A., Watt, P. W. & Hancock, N. A. (1990) J. Exp.
`Biol. 154, 305-320.
`
`10.
`
`11.
`
`12.
`
`13.
`
`14.
`
`15.
`
`16.
`
`17.
`18.
`
`19.
`
`20.
`21.
`
`22.
`
`23.
`24.
`
`25.
`26.
`
`27.
`
`28.
`
`29.
`
`30.
`
`31.
`32.
`
`33.
`
`34.
`
`35.
`
`36.
`
`Friedlander, K. J ., Easton, 0. M. & Nas, M. (1980) J. Comp.
`Physiol. 112, 19—45.
`Tocher, D. R. & Sargent, J. R. (1992) Comp. Biochem. Physiol.
`101, 353—359.
`Kitagawa, S., Matsubayashi, M., Kotani, K., Usui, K. &
`Kametami, F. (1991) J. Membr. Biol. 119, 221-227.
`Kuhry, J. G., Duportail, G., Bronner, C. H. & Laustriat, G.
`(1985) Biochim. Biophys. Acta 845, 60—67.
`Kivelson, D. (1972) in Electron Spin Relaxation in Liquids, eds.
`Muus, L. T. & Atkins, P. W. (Plenum, New York), pp. 213—
`277.
`Jost, P. C., Libertini, L. 1., Herbert, V. C. & Griffith, O. H.
`(1971) J. Mol. Biol. 59, 77-93.
`Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) J. Biol.
`Chem. 226, 497—509.
`Fine, J. B. & Sprecher, H. (1982) J. Lipid Res. 23, 660-663.
`Rouser, G., Fleischer, S. & Yamamoto, H. (1970) Lipids 5,
`494—496.
`Takamura, H., Narita, M., Urade, R. & Kito, H. (1986) Lipids
`21, 356—361.
`Bell, M. V. & Dick, J. R. (1991) Lipids 26, 565—573.
`Chang, M. C. & Roots, B. I.
`(1985) Neurochem. Res. 10,
`355-375.
`Chang, M. C. & Roots, B. I.
`1231—1246.
`Bell, M. V. & Tocher, D. R. (1989) Biochem. J. 264, 909—915.
`Dey, 1., Buda, C., Wiik, T., Halver, J. E. & Farkas, T. (1993)
`Proc. Natl. Acad. Sci. USA 90, 7498—7502.
`Hazel, J. R. &. Zebra, E. (1986) J. Comp. Physiol. 156, 665—674.
`Bazzanti, V., Marenesi, M., Solaini, G. & Turchetto, E. (1990)
`J. Nutr. Biochem. 1, 305—308.
`Alsted, A. L. & Hoy, C. E. (1992) Biochim. Biophys. Acta
`1125, 237-244.
`Takin, Q. S., Blum, M. & Carafoli, E. (1981) Eur. J. Biochem.
`121, 5—13.
`Hargreaves, K. M. & Clandinin, M. T. (1988) Biochim. Bio-
`phys. Acta 962, 98—104.
`Scott, B. L. & Bazan, N. G. (1989) Proc. Natl. Acad. Sci. USA
`86, 2903—2907.
`Farkas, T. & Csengeri, l. (1976) Lipids 11, 401—407.
`Farkas, T. & Roy, R. (1989) Comp. Biochem. Physiol. 93,
`217—222.
`Tocher, D. R., Bell, G. J. & Sargent, J. R. (1992) J. Neuro-
`chem. 57, 2078—2085.
`Tocher, D. R., Mourente, G. & Sargent, J. R. (1992) Lipids 27,
`494—499.
`Evans, R. W., Williams, M. A. & Tinoco, J. (1987) Biochem.
`J. 156, 665—674.
`Coolebar, K. P., Beroe, C. L. & Keough, K. M. W. (1983)
`Biochemistry 13, 2605—2612.
`
`(1985) Neurochem. Res. 10,
`
`000005
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:24)
`
`