Aiming to clarify the mechanism of inhibition of (Na
+
, K
+
)-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic behavior of ...phosphoenzyme-linked partial reactions using a microsomal gill (Na
+
, K
+
)-ATPase from juvenile and adult
M. amazonicum
, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 ± 0.006 s
−1
) in adults than in juveniles (0.053 ± 0.003 s
−1
) for spermidine, but similar to juveniles (0.059 ± 0.004 s
−1
) for putrescine. Maximum phosphointermediate formation for the (Na
+
, K
+
)-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult
M. amazonicum
gill (Na
+
, K
+
)-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.
We characterize the kinetic properties of a gill (Na
+
, K
+
)-ATPase from the pelagic marine seabob
Xiphopenaeus kroyeri.
Sucrose density gradient centrifugation revealed membrane fractions ...distributed mainly into a heavy fraction showing considerable (Na
+
, K
+
)-ATPase activity, but also containing mitochondrial F
0
F
1
- and Na
+
- and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na
+
, K
+
)-ATPase α-subunit with an M
r
of ≈110 kDa. The α-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na
+
, K
+
)-ATPase hydrolyzed ATP obeying Michaelis–Menten kinetics with V
M
= 109.5 ± 3.2 nmol Pi min
−1
mg
−1
and K
M
= 0.03 ± 0.003 mmol L
−1
. Mg
2+
(V
M
= 109.8 ± 2.1 nmol Pi min
−1
mg
−1
, K
0.5
= 0.60 ± 0.03 mmol L
−1
), Na
+
(V
M
= 117.6 ± 3.5 nmol Pi min
−1
mg
−1
, K
0.5
= 5.36 ± 0.14 mmol L
−1
), K
+
(V
M
= 112.9 ± 1.4 nmol Pi min
−1
mg
−1
, K
0.5
= 1.32 ± 0.08 mmol L
−1
), and NH
4
+
(V
M
= 200.8 ± 7.1 nmol Pi min
−1
mg
−1
, K
0.5
= 2.70 ± 0.04 mmol L
−1
) stimulated (Na
+
, K
+
)-ATPase activity following site–site interactions. K
+
plus NH
4
+
does not synergistically stimulate (Na
+
, K
+
)-ATPase activity, although each ion modulates affinity of the other. The enzyme exhibits a single site for K
+
binding that can be occupied by NH
4
+
, stimulating the enzyme. Ouabain (K
I
= 84.0 ± 2.1 µmol L
−1
) and orthovanadate (K
I
= 0.157 ± 0.001 µmol L
−1
) inhibited total ATPase activity by ≈50 and ≈44 %, respectively. Ouabain inhibition increases ≈80 % in the presence of NH
4
+
with a threefold lower K
I
, suggesting that NH
4
+
is likely transported as a K
+
congener.
Aiming to clarify the mechanism of inhibition of (Na super(+), K super(+))-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic ...behavior of phosphoenzyme-linked partial reactions using a microsomal gill (Na super(+), K super(+))-ATPase from juvenile and adult M. amazonicum, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 plus or minus 0.006 s super(-1)) in adults than in juveniles (0.053 plus or minus 0.003 s super(-1)) for spermidine, but similar to juveniles (0.059 plus or minus 0.004 s super(-1)) for putrescine. Maximum phosphointermediate formation for the (Na super(+), K super(+))-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult M. amazonicum gill (Na super(+), K super(+))-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.
We characterize the kinetic properties of a gill (Na super(+), K super(+))-ATPase from the pelagic marine seabob Xiphopenaeus kroyeri. Sucrose density gradient centrifugation revealed membrane ...fractions distributed mainly into a heavy fraction showing considerable (Na super(+), K super(+))-ATPase activity, but also containing mitochondrial F sub(0)F sub(1)- and Na super(+)- and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na super(+), K super(+))-ATPase alpha -subunit with an M sub(r) of approximately 110 kDa. The alpha -subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na super(+), K super(+))-ATPase hydrolyzed ATP obeying Michaelis-Menten kinetics with V sub(M) = 109.5 plus or minus 3.2 nmol Pi min super(-1 )mg super(-1) and K sub(M) = 0.03 plus or minus 0.003 mmol L super(-1). Mg super(2+) (V sub(M) = 109.8 plus or minus 2.1 nmol Pi min super(-1 )mg super(-1), K sub(0.5) = 0.60 plus or minus 0.03 mmol L super(-1)), Na super(+) (V sub(M) = 117.6 plus or minus 3.5 nmol Pi min super(-1 )mg super(-1), K sub(0.5) = 5.36 plus or minus 0.14 mmol L super(-1)), K super(+) (V sub(M) = 112.9 plus or minus 1.4 nmol Pi min super(-1 )mg super(-1), K sub(0.5) = 1.32 plus or minus 0.08 mmol L super(-1)), and NH sub(4) super(+) (V sub(M) = 200.8 plus or minus 7.1 nmol Pi min super(-1 )mg super(-1), K sub(0.5) = 2.70 plus or minus 0.04 mmol L super(-1)) stimulated (Na super(+), K super(+))-ATPase activity following site-site interactions. K super(+) plus NH sub(4) super(+) does not synergistically stimulate (Na super(+), K super(+))-ATPase activity, although each ion modulates affinity of the other. The enzyme exhibits a single site for K super(+) binding that can be occupied by NH sub(4) super(+), stimulating the enzyme. Ouabain (K sub(I) = 84.0 plus or minus 2.1 mu mol L super(-1)) and orthovanadate (K sub(I) = 0.157 plus or minus 0.001 mu mol L super(-1)) inhibited total ATPase activity by approximately 50 and approximately 44 %, respectively. Ouabain inhibition increases approximately 80 % in the presence of NH sub(4) super(+) with a threefold lower K sub(I), suggesting that NH sub(4) super(+) is likely transported as a K super(+) congener.
We investigated modulation by ATP, Mg
2+
, Na
+
, K
+
and NH
4
+
and inhibition by ouabain of (Na
+
,K
+
)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea ...IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps,
Macrobrachium amazonicum
. (Na
+
,K
+
)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (
K
M
= 0.09 ± 0.01 mmol L
−1
) of the decapodid III (Na
+
,K
+
)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na
+
,K
+
)-ATPase activity by K
+
also revealed a threefold greater affinity for K
+
(
K
0.5
= 0.91 ± 0.04 mmol L
−1
) in decapodid III than in other stages; NH
4
+
had no modulatory effect. The affinity for Na
+
(
K
0.5
= 13.2 ± 0.6 mmol L
−1
) of zoea I (Na
+
,K
+
)-ATPase was fourfold less than other stages. Modulation by Na
+
, Mg
2+
and NH
4
+
obeyed cooperative kinetics, while K
+
modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg
2+
stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg
2+
-stimulated ATPases other than (Na
+
,K
+
)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na
+
-ATPase may be involved in the ontogeny of osmoregulation in larval
M. amazonicum.
The NH
4
+
-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.
We investigated modulation by ATP, Mg super(2+), Na super(+), K super(+) and NH sub(4) super(+) and inhibition by ouabain of (Na super(+),K super(+))-ATPa se activity in microsomal homogenates of ...whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na super(+),K super(+))-ATPa se-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K sub(M) = 0.09 plus or minus 0.01 mmol L super(-1)) of the decapodid III (Na super(+),K super(+))-ATPa se, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na super(+),K super(+))-ATPa se activity by K super(+) also revealed a threefold greater affinity for K super(+) (K sub(0.5) = 0.91 plus or minus 0.04 mmol L super(-1)) in decapodid III than in other stages; NH sub(4) super(+) had no modulatory effect. The affinity for Na super(+) (K sub(0.5) = 13.2 plus or minus 0.6 mmol L super(-1)) of zoea I (Na super(+),K super(+))-ATPa se was fourfold less than other stages. Modulation by Na super(+), Mg super(2+) and NH sub(4) super(+) obeyed cooperative kinetics, while K super(+) modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg super(2+) stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg super(2+)-stimulated ATPases other than (Na super(+),K super(+))-ATPa se are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na super(+)-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH sub(4) super(+)-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.
A (Na+,K+)-ATPase presente no tecido branquial dos crustáceos osmorreguladores é um componente essencial de seu sistema de regulação iônica e osmótica. Esta enzima também apresenta um papel relevante ...no processo de excreção ativa de NH4+ através do tecido branquial dos crustáceos. Uma fração microsomal rica em (Na+, K+)ATPase foi preparada por centrifugação diferencial a partir de um homogeneizado do tecido branquial de Callinectes ornatus. A centrifugação em gradiente de sacarose revelou a presença de um unico pico de proteina com atividade ATPase, mas a eletroforese em gel de poliacrilamida em condições desnaturantes revelou a presença de várias bandas protéicas. O uso do anticorpo monoclonal 5 contra a subunidade da (Na+, K+) ATPase, revelou a presença de uma única banda proteica de 110 kDa com atividade (Na+, K+)?ATPase. A (Na+, K+) ATPase hidrolisou o PNPP (V= 52,0 ± 2,0 U/mg e K0,5 = 1,1 ± 0,1 mM) através de interações sítio-sítio (nH= 1,6). A modulação da enzima pelos íons magnésio (V= 52,3 ± 1,3 U/mg e K0,5 = 1,1 ± 0,05 mM), potássio (V= 51,4 ± 1,5 U/mg e K0,5 = 2,3 ± 0,1 mM) e amônio (V= 56,7 ± 2,6 U/mg e K0,5 = 9,8 ± 0,4 mM) ocorreu através de interações sítio-sítio. Os íons sódio atuaram como inibidores da atividade K+-fosfatase da enzima (Ki= 1,7 ± 0,1 mM) e a ouabaína inibiu cerca de 80% a atividade PNPPase independentemente da presença de íons amônio. A (Na+, K+) ATPase hidrolisou o ATP de acordo com cinética de Michelis-Menten, com KM= 0,16 0,01 mM e V= 116,3 5,6 U/mg, enquanto a modulação da atividade da enzima pelos íons magnésio (V= 111,0 ± 5,4 U/mg e K0,5= 0,54 ± 0,03 mM), sódio (V= 110,6 ± 5,3 U/mg e K0,5= 6,3 ± 0,3 mM), potássio (V= 116,0 ± 5,5 U/mg e K0,5= 1,5 ± 0,1 mM) e amônio (V= 173,3 ± 5,4 U/mg e K0,5= 5,4 ± 0,3 mM) ocorreram através de interações sítio-sítio. Também foi observado que na presença de concentrações crescentes de ions amonio, a estimulação da atividade (Na+,K+)-ATPase pelo íons potássio acarretou um aumento de 50% na atividade específica da enzima. A ouabaína inibiu cerca de 86% a atividade (Na+,K+)-ATPase com Ki= 74,5 ?M, sugerindo a presença de 14% de fosfatases e/ou outras ATPase contaminantes. Este é o primeiro trabalho onde se observa uma estimulação sinergística da atividade K-fosfatase da (Na+,K+)-ATPase de crustáceo pelos íons potássio e amônio. Os resultados cinéticos obtidos para a (Na+,K+)-ATPase branquial de Callinectes ornatus, analisados em conjunto com os já descritos para outras espécies de crustáceos poderão abrir novas perspectivas em relação ao papel dessa enzima na adaptação fisiológica-bioquímica, bem como para a sobrevivência desses animais em diferentes ambientes.
(Na+, K+)-ATPase present on branchial tissue osmoregulatory crustaceans is an essential component of their osmotic and ionic regulation system. Apparently, this enzyme also have a relevant role in the active excretion de NH4+ through the branchial crustacean tissue. A (Na+, K+) ATPase-rich microsomal fraction was prepared by differential centrifugation from Callinectes ornatus homogenized branchial tissue. The sucrose gradient sucrose centrifugation showed the presence of a single protein peak with ATPase activity, and SDS-PAGE revealed the presence of several proteins bands. The use of the 5 monoclonal antibody, against the ? subunit, revealed the presence of a unique protein band of 110 kDa corresponding to the (Na+, K+) ATPase. (Na+, K+) ATPase hydrolyzed the PNPP (V= 52.0 2.0 U/mg and K0.5= 1.1 0.1 mM) through the site-site interactions (nH= 1.6). The modulation of (Na+, K+) ATPase by magnesium (V= 52.3 1.3 U/mg and K0.5= 1.1 ? 0.05 mM), potassium (V= 51.4 1.5 U/mg and K0.5= 2.3 0.1 mM) and ammonia ions (V= 56.7 2.7 U/mg and K0.5= 9.8 ? 0.4 mM) followed cooperative kinetics. However, sodium ions inhibited PNPPase activity of (Na+, K+)?ATPase with Ki= 1.7 0.1 mM. Ouabain also inhibited up to 80% the total activity PNPPase independent of the presence of ammonium ions. The hydrolysis of ATP by (Na+, K+) ATPase followed Michaelis-Menten kinetics with Km= 0.16 0.01 mM and V= 116.3 5.6 U/mg, while enzyme modulation by magnesium (V= 111.0 5.4 U/mg and K0.5= 0.54 0.03 mM), sodium (V= 110.6 5.3 U/mg and K0.5= 6.3 0.3 mM), potassium (V= 116.0 5.5 U/mg and K0.5= 1.5 ? 0.1 mM) and ammonium ions (V= 173.3 . Interestingly, the stimulation of (Na+, K+)-ATPase activity by potassium ions in the presence of increasing concentration of ammonium ions to K+ resulted in a 50% higher specific activity. Ouabain inhibited approximately 86% the activity (Na+, K+) ATPase with (Ki= 74.5 M), suggesting the presence of about 14% of phosphatases and/or other ATPases. This is the first work showing synergistic stimulation of crustacean (Na+, K+) ATPase by potassium and ammonium ions when PNPP is used a substrate. The results reported herein for Callinectes ornatus branchial (Na+, K+) ATPase might open new perspectives concerning the physiological adaption and the survival of these animals in different environmental.
