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Biochemical Studies on the Shell Gland of Japanese Quail, Coturnix Coturnix Japonica
Biochemical Studies on the Shell Gland of Japanese Quail, Coturnix Coturnix Japonica 1. EFFECT OF A DEVELOPING EGG LOCATION ON ACTIVITY OF GLYCOLYTIC AND OTHER ENZYMES IN THE SHELL GLAND1 MASAHIRO
YAMADA
Biotron Institute, Kyushu University, Fukuoka City, Japan (Received for publication October 24, 1972) A BSTRA CT Effect of a developing egg location on activity of glucokinase, phosphof ructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, L-a-glycerol-3-phosphate dehydrogenases, lactate dehydrogenase, succinate dehydrogenase, alkaline phosphatase, adenosine triphosphatase and L-a-glycerol-3-phosphatase in the shell gland was examined, and some properties of adenosine triphosphatase in the shell gland mucosa were also investigated. Significant difference of activity of most enzymes examined, due to a developing egg location in the shell gland, was observed in the shell gland and its mucosa. The existence of Mg2+-dependent and Ca 2+ -dependent adenosine triphosphatase in the mucosa was observed. No significant enhanced activity of the enzyme by Na + , K + , or N a + plus K + was observed. The apparent Km value for ATP of the Mg 2+ -dependent adenosine triphosphatase was 0.29mM and that of Ca 2+ dependent adenosine triphosphatase was 0.15 mM. The effect of Ca 2+ on Mg 2+ -dependent adenosine triphosphatase was non competitive, while that of Mg 2+ on Ca 2+ -dependent adenosine triphosphatase was competitive. The activity of L-a-glycerol-3-phosphatase was shown to change during egg formation process. The possible participation of this enzyme in calcification process in the shell gland is discussed. POULTRY SCIENCE 52: 1375-1382, 1973
T
H E avian shell gland, which consists of a mucosal layer, smooth muscle, connective tissue and a sheet of serosal cells, retains a developing egg for 15 t o 20 hours, during which such physiological works as a marked calcium translocation across the mucosa, calcification of the egg shell and pigmentation, especially in Japanese quail are performed (Bernstein et al., 1968; Ehrenspeck et al., 1971; Sturkie, 1965; Woodward and Mather, 1964; Y a m a d a , 1972b). T h e supply of surplus energy to perform the physiological works mentioned above when the shell gland contains a developing egg, i.e., " a c tive" phase, is indispensable, compared with the energy status when there is no egg in the shell gland, i.e., "quiescent" phase. I n studies to elucidate the calcifi-
cation mechanism in the shell gland, it was reported t h a t calcium movement across the shell gland in vitro is in p a r t dependent on metabolic energy derived from oxidative metabolism and requires the generation of phosphate bond energy (Ehrenspeck et al., 1967), and also t h a t a major route of calcium movement across the shell gland involves an active transport mechanism (Ehrenspeck et al., 1971). I n order to understand the oxidative metabolism and active transport mechanism, as the first step, the change of activity of glycolytic and other enzymes corresponding to t h e location of a developing egg in the shell gland a n d some properties of mucosal ATPase were examined and reported in this paper. MATERIALS AND METHODS
x
The following abbreviations have been used: EDTA; ethylenediaminetetraacetate; Km, Michaelis constant; ATPase, adenosine triphosphatase; Pi, inorganic phosphate; PEP, phosphoenolpyruvate; FDP, fructose-l,6-diphosphate.
Animals. Female quail in their first year of lay were kept at 20 + 1°C. under the following lighting program: light period-(0600-1800), dark period-(1800-
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M. YAMADA
0600). All animals were maintained individually in wire cages on a commercial ration and water ad libitum. Well-synchronized animals for egg laying time, which was recorded every day, were employed for experiments. In order to minimize the individual variation, they were decapitated from 08:50 to 09:00. Chemicals. DL-a-glycerol-3-phosphate, succinate, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, phosphoenol-pyruvate, ATP, ADP, cytochrome c(Type III), NAD, NADH and NADP were purchased from Sigma, Saint Louis, U.S.A. All other chemicals were of special reagent grade. Glucose-6phosphate dehydrogenase, aldolase, triose phosphate isomerase, glycerol-3-phosphate dehydrogenase and lactate dehydrogenase were purchased from Boehringer, Mannheim, Germany. Enzyme assays. Enzyme activities were assayed at 30° C. as follows: glucokinase (EC 2.7.1.2) by the method of Walker and Parry (1966), phosphofructokinase (EC 2.7.1.11) by the method of Ling et al. (1966), pyruvate kinase (EC 2.7.1.40) by the methods of Biicher and Pfleiderer (1955), alkaline phosphatase (EC 3.1.3.1) by the method of Malamy and Horecker (1966), ATPase (EC 3.6.1.3) according to the method of Leloir and Cardini (1959) by measuring the amount of liberated inorganic phosphate with the method of Fiske and SubbaRow (1925), glucose6-phosphate dehydrogenase (EC 1.1.1.49) by the method of Kornberg and Horecker (1955). Lactate dehydrogenase (EC 1.1.1.28) and cytoplasmic L-a-glycerol3-phosphate dehydrogenase (EC 1.1.1.8) were assayed with enough of the respective substrate at pH 9.3 of Tris-HCl buffer, following the increase of optical density at 340 nm. (Yamada, 1972a). For the mitochondrial L-a-glycerol-3-phosphate and succinate dehydrogenases (EC 1.1.99.5
and EC 1.3.99.1), the standard reaction mixture of 3.2 ml. contained the following: 64 /jmoles of L-a-glycerol-3-phosphate or succinate, 0.32 jumoles of KCN, 160 ^moles of potassium phosphate buffer (pH 7.5), 0.4 mg. of cytochrome c and 0.2 ml. of enzyme preparation. The initial rate of increase in difference of optical density at 550 nm. and 541 nm. was followed with a Hitachi Two wave length double beam spectrophotometer, Type 356 (Yamada, 1972c). For L- -glycerol-3-phosphatase activity, amount of inorganic phosphate liberated was determined by the method of Fiske and SubbaRow (1925). Activity of the enzymes except "L-aglycerol-3-phosphatase is reported in units per mg. protein. One unit of enzyme was defined as the amount of the preparation which catalyzes the transformation of 3.2 milimcromoles of substrate per min. Specific activity was denned as the number of units per mg. protein. Protein concentration of the preparation was determined according to the methods of Lowry et al. (1951). Preparation of enzymes. After decapitation and exsanguination, fresh shell gland was quickly placed on ice. After the shell gland weight was measured, 1 g. was homogenized for 2 min. in 5 ml. of 0.25M sucrose solution and then centrifuged at 900 X g for 30 min. to eliminate cell debris and blood cells. The supernatant solution was passed through a glass wool plug in a funnel to remove fat and, after the glass wool plug was washed twice with 0.25M sucrose solution, the combined supernatant solution was adjusted with 0.25M sucrose solution up to 100 ml. which was used as the soluble enzyme preparation. The obtained precipitate by 10,000 X g centrifugation was dissolved in a cold distilled water and homogenized with Teflon homogenizer and was used as the enzyme preparation for particulate
SHELL GLAND ENZYMES
enzymes (Yamada, 1972a,b,c). For the preparation of the mucosal enzymes, the shell gland mucosa separated with an acryl board was homogenized for 2 min. and centrifuged for 30 min. at 10,000 X g. The obtained supernatant solution was used for the enzyme assays. To examine some properties of ATPase of the shell gland mucosa, the supernatant solution obtained by 10,000 X g centrifugation, was centrifuged at 105,000 X g for 2 hours. The obtained precipitate was suspended with Teflon homogenizer homogeneously in distilled water and used as the enzyme preparation.
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TABLE 1.—Fresh weights of parts of oviduct in the presence or absence of a developing egg in shell gland Fresh weight (grams)*
Body weight Oviduct weight Magnum Isthmus Shell gland
With an egg
With no egg
130.0 ± 3 . 0 5.5 ± 0 . 2 3.3 ± 0 . 2 0.63±0.02 1.7 ± 0 . 0 4
129.8 ± 2 . 6 5.5 ± 0 . 4 3.0 ± 0 . 2 0.64±0.02 1.7 ± 0 . 0 8
* Values are expressed as Mean ± S.E. of seven quail.
drogenase was obtained. Lactate dehydrogenase in the isthmus had higher specific activity per mg. protein than those in the other two portions. In the shell RESULTS gland, the significant differences between Activity of glycolytic and other enzymes the two groups, with respect to activity of in the shell gland. In spite of no significant phosphofructokinase, pyruvate kinase, difference of oviduct weight between quail alkaline phosphatase, succinate dehydrowith an egg and quail with no egg in the genase, cytoplasmic L-a-glycerol-3-phosshell gland, significant differences in ac- phate dehydrogenase, lactate dehydrogetivity of the enzymes in the oviduct were nase and glucose-6-phosphate dehydroobtained, as shown in Table 1 and Table genase, was obtained. 2. In the magnum, no significant differActivity of the mucosal enzymes. The ence of activity of the enzymes except activity of pyruvate kinase, glucose-6pyruvate kinase was obtained between the phosphate dehydrogenase, cytoplasmic two group. In the isthmus, significant dif- L-a-glycerol-3-phosphate dehydrogenase, ference of activity of pyruvate kinase, alkaline phosphatase and Mg 2+ -dependent succinate dehydrogenase and the cyto- ATPase in the shell gland mucosa was plasmic L-a-glycerol-3-phosphate dehy- significantly affected with location of a TABLE 2.—Effect of a developing egg location in shell gland on activity of several enzymes in respective part of oviduct of Japanese quail, Coturnix coturnix japonica Specific activity (Units/mg. protein)a M a g n u m portion With and egg With no egg
Shell gland portion
Isthmus portion With an egg
With no egg
With an egg
2.8 + 0 . 1 2.6 + 0.3 5.6 1.7 + 0 . 1 1.8 + 0 . 1 Glucokinase 0 . 6 9 + 0.03 0.76 + 0.07 1.67 Phosphofructokinase 0.41 + 0 . 0 3 0 . 4 0 + 0 . 0 3 Pyruvate kinase 2 . 0 5 + 0.15* 1.25 + 0 . 0 5 22.25 4.50 ± 0.05** 2 . 3 0 + 0.2 Alkaline phosphatase 122.6 + 11.7 112.1 + 12.1 200.5 60.6 ± 3.4 5 6 . 2 + 3.0 Succinate dehydrogenase 0 . 3 3 + 0.06** 0.51 + 0 . 0 4 6.0 2.6 + 0.3 2 . 7 + 0.3 Mitochondrial a-glycerol-30.31 + 0 . 0 6 0 . 4 3 + 0 . 0 7 15.6 8.1 ± 0 . 6 8.0 ± 0.6 phosphate dehydrogenase 0 . 7 2 + 0.11 3.32 Cytoplasmic a-glycerol-31.15 ± 0.15 1.26 + 0.15 4 . 6 4 + 0.58** phosphate dehydrogenase 195.6 ± 8.5 173.4 Lactate dehydrogenase 28.6 ± 2 . 8 2 9 . 5 + 2.7 223.2 ± 14.5 Glucose-6-phosphate 0 . 8 8 ± 0.17 0.80 ± 0 . 1 3 15.30 5.15 ± 0.46 4.66 ± 1.03 dehydrogenase
+ ± + ± + ±
With no egg
0.2 5 . 1 ± 0.2 0.14** 0.64 ± 0 . 0 8 1.3** 12.4 + 0.95 12.7** 134.4 ± 3.7 0.6* 4.5 + 0.4 1.6 17.2 ± 1.3
+ 0.47*
1 . 3 4 + 0.2
± 12.8** 9 3 . 1 ± 6 . 4 ± 1.70** 9 . 6 8 ± 0.70
*p <0.05. **p <0.01. 8 Enzymes were assayed as described in Materials and Methods. Values are expressed as Mean : S.E. of seven quail.
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TABLE 3.—Effect of a developing egg location in the shell gland on activity of several enzymes in the shell gland mucosa
TABLE 4.—Effect of metal ions on activity of A TPase from Japanese quail shell gland mucosa
Specific activity (Units/mg. protein) With an egg 1.25± 0.12 6.07± 0.49*»
Phosphofructokinase Pyruvate kinase Glucose-6-phosphate dehydrogenase 8.85± 0 . 1 3 " Cytoplasmic L-a-glycerol-3-phosphate dehydrogenase 1.16± 0.08» Lactate dehydrogenase 76.51± 3.63* Alkaline phosphatase 127.3 ± 1 1 . 6 " 2+ Mg -dependent ATPase 6515.2 ±153.6**
Relative activity
With no egg 1.20± 0.13 2.27+ 0.11 6.36±
0.19
0.79+ 0.07 56.96+ 4.26 180.3 + 18.4 2342.4 +102.4
Values are expressed as mean± S.E. of seven animals. *p<0.05. "p<0.01.
developing egg in the shell gland, as shown in Table 3. Some properties of the mucosal A TPase. As various mono- and divalent ions contained in the shell gland fluid (Schraer and Schraer, 1965; EL Jack and Lake, 1967) appear to affect the activity of many enzymes, especially of ATPase which is closely related with active transport mechanism, some basic properties of particle bound ATPase obtained by 105,000 X g centrifugation for 2 hours were examined. As shown in Table 4, Mg 2+ , compared with other metals, showed the highest activity and was followed by Ca2+ and Mn ! + in the order. No activating effect of Na+, K+, Na+ plus K+, Ni2+, Co2+, Zn2+ and EDTA was obtained. The highest activation of Mg2+-dependent ATPase and Ca2+-dependent ATPase was observed at 5mM Mg 2+ and at 5mM Ca2+ concentration, respectively, as shown in Fig. 1. The optimum pH values of Mgdependent and Ca-dependent ATPase was pH 8 (Fig. 2). Respective Km value (Km: Miehaelis constant, Concentration of substrate at which v = V/2) for ATP of Mg2+-dependent ATPase and Ca2+-dependent ATPase was 0.29mM and 0.14mM at lower ATP concentration than 0.6mM, but at higher ATP concentration than 0.6mM, larger Km value for ATP of these
(%) + H 2 0 (Control) + 5 m M MgCl 2 H-SmM CaCl2 + 5 m M MgCl 2 +5mM CaCl2 + 5 m M NiCl 2 + 5 m M MnCl 2 + 5 m M CoCl2 + 5 m M ZnCb + 5 m M KC1 + 5 m M NaCl + S m M KCl+5mM NaCl + 5 m M EDTA
100 615 460 530 30 229 82 64 105 97 95 70
+ 5 m M MgCl2 100 + 5 m M MgCl 2 +lOOmM KC1 84 +5mMMgCI 2 -|-100mMNaCl 87 + 5 m M M g C l 2 + 20mMKCl+100mMNaCl 98 + 5 m M MgCl 2 +lOOmM KC1+lOOmM NaCl 94 + 5 m M CaCl2 100 -r-5mMCaCl 2 +100mMKCl 94 +5mMCaCl 2 +100mMNaCl 90 + 5 m M CaCl 2 + 20mMKCl+100mMNaCl 87 +5mMCaCl 2 +100mMKCl+100mMNaCl 86 + 5 m M MgCl 2 +5mM CaCl2 + 5 m M MgCl 2 +5mM CaCl 2 +100mM KC1 + 5 m M MgCl 2 +5mM CaCl 2 +100mM NaCl + 5 m M MgCl 2 +5mM CaCl 2 +100mM KC1 +100mM NaCl + 5 m M MgCl2-|-5mM CaCl 2 +20mM KC1 +100mM NaCl
100 100 98 96 84
CaCl2(mH)
FIG. 1. Respective effect of MgCl 2 and CaCl 2 concentration on activity of adenosine triphosphatase from the shell gland mucosa in Japanese quail. • •,MgCl2 O O.CaCls
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SHELL GLAND ENZYMES i.o
Mg -ATPase 0.8
Ca -ATPase
//*>»"> 0.4
FIG. 3. Effect of adenosine triphosphate concentration on activity of Mg2+-dependent and Ca2+dependent adenosine triphosphatases from the shell gland mucosa in Japanese quail. • • , Mg2+-dependent ATPase, O O, Ca2+-dependent ATPase
•10 pH
FIG. 2. Effect of pH on activity of Mg2+-dependent- and Ca2+-dependent adenosine triphosphatases from the shell gland mucosa in Japanese quail. • • , Mg2+-dependent ATPase, O O, Ca2+-dependent ATPase
phosphatase in the ATPase preparation was considered. Inorganic phosphate amount in the shell gland. T h e amount of inorganic phosphate in the shell gland was determined, b u t n o significant difference owing to a developing egg location in the shell gland was
enzymes were obtained with smaller F m a x , as shown in Fig. 3. Fig. 4 and Fig. 5 show the inhibition extent and inhibitive way of Mg 2 + on Ca 2 + -dependent ATPase and Ca 2 + on Mg 2 + -dependent ATPase. Inhibition of Mg 2 + -dependent ATPase by Ca 2 + was noncompetitive and t h a t of Ca 2 + -dependent ATPase by Mg 2 + was competitive, as shown in Fig. 5a and Fig. 5b. I n studies of effect of such metabolic intermediates as A D P , A M P , P E P , N A D and N A D H on the activity of Mg 2 + dependent and Ca 2 + -dependent ATPases, no significant activation by these compounds except A D P was observed. A D P was hydrolyzed at about one-third to onefourth of A T P , by the both ATPases. The contamination of the nucleoside pyro-
Ca
(mM)
Mg
(mM)
FIG. 4. Respective effect of CaC^ and MgCk concentration on activity of Mg2+-dependent and Ca2+dependent adenosine triphosphatases from the shell gland mucosa in Japanese quail. • • , Mg2+-dependent ATPase, O O , Ca2+-dependent ATPase
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M. YAMADA
M^
"with
20
30
th
80mM
MgCl2
With
40mM
HgCl2
wate
40
FIG. 5a. Reciprocal plots of initial hydrolysis rate of ATP by Mg 2+ -dependent adenosine triphosphatase at various Ca 2+ concentrations.
obtained between the two groups. L-aglycerol-3-phosphate was hydrolyzed by a phosphatase (L-a-glycerol-3-phosphatase) at pH 8 and the activity of this enzyme was influenced by location of a developing egg in the shell gland, as shown in Table 5. /J-glycerol-3-phosphate was also hydrolyzed by this enzyme to the same extent. DISCUSSION
The data in the present paper show that the existence of a developing egg in the shell gland affects markedly the activities of the most of glycolytic enzymes in the shell gland and in the mucosal layer, and that the physiological function of the mucosal ATPase is complicated
FIG. 5b. Reciprocal plots of initial hydrolysis rate of ATP by Ca 2+ -dependent adenosine triphosphatase at various Mg 2+ concentration.
and appears to be influenced by coexisting metal ions in the tissue. It is recognized that glucokinase, phosphofructokinase and pyruvate kinase are the key glycolytic enzymes in the conversion of glucose into lactate in the liver (Weber et al., 1966). These three enzymes govern one way reaction coordinating2 with low activity and show little organ specificity, being involved in ATP-utilizing or producing reactions (Weber et al., 1966). Activities of these enzymes in 2 The apparent parallel change of ratio of activity: pyruvate kinase activity vs. phosphofructokinase activity, independent of the presence or absence of a developing egg in the shell gland.
TABLE 5.—Effect of a developing egg location on amount of inorganic phosphate a on activity of L-a-glycerol-3-phosphatase in the shell gland of Japanese quail Shell gland (g.) No developing egg in the oviduct A developing egg in magnum portion A developing egg in shell gland portion Before pigmentation After pigmentation
L-a-glycerol-3-phosphatase activity Inorganic phosphate (jumoles./g. tissue) (jumoles/h./mg. protein) (Mmoles/h./g. tissue)
1.7 + 0.03
291+30
45.2 + 7.7
498 + 82
1.9 + 0.07
314 + 43
43.1±4.7
516 + 95
1.8 + 0.04 1.7 + 0.02
212 + 20 296 + 26
30.5+1.7 22.8 + 3.4
374 + 29 270 + 43
SHELL GLAND ENZYMES
Table 2 seem to change in a coordinating way, independently of the presence of a developing egg in the shell gland. These three enzymes in the shell gland mucosa did not show the coordinating activity, as observed for the whole shell gland. However, no d a t a to connect the obtained activities of these enzymes (Table 2 and Table 3) with the route of energy supply for the performance of the physiological works is available. Further investigation on the energy supply route is required to elucidate this problem. Participation of active transport mechanism is reported in the translocation of Ca 2 + across the shell gland membrane (Ehrenspeck et al., 1971). Active transport mechanism, in general, is considered to be associated with the function of the membrane bound ATPase. The mucosal ATPase in the quail shell gland was activated by Mg 2 + a n d / o r Ca 2+ , b u t not by Na+ a n d / o r K+. These facts seem to suggest the presence of two ATPases: Mg 2 + dependent ATPase and Ca 2 + -dependent ATPase. More data to confirm the presence of these two ATPases are shown in Fig. 1 to Fig. 5. Concerning the K m value for A T P of Mg 2 + -dependent- or Ca 2 + -dependent ATPase in the shell gland mucosa, two K m values were obtained, respectively, depending on A T P concentration (Fig. 3 and Fig. 5). This phenomenon of two K m values for A T P was also reported in other work (Neufeld and Levy, 1969, 1970), and suggests t h a t A T P might act as a substrate and act as one of regulators for the enzyme action. The physiological meaning of noncompetitive inhibition of Ca 2 + on Mg 2 + -dependent ATPase and competitive inhibition of M g 2 + on Ca 2 + -dependent ATPase is not clear (Fig. 5). Although the existence of two ATPases: Mg 2 + -dependent ATPase and Ca 2 + -dependent ATPase, is described, it is still probable
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t h a t the mucosal ATPase could exist in a single component with two separate binding sites for Mg 2 + and Ca 2+ , respectively. No d a t a to decide one of these two alternatives is available. I t was reported t h a t calcium carbonate precipitation was prevented by the presence of phosphate ions at concentration too low for the precipitation of calcium phosphate and the composition of most biological fluids does not allow the deposition of calcium carbonate (Bachra et al., 1963). I n the avian shell gland, calcification of calcium carbonate is performed quite evidently, in spite of presence of inorganic phosphate in the tissue. As shown in Table 5, the amount of inorganic phosphate in the shell gland is not significantly affected by location of a developing egg in the shell gland, b u t activity of L-aglycerol-3-phosphatase as well as /3-glycerol-3-phosphatase in the shell gland was influenced by location of a developing egg in the shell gland. High activity of the cytoplasmic L-a-glycerol-3-phosphate dehydrogenase was observed in the shell gland (Table 2 and Table 3). If this high activity suggests the ample presence of L-a-glycerol-3-phosphate amount in the tissue, the possibility for this substrate to act as one of the inorganic phosphate donor remains. T h e function of the cytoplasmic and mitochondrial L-a-glycerol-3-phosphate dehydrogenases might also contribute as one of the regulatory steps in the carbohydrate metabolism b y the a-glycero-phosphate cycle (Estabrook and Sacktor, 1958; Zebe et al., 1959). I n the quail shell gland, the presence of two allosteric components of pyruvate kinase was obtained b y isoelectric focusing. The enzymic properties of these two components and their physiological significance in the regulation of carbohydrate metabolism in the shell gland during egg
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formation process in the oviduct will be reported in near future. REFERENCES Bachra, B. N., 0 . R. Trautz and S. L. Simon, 1963. Precipitation of calcium carbonates and phosphates. I. Spontaneous precipitation of calcium carbonates and phosphates under physiological conditions. Arch. Biochem. Biophys. 103: 124138. Bernstein, R. S., T. Navalainen, R. Schraer and H. Schraer, 1968. Intracellular distribution and role of carbonic anhydrase in the avian (Gallus Domesticus) shell gland mucosa. Biochim. Biophys. Acta, 159: 367-376. Biicher, T., and G. Pfieiderer, 1955. Pyruvate kinase from muscle. In: Methods in Enzymology, Vol. 1: pp. 435-440, Academic Press, New York. Ehrenspeck, G., H. Schraer and R. Schraer, 1967. Some metabolic aspects of calcium movement across the isolated avian shell gland. Pro. Soc. Exp. Biol. Med. 126: 392-395. Ehrenspeck, G., H. Schraer and R. Schraer, 1971. Calcium transfer across isolated avian shell gland. Am. J. Physiol. 220: 967-972. KL Jack, M. H., and P. E. Lake, 1967. The content of the principal inorganic ions and carbon dioxide in uterine fluids of the domestic hen. J. Reprod. Fert. 13: 127-132. F.stabrook, R. W. and B. Sacktor, 1958. a-glycerophosphate oxidase of flightmuscle mitochondria. J. Biol. Chem. 233: 1014-1019. Fiske, C. H., and Y. Subba Row, 1925. The calorimetric determination of phosphorus. J. Biol. Chem. 66: 375-400. Kornberg, A., and B. L. Horecker, 1955. Glucose-6phosphate dehydrogenase. In: Methods in Enzymology, Vol. 1: pp. 323-327. Academic Press, New York. Leloir, L. F., and C. E. Cardini, 1959. Characterization of phosphorus compounds by acid lability. In: Methods in Enzymology, Vol. 3: pp. 840850. Academic Press, New York. Ling, K. H., V. Peatkau, F. Marcus and H. A. Lardy, 1966. Phosphofructokinase (I. skeletal muscle). In: Methods in Enzymology, vol. 9: pp. 425-429, Academic Press, New York. Lowry, O. H., N. J. Rosenbrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Malamy, M., and B. 1. Horecker, 1966. Alkaline
phosphatase. In: Methods in Enzymology, Vol. 9: pp. 639-642. Academic Press, New York. Neufeld, A. H., and H. M. Levy, 1969. A second ouabain-sensitive sodium-dependent adenosine triphosphatase in brain microsomes. J. Biol. Chem. 244: 6493-6497. Neufeld, A. H., and H. M. Levy, 1970. The steady state level of phosphorylated intermediate in relation to the two sodium-dependent adenosine triphosphatases of calf brain microsomes. J. Biol. Chem. 245: 4962-4967. Schraer, R., and H. Schraer, 1965. Changes in metal distribution of the avian oviduct during the ovulation cycle. Pro. Soc. Exp. Biol. Med. 119: 937942. Sturkie, P. D., 1965. Avian Physiology, 2nd ed.: 452, Comstock Publishing Associates, Cornell Univ. Press, Ithaca. Walker, D. G., and M. J. Parry, 1966. Glucokinase 1. Liver. In: Methods in Enzymology, Vol. 9: pp. 381-388, Academic Press, New York. Weber, G., R. L. Singhal, N. B. Stamm, M. A. Lea and E. A. Fisher, 1966. Synchronous behaviour pattern of key glycolytic enzymes: glucokinase, phosphofructokinase, and pyruvate kinase. Advan. Enzyme Regul. 4: 50-81. Woodward, A. E., and F. B. Mather, 1964. The timing of ovulation, movement of the ovum through the oviduct, pigmentation and shell deposition in Japanese quail {Colurnix coturnix japonica). Poultry Sci. 43: 1427-1432. Yamada, M., 1972a. Studies on L-glycerol-3-phosphate dehydrogenases in Japanese quail, Coturnix Coturnix Japonica. I. Multiple components and some enzymic properties of nicotinamideadenine dinucleotide linked L-glycerol-3-phosphate dehydrogenase from liver and testes. J. Biochem. 72: 1081-1086. Yamada, M., 1972b. S-Aminolevulinic acid dehydratases from shell gland and liver of Japanese quail, Coturnix Coturnix Japonica. I. Purification, properties and hormonal induction. Biochim. Biophys. Acta, 279: 535-543. Yamada, M., 1972c. Studies on L-glycerol-3-phosphate dehydrogenases in Japanese quail, Coturnix Coturnix Japonica. I I . Comparison of activity of mitochondrial and cytoplasmic L-glycerol-3-phosphate dehydrogenases in testes and liver of a developing male Japanese quail. J. Biochem. In press. Zebe, E., A. Delbruck and T. H. Biicher, 1959. tiber den GIycerin-1-p Cyclus in Flugmuskel von locusta migratoria. Biochem. Z. 331: 254-272.
YAMADA
Biotron Institute, Kyushu University, Fukuoka City, Japan (Received for publication October 24, 1972) A BSTRA CT Effect of a developing egg location on activity of glucokinase, phosphof ructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, L-a-glycerol-3-phosphate dehydrogenases, lactate dehydrogenase, succinate dehydrogenase, alkaline phosphatase, adenosine triphosphatase and L-a-glycerol-3-phosphatase in the shell gland was examined, and some properties of adenosine triphosphatase in the shell gland mucosa were also investigated. Significant difference of activity of most enzymes examined, due to a developing egg location in the shell gland, was observed in the shell gland and its mucosa. The existence of Mg2+-dependent and Ca 2+ -dependent adenosine triphosphatase in the mucosa was observed. No significant enhanced activity of the enzyme by Na + , K + , or N a + plus K + was observed. The apparent Km value for ATP of the Mg 2+ -dependent adenosine triphosphatase was 0.29mM and that of Ca 2+ dependent adenosine triphosphatase was 0.15 mM. The effect of Ca 2+ on Mg 2+ -dependent adenosine triphosphatase was non competitive, while that of Mg 2+ on Ca 2+ -dependent adenosine triphosphatase was competitive. The activity of L-a-glycerol-3-phosphatase was shown to change during egg formation process. The possible participation of this enzyme in calcification process in the shell gland is discussed. POULTRY SCIENCE 52: 1375-1382, 1973
T
H E avian shell gland, which consists of a mucosal layer, smooth muscle, connective tissue and a sheet of serosal cells, retains a developing egg for 15 t o 20 hours, during which such physiological works as a marked calcium translocation across the mucosa, calcification of the egg shell and pigmentation, especially in Japanese quail are performed (Bernstein et al., 1968; Ehrenspeck et al., 1971; Sturkie, 1965; Woodward and Mather, 1964; Y a m a d a , 1972b). T h e supply of surplus energy to perform the physiological works mentioned above when the shell gland contains a developing egg, i.e., " a c tive" phase, is indispensable, compared with the energy status when there is no egg in the shell gland, i.e., "quiescent" phase. I n studies to elucidate the calcifi-
cation mechanism in the shell gland, it was reported t h a t calcium movement across the shell gland in vitro is in p a r t dependent on metabolic energy derived from oxidative metabolism and requires the generation of phosphate bond energy (Ehrenspeck et al., 1967), and also t h a t a major route of calcium movement across the shell gland involves an active transport mechanism (Ehrenspeck et al., 1971). I n order to understand the oxidative metabolism and active transport mechanism, as the first step, the change of activity of glycolytic and other enzymes corresponding to t h e location of a developing egg in the shell gland a n d some properties of mucosal ATPase were examined and reported in this paper. MATERIALS AND METHODS
x
The following abbreviations have been used: EDTA; ethylenediaminetetraacetate; Km, Michaelis constant; ATPase, adenosine triphosphatase; Pi, inorganic phosphate; PEP, phosphoenolpyruvate; FDP, fructose-l,6-diphosphate.
Animals. Female quail in their first year of lay were kept at 20 + 1°C. under the following lighting program: light period-(0600-1800), dark period-(1800-
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M. YAMADA
0600). All animals were maintained individually in wire cages on a commercial ration and water ad libitum. Well-synchronized animals for egg laying time, which was recorded every day, were employed for experiments. In order to minimize the individual variation, they were decapitated from 08:50 to 09:00. Chemicals. DL-a-glycerol-3-phosphate, succinate, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, phosphoenol-pyruvate, ATP, ADP, cytochrome c(Type III), NAD, NADH and NADP were purchased from Sigma, Saint Louis, U.S.A. All other chemicals were of special reagent grade. Glucose-6phosphate dehydrogenase, aldolase, triose phosphate isomerase, glycerol-3-phosphate dehydrogenase and lactate dehydrogenase were purchased from Boehringer, Mannheim, Germany. Enzyme assays. Enzyme activities were assayed at 30° C. as follows: glucokinase (EC 2.7.1.2) by the method of Walker and Parry (1966), phosphofructokinase (EC 2.7.1.11) by the method of Ling et al. (1966), pyruvate kinase (EC 2.7.1.40) by the methods of Biicher and Pfleiderer (1955), alkaline phosphatase (EC 3.1.3.1) by the method of Malamy and Horecker (1966), ATPase (EC 3.6.1.3) according to the method of Leloir and Cardini (1959) by measuring the amount of liberated inorganic phosphate with the method of Fiske and SubbaRow (1925), glucose6-phosphate dehydrogenase (EC 1.1.1.49) by the method of Kornberg and Horecker (1955). Lactate dehydrogenase (EC 1.1.1.28) and cytoplasmic L-a-glycerol3-phosphate dehydrogenase (EC 1.1.1.8) were assayed with enough of the respective substrate at pH 9.3 of Tris-HCl buffer, following the increase of optical density at 340 nm. (Yamada, 1972a). For the mitochondrial L-a-glycerol-3-phosphate and succinate dehydrogenases (EC 1.1.99.5
and EC 1.3.99.1), the standard reaction mixture of 3.2 ml. contained the following: 64 /jmoles of L-a-glycerol-3-phosphate or succinate, 0.32 jumoles of KCN, 160 ^moles of potassium phosphate buffer (pH 7.5), 0.4 mg. of cytochrome c and 0.2 ml. of enzyme preparation. The initial rate of increase in difference of optical density at 550 nm. and 541 nm. was followed with a Hitachi Two wave length double beam spectrophotometer, Type 356 (Yamada, 1972c). For L- -glycerol-3-phosphatase activity, amount of inorganic phosphate liberated was determined by the method of Fiske and SubbaRow (1925). Activity of the enzymes except "L-aglycerol-3-phosphatase is reported in units per mg. protein. One unit of enzyme was defined as the amount of the preparation which catalyzes the transformation of 3.2 milimcromoles of substrate per min. Specific activity was denned as the number of units per mg. protein. Protein concentration of the preparation was determined according to the methods of Lowry et al. (1951). Preparation of enzymes. After decapitation and exsanguination, fresh shell gland was quickly placed on ice. After the shell gland weight was measured, 1 g. was homogenized for 2 min. in 5 ml. of 0.25M sucrose solution and then centrifuged at 900 X g for 30 min. to eliminate cell debris and blood cells. The supernatant solution was passed through a glass wool plug in a funnel to remove fat and, after the glass wool plug was washed twice with 0.25M sucrose solution, the combined supernatant solution was adjusted with 0.25M sucrose solution up to 100 ml. which was used as the soluble enzyme preparation. The obtained precipitate by 10,000 X g centrifugation was dissolved in a cold distilled water and homogenized with Teflon homogenizer and was used as the enzyme preparation for particulate
SHELL GLAND ENZYMES
enzymes (Yamada, 1972a,b,c). For the preparation of the mucosal enzymes, the shell gland mucosa separated with an acryl board was homogenized for 2 min. and centrifuged for 30 min. at 10,000 X g. The obtained supernatant solution was used for the enzyme assays. To examine some properties of ATPase of the shell gland mucosa, the supernatant solution obtained by 10,000 X g centrifugation, was centrifuged at 105,000 X g for 2 hours. The obtained precipitate was suspended with Teflon homogenizer homogeneously in distilled water and used as the enzyme preparation.
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TABLE 1.—Fresh weights of parts of oviduct in the presence or absence of a developing egg in shell gland Fresh weight (grams)*
Body weight Oviduct weight Magnum Isthmus Shell gland
With an egg
With no egg
130.0 ± 3 . 0 5.5 ± 0 . 2 3.3 ± 0 . 2 0.63±0.02 1.7 ± 0 . 0 4
129.8 ± 2 . 6 5.5 ± 0 . 4 3.0 ± 0 . 2 0.64±0.02 1.7 ± 0 . 0 8
* Values are expressed as Mean ± S.E. of seven quail.
drogenase was obtained. Lactate dehydrogenase in the isthmus had higher specific activity per mg. protein than those in the other two portions. In the shell RESULTS gland, the significant differences between Activity of glycolytic and other enzymes the two groups, with respect to activity of in the shell gland. In spite of no significant phosphofructokinase, pyruvate kinase, difference of oviduct weight between quail alkaline phosphatase, succinate dehydrowith an egg and quail with no egg in the genase, cytoplasmic L-a-glycerol-3-phosshell gland, significant differences in ac- phate dehydrogenase, lactate dehydrogetivity of the enzymes in the oviduct were nase and glucose-6-phosphate dehydroobtained, as shown in Table 1 and Table genase, was obtained. 2. In the magnum, no significant differActivity of the mucosal enzymes. The ence of activity of the enzymes except activity of pyruvate kinase, glucose-6pyruvate kinase was obtained between the phosphate dehydrogenase, cytoplasmic two group. In the isthmus, significant dif- L-a-glycerol-3-phosphate dehydrogenase, ference of activity of pyruvate kinase, alkaline phosphatase and Mg 2+ -dependent succinate dehydrogenase and the cyto- ATPase in the shell gland mucosa was plasmic L-a-glycerol-3-phosphate dehy- significantly affected with location of a TABLE 2.—Effect of a developing egg location in shell gland on activity of several enzymes in respective part of oviduct of Japanese quail, Coturnix coturnix japonica Specific activity (Units/mg. protein)a M a g n u m portion With and egg With no egg
Shell gland portion
Isthmus portion With an egg
With no egg
With an egg
2.8 + 0 . 1 2.6 + 0.3 5.6 1.7 + 0 . 1 1.8 + 0 . 1 Glucokinase 0 . 6 9 + 0.03 0.76 + 0.07 1.67 Phosphofructokinase 0.41 + 0 . 0 3 0 . 4 0 + 0 . 0 3 Pyruvate kinase 2 . 0 5 + 0.15* 1.25 + 0 . 0 5 22.25 4.50 ± 0.05** 2 . 3 0 + 0.2 Alkaline phosphatase 122.6 + 11.7 112.1 + 12.1 200.5 60.6 ± 3.4 5 6 . 2 + 3.0 Succinate dehydrogenase 0 . 3 3 + 0.06** 0.51 + 0 . 0 4 6.0 2.6 + 0.3 2 . 7 + 0.3 Mitochondrial a-glycerol-30.31 + 0 . 0 6 0 . 4 3 + 0 . 0 7 15.6 8.1 ± 0 . 6 8.0 ± 0.6 phosphate dehydrogenase 0 . 7 2 + 0.11 3.32 Cytoplasmic a-glycerol-31.15 ± 0.15 1.26 + 0.15 4 . 6 4 + 0.58** phosphate dehydrogenase 195.6 ± 8.5 173.4 Lactate dehydrogenase 28.6 ± 2 . 8 2 9 . 5 + 2.7 223.2 ± 14.5 Glucose-6-phosphate 0 . 8 8 ± 0.17 0.80 ± 0 . 1 3 15.30 5.15 ± 0.46 4.66 ± 1.03 dehydrogenase
+ ± + ± + ±
With no egg
0.2 5 . 1 ± 0.2 0.14** 0.64 ± 0 . 0 8 1.3** 12.4 + 0.95 12.7** 134.4 ± 3.7 0.6* 4.5 + 0.4 1.6 17.2 ± 1.3
+ 0.47*
1 . 3 4 + 0.2
± 12.8** 9 3 . 1 ± 6 . 4 ± 1.70** 9 . 6 8 ± 0.70
*p <0.05. **p <0.01. 8 Enzymes were assayed as described in Materials and Methods. Values are expressed as Mean : S.E. of seven quail.
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M. YAMADA
TABLE 3.—Effect of a developing egg location in the shell gland on activity of several enzymes in the shell gland mucosa
TABLE 4.—Effect of metal ions on activity of A TPase from Japanese quail shell gland mucosa
Specific activity (Units/mg. protein) With an egg 1.25± 0.12 6.07± 0.49*»
Phosphofructokinase Pyruvate kinase Glucose-6-phosphate dehydrogenase 8.85± 0 . 1 3 " Cytoplasmic L-a-glycerol-3-phosphate dehydrogenase 1.16± 0.08» Lactate dehydrogenase 76.51± 3.63* Alkaline phosphatase 127.3 ± 1 1 . 6 " 2+ Mg -dependent ATPase 6515.2 ±153.6**
Relative activity
With no egg 1.20± 0.13 2.27+ 0.11 6.36±
0.19
0.79+ 0.07 56.96+ 4.26 180.3 + 18.4 2342.4 +102.4
Values are expressed as mean± S.E. of seven animals. *p<0.05. "p<0.01.
developing egg in the shell gland, as shown in Table 3. Some properties of the mucosal A TPase. As various mono- and divalent ions contained in the shell gland fluid (Schraer and Schraer, 1965; EL Jack and Lake, 1967) appear to affect the activity of many enzymes, especially of ATPase which is closely related with active transport mechanism, some basic properties of particle bound ATPase obtained by 105,000 X g centrifugation for 2 hours were examined. As shown in Table 4, Mg 2+ , compared with other metals, showed the highest activity and was followed by Ca2+ and Mn ! + in the order. No activating effect of Na+, K+, Na+ plus K+, Ni2+, Co2+, Zn2+ and EDTA was obtained. The highest activation of Mg2+-dependent ATPase and Ca2+-dependent ATPase was observed at 5mM Mg 2+ and at 5mM Ca2+ concentration, respectively, as shown in Fig. 1. The optimum pH values of Mgdependent and Ca-dependent ATPase was pH 8 (Fig. 2). Respective Km value (Km: Miehaelis constant, Concentration of substrate at which v = V/2) for ATP of Mg2+-dependent ATPase and Ca2+-dependent ATPase was 0.29mM and 0.14mM at lower ATP concentration than 0.6mM, but at higher ATP concentration than 0.6mM, larger Km value for ATP of these
(%) + H 2 0 (Control) + 5 m M MgCl 2 H-SmM CaCl2 + 5 m M MgCl 2 +5mM CaCl2 + 5 m M NiCl 2 + 5 m M MnCl 2 + 5 m M CoCl2 + 5 m M ZnCb + 5 m M KC1 + 5 m M NaCl + S m M KCl+5mM NaCl + 5 m M EDTA
100 615 460 530 30 229 82 64 105 97 95 70
+ 5 m M MgCl2 100 + 5 m M MgCl 2 +lOOmM KC1 84 +5mMMgCI 2 -|-100mMNaCl 87 + 5 m M M g C l 2 + 20mMKCl+100mMNaCl 98 + 5 m M MgCl 2 +lOOmM KC1+lOOmM NaCl 94 + 5 m M CaCl2 100 -r-5mMCaCl 2 +100mMKCl 94 +5mMCaCl 2 +100mMNaCl 90 + 5 m M CaCl 2 + 20mMKCl+100mMNaCl 87 +5mMCaCl 2 +100mMKCl+100mMNaCl 86 + 5 m M MgCl 2 +5mM CaCl2 + 5 m M MgCl 2 +5mM CaCl 2 +100mM KC1 + 5 m M MgCl 2 +5mM CaCl 2 +100mM NaCl + 5 m M MgCl 2 +5mM CaCl 2 +100mM KC1 +100mM NaCl + 5 m M MgCl2-|-5mM CaCl 2 +20mM KC1 +100mM NaCl
100 100 98 96 84
CaCl2(mH)
FIG. 1. Respective effect of MgCl 2 and CaCl 2 concentration on activity of adenosine triphosphatase from the shell gland mucosa in Japanese quail. • •,MgCl2 O O.CaCls
1379
SHELL GLAND ENZYMES i.o
Mg -ATPase 0.8
Ca -ATPase
//*>»"> 0.4
FIG. 3. Effect of adenosine triphosphate concentration on activity of Mg2+-dependent and Ca2+dependent adenosine triphosphatases from the shell gland mucosa in Japanese quail. • • , Mg2+-dependent ATPase, O O, Ca2+-dependent ATPase
•10 pH
FIG. 2. Effect of pH on activity of Mg2+-dependent- and Ca2+-dependent adenosine triphosphatases from the shell gland mucosa in Japanese quail. • • , Mg2+-dependent ATPase, O O, Ca2+-dependent ATPase
phosphatase in the ATPase preparation was considered. Inorganic phosphate amount in the shell gland. T h e amount of inorganic phosphate in the shell gland was determined, b u t n o significant difference owing to a developing egg location in the shell gland was
enzymes were obtained with smaller F m a x , as shown in Fig. 3. Fig. 4 and Fig. 5 show the inhibition extent and inhibitive way of Mg 2 + on Ca 2 + -dependent ATPase and Ca 2 + on Mg 2 + -dependent ATPase. Inhibition of Mg 2 + -dependent ATPase by Ca 2 + was noncompetitive and t h a t of Ca 2 + -dependent ATPase by Mg 2 + was competitive, as shown in Fig. 5a and Fig. 5b. I n studies of effect of such metabolic intermediates as A D P , A M P , P E P , N A D and N A D H on the activity of Mg 2 + dependent and Ca 2 + -dependent ATPases, no significant activation by these compounds except A D P was observed. A D P was hydrolyzed at about one-third to onefourth of A T P , by the both ATPases. The contamination of the nucleoside pyro-
Ca
(mM)
Mg
(mM)
FIG. 4. Respective effect of CaC^ and MgCk concentration on activity of Mg2+-dependent and Ca2+dependent adenosine triphosphatases from the shell gland mucosa in Japanese quail. • • , Mg2+-dependent ATPase, O O , Ca2+-dependent ATPase
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M. YAMADA
M^
"with
20
30
th
80mM
MgCl2
With
40mM
HgCl2
wate
40
FIG. 5a. Reciprocal plots of initial hydrolysis rate of ATP by Mg 2+ -dependent adenosine triphosphatase at various Ca 2+ concentrations.
obtained between the two groups. L-aglycerol-3-phosphate was hydrolyzed by a phosphatase (L-a-glycerol-3-phosphatase) at pH 8 and the activity of this enzyme was influenced by location of a developing egg in the shell gland, as shown in Table 5. /J-glycerol-3-phosphate was also hydrolyzed by this enzyme to the same extent. DISCUSSION
The data in the present paper show that the existence of a developing egg in the shell gland affects markedly the activities of the most of glycolytic enzymes in the shell gland and in the mucosal layer, and that the physiological function of the mucosal ATPase is complicated
FIG. 5b. Reciprocal plots of initial hydrolysis rate of ATP by Ca 2+ -dependent adenosine triphosphatase at various Mg 2+ concentration.
and appears to be influenced by coexisting metal ions in the tissue. It is recognized that glucokinase, phosphofructokinase and pyruvate kinase are the key glycolytic enzymes in the conversion of glucose into lactate in the liver (Weber et al., 1966). These three enzymes govern one way reaction coordinating2 with low activity and show little organ specificity, being involved in ATP-utilizing or producing reactions (Weber et al., 1966). Activities of these enzymes in 2 The apparent parallel change of ratio of activity: pyruvate kinase activity vs. phosphofructokinase activity, independent of the presence or absence of a developing egg in the shell gland.
TABLE 5.—Effect of a developing egg location on amount of inorganic phosphate a on activity of L-a-glycerol-3-phosphatase in the shell gland of Japanese quail Shell gland (g.) No developing egg in the oviduct A developing egg in magnum portion A developing egg in shell gland portion Before pigmentation After pigmentation
L-a-glycerol-3-phosphatase activity Inorganic phosphate (jumoles./g. tissue) (jumoles/h./mg. protein) (Mmoles/h./g. tissue)
1.7 + 0.03
291+30
45.2 + 7.7
498 + 82
1.9 + 0.07
314 + 43
43.1±4.7
516 + 95
1.8 + 0.04 1.7 + 0.02
212 + 20 296 + 26
30.5+1.7 22.8 + 3.4
374 + 29 270 + 43
SHELL GLAND ENZYMES
Table 2 seem to change in a coordinating way, independently of the presence of a developing egg in the shell gland. These three enzymes in the shell gland mucosa did not show the coordinating activity, as observed for the whole shell gland. However, no d a t a to connect the obtained activities of these enzymes (Table 2 and Table 3) with the route of energy supply for the performance of the physiological works is available. Further investigation on the energy supply route is required to elucidate this problem. Participation of active transport mechanism is reported in the translocation of Ca 2 + across the shell gland membrane (Ehrenspeck et al., 1971). Active transport mechanism, in general, is considered to be associated with the function of the membrane bound ATPase. The mucosal ATPase in the quail shell gland was activated by Mg 2 + a n d / o r Ca 2+ , b u t not by Na+ a n d / o r K+. These facts seem to suggest the presence of two ATPases: Mg 2 + dependent ATPase and Ca 2 + -dependent ATPase. More data to confirm the presence of these two ATPases are shown in Fig. 1 to Fig. 5. Concerning the K m value for A T P of Mg 2 + -dependent- or Ca 2 + -dependent ATPase in the shell gland mucosa, two K m values were obtained, respectively, depending on A T P concentration (Fig. 3 and Fig. 5). This phenomenon of two K m values for A T P was also reported in other work (Neufeld and Levy, 1969, 1970), and suggests t h a t A T P might act as a substrate and act as one of regulators for the enzyme action. The physiological meaning of noncompetitive inhibition of Ca 2 + on Mg 2 + -dependent ATPase and competitive inhibition of M g 2 + on Ca 2 + -dependent ATPase is not clear (Fig. 5). Although the existence of two ATPases: Mg 2 + -dependent ATPase and Ca 2 + -dependent ATPase, is described, it is still probable
1381
t h a t the mucosal ATPase could exist in a single component with two separate binding sites for Mg 2 + and Ca 2+ , respectively. No d a t a to decide one of these two alternatives is available. I t was reported t h a t calcium carbonate precipitation was prevented by the presence of phosphate ions at concentration too low for the precipitation of calcium phosphate and the composition of most biological fluids does not allow the deposition of calcium carbonate (Bachra et al., 1963). I n the avian shell gland, calcification of calcium carbonate is performed quite evidently, in spite of presence of inorganic phosphate in the tissue. As shown in Table 5, the amount of inorganic phosphate in the shell gland is not significantly affected by location of a developing egg in the shell gland, b u t activity of L-aglycerol-3-phosphatase as well as /3-glycerol-3-phosphatase in the shell gland was influenced by location of a developing egg in the shell gland. High activity of the cytoplasmic L-a-glycerol-3-phosphate dehydrogenase was observed in the shell gland (Table 2 and Table 3). If this high activity suggests the ample presence of L-a-glycerol-3-phosphate amount in the tissue, the possibility for this substrate to act as one of the inorganic phosphate donor remains. T h e function of the cytoplasmic and mitochondrial L-a-glycerol-3-phosphate dehydrogenases might also contribute as one of the regulatory steps in the carbohydrate metabolism b y the a-glycero-phosphate cycle (Estabrook and Sacktor, 1958; Zebe et al., 1959). I n the quail shell gland, the presence of two allosteric components of pyruvate kinase was obtained b y isoelectric focusing. The enzymic properties of these two components and their physiological significance in the regulation of carbohydrate metabolism in the shell gland during egg
1382
M. YAMADA
formation process in the oviduct will be reported in near future. REFERENCES Bachra, B. N., 0 . R. Trautz and S. L. Simon, 1963. Precipitation of calcium carbonates and phosphates. I. Spontaneous precipitation of calcium carbonates and phosphates under physiological conditions. Arch. Biochem. Biophys. 103: 124138. Bernstein, R. S., T. Navalainen, R. Schraer and H. Schraer, 1968. Intracellular distribution and role of carbonic anhydrase in the avian (Gallus Domesticus) shell gland mucosa. Biochim. Biophys. Acta, 159: 367-376. Biicher, T., and G. Pfieiderer, 1955. Pyruvate kinase from muscle. In: Methods in Enzymology, Vol. 1: pp. 435-440, Academic Press, New York. Ehrenspeck, G., H. Schraer and R. Schraer, 1967. Some metabolic aspects of calcium movement across the isolated avian shell gland. Pro. Soc. Exp. Biol. Med. 126: 392-395. Ehrenspeck, G., H. Schraer and R. Schraer, 1971. Calcium transfer across isolated avian shell gland. Am. J. Physiol. 220: 967-972. KL Jack, M. H., and P. E. Lake, 1967. The content of the principal inorganic ions and carbon dioxide in uterine fluids of the domestic hen. J. Reprod. Fert. 13: 127-132. F.stabrook, R. W. and B. Sacktor, 1958. a-glycerophosphate oxidase of flightmuscle mitochondria. J. Biol. Chem. 233: 1014-1019. Fiske, C. H., and Y. Subba Row, 1925. The calorimetric determination of phosphorus. J. Biol. Chem. 66: 375-400. Kornberg, A., and B. L. Horecker, 1955. Glucose-6phosphate dehydrogenase. In: Methods in Enzymology, Vol. 1: pp. 323-327. Academic Press, New York. Leloir, L. F., and C. E. Cardini, 1959. Characterization of phosphorus compounds by acid lability. In: Methods in Enzymology, Vol. 3: pp. 840850. Academic Press, New York. Ling, K. H., V. Peatkau, F. Marcus and H. A. Lardy, 1966. Phosphofructokinase (I. skeletal muscle). In: Methods in Enzymology, vol. 9: pp. 425-429, Academic Press, New York. Lowry, O. H., N. J. Rosenbrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Malamy, M., and B. 1. Horecker, 1966. Alkaline
phosphatase. In: Methods in Enzymology, Vol. 9: pp. 639-642. Academic Press, New York. Neufeld, A. H., and H. M. Levy, 1969. A second ouabain-sensitive sodium-dependent adenosine triphosphatase in brain microsomes. J. Biol. Chem. 244: 6493-6497. Neufeld, A. H., and H. M. Levy, 1970. The steady state level of phosphorylated intermediate in relation to the two sodium-dependent adenosine triphosphatases of calf brain microsomes. J. Biol. Chem. 245: 4962-4967. Schraer, R., and H. Schraer, 1965. Changes in metal distribution of the avian oviduct during the ovulation cycle. Pro. Soc. Exp. Biol. Med. 119: 937942. Sturkie, P. D., 1965. Avian Physiology, 2nd ed.: 452, Comstock Publishing Associates, Cornell Univ. Press, Ithaca. Walker, D. G., and M. J. Parry, 1966. Glucokinase 1. Liver. In: Methods in Enzymology, Vol. 9: pp. 381-388, Academic Press, New York. Weber, G., R. L. Singhal, N. B. Stamm, M. A. Lea and E. A. Fisher, 1966. Synchronous behaviour pattern of key glycolytic enzymes: glucokinase, phosphofructokinase, and pyruvate kinase. Advan. Enzyme Regul. 4: 50-81. Woodward, A. E., and F. B. Mather, 1964. The timing of ovulation, movement of the ovum through the oviduct, pigmentation and shell deposition in Japanese quail {Colurnix coturnix japonica). Poultry Sci. 43: 1427-1432. Yamada, M., 1972a. Studies on L-glycerol-3-phosphate dehydrogenases in Japanese quail, Coturnix Coturnix Japonica. I. Multiple components and some enzymic properties of nicotinamideadenine dinucleotide linked L-glycerol-3-phosphate dehydrogenase from liver and testes. J. Biochem. 72: 1081-1086. Yamada, M., 1972b. S-Aminolevulinic acid dehydratases from shell gland and liver of Japanese quail, Coturnix Coturnix Japonica. I. Purification, properties and hormonal induction. Biochim. Biophys. Acta, 279: 535-543. Yamada, M., 1972c. Studies on L-glycerol-3-phosphate dehydrogenases in Japanese quail, Coturnix Coturnix Japonica. I I . Comparison of activity of mitochondrial and cytoplasmic L-glycerol-3-phosphate dehydrogenases in testes and liver of a developing male Japanese quail. J. Biochem. In press. Zebe, E., A. Delbruck and T. H. Biicher, 1959. tiber den GIycerin-1-p Cyclus in Flugmuskel von locusta migratoria. Biochem. Z. 331: 254-272.