Fowl Blood Plasma Research Articles

Thermodynamics of Enzymatic Transamination of Corticoid-Induced Aminotransferases of F-GOT System in Fowl Blood Plasma : I. Relationship between Reaction Velocity and Substrate Concentration in F-GOT Reaction

Kinetical analysis on the self-control mechanism of corticoid-induced F-GOTgave the following results.Corticoid-induced F-GOT might be one of K class allosteric enzymes and a typical ex-ample of oligomeric enzyme. This enzyme catalyzes the reaction of the forward directionin the presence of L-aspartate and a-ketoglutarate for the following three reasons:(a) F-GOT, particularly homotropic one showing positive cooperativity, shows asigmoid curve in relation to the initial steady-state velocity (Vi) to substrate concentra-tion ([S]), rather than a rectangular hyperbola to be expected from the Michaelis-Mentenrelationship. Apparently, the allosteric transition is also recognized in a cturve of doublereciprocal (Lineweaver-Burk) plot of Vi vs. [S], (b) F-GOT responds to a positive modula-tor with a change in apparent Km(:[84 ;), but without a change in maximum velocity(V=..). This fact proves it to be K enzyme, and (c) Desensitized allosteric F-GOT whichhas lost its previous sensitivity to modulators shows normal Michaelis-Menten kinetics.In this case, a change in V.... in response to the substrate without a change in Michaelisconstant (Km:[5]..), is recognized. This fact proves the catalytic site to be the V form.Such sites are similar to one another. The following consideraiton is put forth: Funda-mentally, F-GOT at a normal steady-state level may exist in a desensitized form ofallosteric F-GOT which has been released by corticoid treatment during the period ofsecondary adaptable responses.Although an excess in substrate resulted in an inhibition of the enzyme activity, thesigmoidal inhibitory curve showed that the kinetic characteristic was not necessarily thesame among enzymes, including an atypical one. It was considered that each enzymemight have been affected differently with its substrate by virtue of its own characteristics.

Influence of Corticoid on Induction and Activation of Transaminases of F-GOT System in Fowl Blood Plasma

合成 corticoid, betamethasone disodium phosphate (β-MDP)の連続投与により惹惹された蛋白質異化作用の発現時にほぼ一致する, β-MDP投与後3日(Ct3)および10日(Ct10)時の二度にわたって, 基本活性(St.)に対して約2倍相当の血漿中F-GOT系アミノ基転移酵素(T-ase)の活性上昇が認められた. これら二活性区での反応速度定数(k)から算定された当該T-aseの半減期(t1/2)は, いずれも同日対照の同系T-ase(N)のt1/2よりも短い. これらのことから, corticoid作用下の血漿中F-GOT系酵素は, 定常態下の同系T-aseと, 代謝回転速度上, 異種の酵素蛋白質から成り, 投与corticoid に由来する生体側の二次的順応現象によって, 組織内に誘導を受け, 血漿中に逸脱した, いわゆるcorticoid誘導酵素と考えられた. そしてこの点は, 副腎摘除と非摘除対照の各鶏群に対して, 合成ACTH, 酢酸tetracosactide (T-cosac.), 合成corticoid, β-MDPのいずれかを, 一次または二次のインデューサーとして投与した場合の系上分泌corticoidsまたは投与corticoid, それぞれに由来する活性発現の傾向からも支持された. その他, corticoid作用下における被誘導性に関して, 比較・対照酵素としたR-GOT, F-GPTおよびR-GPTら3系のT-ase群も, F-GOT系酵素と同様, corticoid誘導酵素であることが認められた.

Thermodynamics of enzymatic transamination of F-GOT in normal fowl blood plasma. III. Relationship between temperature and velocity in F-GOT reaction (author's transl)

4-60℃間各区の耐熱処理血漿試料F-GOTの活性曲線は, 50℃区処理の試料活性をピークとする山型曲線として示される. また, これらの試料に対する定量範囲のPyridoxal 5'-phosphate (PALP) 添加の効果は, PALP濃度に比例的な活性の賦活, もしくは復活傾向として示され, そのうち, 最高活性の発現は, 20℃区の処理試料において認められた. このように, 補体的要素としてPALPをとるF-GOTは, 形式上, 一次の反応に属する反応次数を持ち, 熱失活を無視することができる温度範囲内の4-40℃区において, 反応速度定数(k)の温度変化から求められたこれら酵素の見掛けの賦活エネルギー(EA)は, 平均9,024.18cal/Mである. また, Arrhenius式に適合するこれらF-GOTのEA算定の基準となった, 反応初速度(Vi)から測定された最大反応速度(Vmax)と絶対温度(T)間の関係を示す, (log Vmax-1/T)プロット上の, 20℃区を境とする折線グラフは, 当該温度の両側温度区では, 温度上昇とともに, 本系反応速度が律速的に変化・加速され, その反応最適温度は40℃区にあることを示している. この場合, 0℃区の温度係数(Qt)を基準に算定した, 10-50℃区間にあって, 10℃差のQtを示すQ10値範囲は1-4である. なお, これらの反応に, 系上, 対応するR-GOT系酵素由来のR-GOT反応との間には, 直接的な平衡関係は存在しない.

Thermodynamics of enzymatic transamination of F-GOT in normal fowl blood plasma. I. Relationship between reaction velocity and substrate concentration in F-GOT reaction (author's transl)

鶏血漿中 F-GOT 反応の Lineweaver-Burk (L-B) プロットから求められた, 系上の基質(Sub.)成分 α-Ketoglutarate (KG) および L-Aspartate (Asp) の見掛けの Michaelis 定数(Km)は, それぞれ0.72±0.03 mM および7.19±0.28 mM で, 基質 α-KG による最大反応速度 (Vmax) は (2.00±0.08)×10-2 mM であった. これらの Km-Vmax 関係によって表わされた, 当該基質成分の F-GOT に対する親和性の程度は, L-Asp より α-KG においてはるかに高く, その過剰量を含む "Excess Sub." によって, 当該反応は, 一次から零次への反応移行期においては双曲線型の活性傾向を示すのに対して, シグモイド型曲線で表わされる活性阻害の傾向を見せた. その場合の抑制物質定数(K'i)は, (1.78±0.07)×10-5 であった. また, これらの関係は, 本 F-GOTが, α-KG および L-AsP を特異的な基質としてとる Michaelis-Menten (M-M) 型酵素であることを示しており, このことは, 基質濃度と零時の反応速度 (Vi) との関係から作図して得た M-M および L-B プロット上の反応型式からも立証された.

Activities of aspartate- and alanine-aminotransferases isolated from plasma globulins of the chicken and their physicochemical properties. II

In recent years, some trials for separating the plasma protein components have beenput in practice by the author33=3) to explain the behavior of transaminases in fowl bloodplasma and their physicochemical properties.As a result, it was pointed out that several components obtained by subfractionationof albumin37) and beta-globulins36) were regarded satisfactorily as carriers of reverse (R)-aspartate aminotransferase (GOT), R-alanine aminotransferase (GPT), and reversibleGPT. Several physicochemical determinations laave already been performed to clarifythe properties of these components36?37).On the contrary, it is known that the fairly effective purification of ant active material?on1y for forward (F)-GOT substrate was accomplished as fraction IV-73?35). Moreover, aprotein component with an active fragment of F-GOT has frequently been found in acertain globulin group3?35). Notwithstanding, that fraction has not yet been subjectedto a highly effective purification, because the problem of how to separate and purify itremains unsettled. In order to obtain a basic solution for this problem, it is necessary to?devise a method for systematic separation of blood plasma globulins.The present report deals with a method developed and introduced into practical, application by the author.The results of experiments with this method are summarized as follows.I. On the basis of the relations among several quantities listed in some tables, acomponent distinguishable by disc electrophoresis that had migrated to position 5-b wasobtained by subfractionation from the original fraction, IV-4, as fraction IV-4(d). It wasfound to be F-GOT. Moreover, one of the R-GPT samples existed among the conjugatedprotein components with the relative position, 6-a to b. This conjugated component wasobtained from the original fraction, IV-l, as fraction IV-1(f).2. These components were found to be globulin with the same relative position asgamma.-globulins by the use of cellulose acetate electrophoresis"9?33). This result was thesame as obtained from components 4-b and c36).These data lend support to the conclusion that a few gammax-globulins are constantlypresent in fowl blood plasma and that their fractionation can be carried out. It is im-portant to test tavailable are carried only by the GOT group. It became evident that components 5-band 4-c were derixzed from the reaction of transamination with F-GOT and R-GOT, respectively.If relations among several isozymes of R-GPT or CRT-ase36?37) are examined, it willbe noted that the carriers of these isozymes are evenly distributed among several compo-nents obtained from albumin and globulins. This does not necessarily mean, however, all the isozymes show the same distribution.As mentioned above, on certain reverse transaminases, especially those of the R-GPTgroup, these isozymes seem to have an important action. This considration is ttseful, especially when the physiological function of the GPT group is studied.For this reason, it is interesting to obtain more detailed information on this aspectfrom future investigation.

Aspartate and alanine aminotransferase activities and physiochemical properties of albumin components subfractionated from fowl plasma

In view of the results of experirnents34 36) conducted to fractionate the transaminasesin fowl blood plasma, some of these enzymes called forward (F)-aspartate aminotransferase(GOT), reverse (R)-GOT, F-alanine aminotransferase (GPT), and R-GPT were presumedto be contained in a limited number of albumin components35?36). No effective verifi-cation of this conjecture, however, could be made for lack of detailed information on anysystematic method for the subfractionation of fowl blood plasma albumin. Naturally, the physiochemical properties of those enzymes have been left unknown due to insuf-ftcient data.In the present investigation, a practical solution was reached for these problems bymeans of the authors method introduced. This report gives an outline of the methodused for subfractionation of plasma albumin. The results obtained are summarized asfollows. l. Disc electrophoretic analysis revealed tlaat plasma albumin consisted of six differ-ent components classified into four large divisions. The components were called I-a, [-b., I-b., I-(b, c), I-c., and I-c.. Their relative positions are shown in detail in theremarks of Table 3.The major part of the albumin was occupied by two components, I-a and I-(b, c).The ratio of both components to the total albumin was approximately 93%. The yieldper total albumin was maximum.2. The albumins subfractionated were characterized by the following physicochemi-cal properties.(a) Their isoe[ectric points were in a range of pH from 4.4 to 6.0. The isoelectricpoints of components I-a and I-(b, c) were found to be 4.7 and 4.8, respectively.(b) Their sedimentation constants ranged from a minimum value of 4.1 to amaximum value of 4, 9. The sedimentation constants of the two comopnents mentionedabove were found to be 4.4 and 4.6, respectively.(c) The different absorbancy of each component has been interpreted as indicatingthe presence of six different types of conjugated protein. The greater part of each corn-ponent, however, consisted of protein.(d) The albumins were classified roughly into a group of glucoprotein. The con-clusion was indicated by the hexosamine content included in each of the components.3. When the criteria described in Tables 3, 4-A, and 4-B were applied, a more de-tailed classification was made for the transaminases which were usually classified into fourlarge divisions, The classification finally reached is illustrated as follows.On the basis of this classification, the following conclusions could be drawn, (a) The albumin counponents were positiwe for the transamination reaction, as wasexpected. None of them reacted as a self-sttpporting transaminase, as pointecl out fromtlae observation on F-GOT, R-GOT, reversible GOT, and F-GPT mentioned above.(b) The albumin components subfractionated supported all the actixze fragments ofR-GPT.(c) The albumins contained the following transamisases: three isozymes of tlaegrou

Purification of immunoglobulins, IgG, IgA and IgM, from the pig

This paper deals with a procedure of purification of three classes of immunoglobulinfrom porcine serum and sows colostrum. The procedure was summarized as follows(Charts 1 to 3).I. For the purification of immunoglobulins IgG and IgM, pooled porcine serumwas treated with (NH4)2804 to a concentration of ? saturation. Then precipitation wascarried out with a DEAF, -cellulose column (anion-exchange chromatography). The step-wise elution entailed tlte use of seven buffers: (J) 0.01 M NaH:P0+ adjusted to pH 7.6with 0.01 M NaOII, (2) 0.02 M NaH.P0. adjusted to pH 6.3 with 0.02 M Na0H, (3) 0.05 MN1H204, (4) 0.1 M N3lH2PO4, (5) 0.15 M NlH2P04, (6) 0.3 M NlH2P04, and (7) 0.4 MNaH, .P0+. IgG was seen in an elution obtained with buffers (l) and (2). This IgG waspurified by chromatography on CM-cellulose (cation-exchange chromatography) and getfiltration on Sephadex G-200. IgM could be obtained with buffers (4) to (6). The proteincontaining IgM was also fractionated by repetition at get filtration three times onSpehadex G-200 using 0.2 M Tris-HCI and 0.15 M Nail buffer (pH 8.1) for purification, From 100 ml of pooled porcine serum, about 675 rug of IgG was collected, and IgMwas estimated to be 117 mg.2. Colostrum was collected from sows for the purification of IgA. Fat was removedfrom the colostrum, which was then subjected to decaseination and delipoproteinization.The whey was fractionated with Sephadex G-200 against 0.2 M Tris-HCI saline buffer(pH 8.1) which had been used for elution. The resultant fraction was then applied to acolumn of DEAF, -cellulose by stepwise elution. The protein that was assumed to be IgAwas eluted with 0.125 M and 0.15 M Tris-HCI buffer (pH 7.4). This crude IgA was fractionated by repetition of get filtration three times on Sephadex G-200 with Tris-HCIsaline buffer (pH 8.1).From TOO ml of colostrum, about 192 mg of IgA was finally collected.3. Antisera were prepared by injection of rabbits with the three classes of immuno-globulin in Freunds complete adjuvant. Alpha- or beta-globulin was seen in ant In recent years, some trials for separating the plasma protein components have beenput in practice by the author33=3) to explain the behavior of transaminases in fowl bloodplasma and their physicochemical properties. As a result, it was pointed out that several components obtained by subfractionationof albumin37) and beta-globulins36) were regarded satisfactorily as carriers of reverse (R)-aspartate aminotransferase (GOT), R-alanine aminotransferase (GPT), and reversibleGPT. Several physicochemical determinations laave already been performed to clarifythe properties of these components36?37).On the contrary, it is known that the fairly effective purification of ant active material?on1y for forward (F)-GOT substrate was accomplished as fraction IV-73?35). Moreover, aprotein component with an active fragment of F-GOT has frequently been found in acertain globulin group3?35). Notwithstanding, that fraction has not yet been subjectedto a highly effective purification, because the problem of how to separate and purify itremains unsettled. In order to obtain a basic solution for this problem, it is necessary to?devise a method for systematic separation of blood plasma globulins.The present report deals with a method developed and introduced into practical, application by the author.The results of experiments with this method are summarized as follows.I. On the basis of the relations among several quantities listed in some tables, acomponent distinguishable by disc electrophoresis that had migrated to position 5-b wasobtained by subfractionation from the original fraction, IV-4, as fraction IV-4(d). It wasfound to be F-GOT. Moreover, one of the R-GPT samples existed among the conjugatedprotein components with the relative position, 6-a to b. This conjugated component wasobtained from the original fraction, IV-l, as fraction IV-1(f).2. [the rest omitted]

Method for separating aspartate and alanine-aminotransferases in fowl plasma

As was reported previously31=34?36), aspartate aminotransferase (GOT) and alanineaminotransferase (GPT) in fowl blood plasma can be classified roughly into four typesunder the general terms of forward(F)-GOT, reverse (R)-GOT, F-GPT, and R-GPT.The author36) succeeded considerably in overcoming the difficulties of fractionatingthese transaminases by using an ingenious method introduced by COHNIO) and 0NCLEY25).Some of the transaminases fractionated were rich in lipids and contained such proteincomponents of particular kind as beta.-metal combining protein28, 35) and heme prote-j.35)In order fully to purify these transaminases, it was necessary to remove lipids andparticular components. For the purpose of meeting this necessity, the author devised amethod. The following diagram is a schematic illustration of this method for separationof the four fractions involved in the transamination reaction.A.B.C.Removal of lipids from fowl blood plasma with n-butanol.To I volume of plasma was added I volume of n-butanol. A mixture wassturred at 0C [01 30 II11I1UtCS and centrufuged It 700 X g at OC for 30 minutes.The supernatant and the insoluble middle layer were discarded. The solublelowermost layer at pH 8.4 was saved as source sample I.Fractional precipitation with ammonium sulfate.Source sample I was used for a partial fractionation of the transaminases bystepwise additions of ammonium sulfate. Some of the filtrates were pooledas source sample 2.In addition, salting out was performed by Osbornes method.Sephadex get fractionation of source sample 2.Sourse sample 2 was get filtrated through Sephadex G-100 and with phosphatebuffer at pH 7.4 as eluant.Some of the filtrates were pooled as source sample 3. Samples were usually maintained at 0C throughout an experiment for purification.The results obtained are summarized as follows.l. It was impossible to recover fractiorns I, [14111, and IV-I from source sample 3by the 6th method of COIN. These fractions could be removed by means of a methoddevised by the author (n-butanol extraction, salting out, and Sephadex get fractionation).Source sample 3 also contained fractions IV-4, V, and V-l.2. Fraction IV-5 was separated by the authors method. It was useful for studieson the possible role of lipoprotein in the transaminatioru reaction of particular kind.3. There was 110 good agreement among the transaminases in the mode of fractionalprecipitation. R-GOT and R-GPT, however, agreed verv well with each other in thismode and partially with F-GPT. It is of interest to speculate from these results thatF-GPT, R-GOT, and R-GPT may have a common ancestry. To lend support to thisspeculation, discussion will be made in a future report on the possible effect of fractionIV-8 on the reversible transamination reaction of GPT.From the results described above, it seems possible to reocver this reversible GPT.It is clear that there is no correlation betweern F-GOT and any other transaminase.

Activities and physicochemical properties of aspartate and alanine aminotransferases isolated from fowl blood plasma globulins

As was reported prexziously30?31), the results of the dissociation of transaminases, wlticlt were rouglaly classified into forward (F)-aspartate aminotransferase (GOT), reverse(R)-GOT, F-alanine aminotransferae (GPT), and [-GPT"?", haxre led to the suggestionthat some of the globulin components in fowl blood plasma may play a significant rolein tlte vehicle function of all the transaminases mentioned above, except F-GPT. It isdifficult at present to verify this suggestion. It is of interest, however, to investigate thesuggested situation in more detail.Tlae author tried to fractionate components 4-b and c belonging to tlae beta-globulins") distinguishable by disc electrophoresis. Judging from the experimentalevidence30?") collected in recent years, both components seemed to be carriers of thereverse transaminases.Cltarts I-(A) and (B) slaow tI?e authors method for separating these components.In addition, the transaminase activity values were determined by the authorsmethod25?8). The results obtained are summarized as follows.l. Components 4-b and c coulcl be separated in fractions of IV-7(e) and IV-5(e), respectively.2. The relationslaip between the components separated and their transaminationreaciton led to the conclusion that both substrates of R-GOT and R-GPT were catalyzedby component 4-b. The activity value of R-GPT was much larger than that of R-GOT.This kind of transaminase was introduced already under the name of CRT-ase32).On the contrary, component 4-c catalyzed only R-GOT substrate. Neither corn-pontent had any actixze fragment closely related to F-GOT, F-GPT, R-GPT, reversibleGOT, or GPT.3. From the physicoclaemical properties of components 4-b and c, the conclusionsreaclaed can be expressed as follows.The involving fractions for botlt components showed much the same mode offractional precipitation as fractions IV-4, IV-5, and IV-7 indicated by C0HN5) andONCLEY15). It was pointed out by them5?15) that tlte following components of beta-globulins were involved in the fractions just described. Someisoelectric poiu?t were 5.4 auad 4.9 of pH, respectively.Besides, from the hexosamine content10?12), it is probable that both componentsnaay be of mucoprotein19?2=2). The asterisk placed after the term mucoprotein indi-cates that the components are beta-mucog1obu1ins21).4. Both components represented the same relative position as gamma.-globulinson cellulose acetate electrophoretic analysis"9).For a general understanding of immunizing process in living materia1s20), thisfinding that some of the gamma.-globulins in fowl blood plasma consist of componentswith a sedimentation constant within a range of 5.0 to 5.4 is of extreme importance.

Methods for the determination of aspartate and alanine aminotransferase activity in fowl blood plasma

A. 鶏血漿中 Aspartate- および alanine-amino-transferase (GOT および GPT とそれぞれ略記する) の Forward (F), Reverse (R) 系 T-ase の活性は, REITMAN & FRANKEL (R・F) 変法によって, 測定することができる. B. 鶏血漿中 GOT, GPT の F, R 系 Transaminase (T-ase) 活性を, 産生α-ケト酸の Hydra-zone 化合物量で比較してみると, F-GOT>R-GOT, F-GPT R-GPT≧R-GOT>F-GPT である場合が多い. また, 本報告で用いた3カ月令ビナ, および12カ月令成鶏の, 各血漿中, 4種の T-ase 活性を, 同じく, その蛋白質 N量を単位として比較すると, いずれの T-ase 活性も, ヒナ>成鶏の関係を示す. さらに, 性別上から活性関係を比べてみると, ヒナの場合には, GOT, GPT の F系 T-ase は, 雄≦雌であるが, R系 T-ase は明らかに雄>雌である. これに対して成鶏の場合には, F, R系 T-ase はともに雄>雌である. C. 本報告で用いた3カ月令ビナおよび12カ月令成鶏の F-GOT/F-GPT 比は, ヒナの雄7.34±0.82, 雌7.46±1.37, 成鶏の雄 15.97±4.86, 雌19.04±5.64である. これに対応する R-GOT/R-GPT 比は, ヒナの雄0.63±0.10, 雌0.39±0.12, 成鶏の雄2.06±0.18, 雌0.81±0.01である.

The effect of excess vitamin A on cultures of embryonic chicken skin explanted at different stages of differentiation.

In earlier experiments, Fell & Mellanby (1953) showed that the simple, two-layered epidermis of the 7-day embryonic chick underwent mucous metaplasia when grown in medium containing excess vitamin A. The present investigation was undertaken to see whether epidermis at more advanced stages of development would undergo a similar transformation in vitro under the influence of vitamin A. Skin from the shank and foot of 13-, 14- and 18-day chick embryos was grown on rayon acetate cloth by Shaffer’s modification of the watch-glass method, in medium (cock plasma and embryo extract) to which natural or synthetic vitamin A alcohol had been added. For purposes of comparison, one experiment was made with skin from the trunk and limbs of 7-day embryos. A dose of 1500 i. u. vitamin A /100 ml. of culture medium completely inhibited keratinization in all the + A explants, whatever the age of the embryo from which they were obtained. This concentration induced mucous metaplasia in all the explants from 7- and 13-day chicks, and in a minority of those from 18-day embryos. In the 13- and 18-day explants, the outer strata of epidermal cells degenerated and were sloughed, and the secretory epithelium was formed from the deepest and least differentiated layers. The dermis also was affected by the vitamin. When the explants were transferred from + A to normal medium, mucin secretion at first increased, often becoming astonishingly copious; later the mucous tissue was shed and the deeper cells regenerated a squamous, keratinizing epidermis. In all the controls grown on normal medium, the epidermis retained its squamous structure and formed increasing amounts of keratin, except at the margin of the 7- and 13-day cultures; here the newly formed epithelium, which had spread beyond or below the dermis, often failed to cornify and in one 7-day control, which elsewhere was heavily keratinized, it even developed some secretory cells. This peripheral effect is thought to be due to the close and prolonged contact of the outwandering epithelium with the fairly high level of vitamin A normally present in fowl blood plasma. The concentrations of vitamin A used in the present experiments were much less than those that can be produced in the blood of fowls fed on a high vitamin A diet. The vitamin A in the culture medium, however, may be much more readily available to the epidermis than the same concentrations of vitamin in the blood stream of a hypervitaminotic bird. It is also probable that the vitamin is in a more active state in the culture medium than it is in vivo (cf. Fell & Mellanby 1952).