Calcium-stimulated ATPase of dog submandibular gland

Arch, o).uiBwl.Vol. 19. pp. 13 to 16. PergamonPress. 1974 Printedm Grrat Brltam

CALCIUM-STIMULATED SUBMANDIBULAR

ATPase OF DOG GLAND

EILEEN L. WATSON, K. T. IZUTSU

Departments

and I. A.

SIEGEL

of Oral Biology and Pharmacology and Centre for Research in Oral Biology, University of Washington, Seattle, Washington 98195, U.S.A.

Summary-A Ca2- -stimulated ATPase present in a microsomal fraction prepared from canine submandibular glands was investigated. The CaZt concentration for half maximal activation of the enzyme was about 0.3 mM. Addition of Mg’+ to incubation media containing Ca ‘+ decreased the ATPase activity. of the enzyme. Also, Ca2+ will not The presence of neither Na’ nor K’ is required for Ca’+ -activation substitute for Mg* + in the Mg2+ -dependent (Na+ + K+)-ATPase reaction. The Ca’+-activation was not appreciably affected by ouabain (10-4M), but was inhibited by about 50 per cent by 5 x 10e3 M ethacrynic acid. These studies provide a possible enzymatic basis for the calcium uptake by salivary gland microsomes that has been reported by other workers.

INTRODUCTION

The (Na+ + K+)-ATPase activity was determined in a medium which contained 0.1 ml of microsomal suspension (approx. 005-0.1 mg of protein), 80 mM NaCl, 15 mM KCl, O-10 mM MgCl,, O-10 mM CaCl,, 4 mM ethyleneglycolbis-(B-aminoethyl ether) N,N’-tetraacetic acid (EGTA), and 30 mM Tris-HCl at a pH of 7.1. Magnesium-dependent (Na+ + K+)-ATPase activity was calculated as the difference between activities in the presence and absence of ouabain. Ca’+-ATPase, was calculated as the difference produced by the addition of Ca’+ either in the presence of ouabain, when Na+ and Kf were replaced by choline, or when K’ was replaced by Na’. Tris-ATP (3 mM) was added to start each reaction and the mixtures were incubated at 37°C for 1 hr. Under these conditions, ATP hydrolysis was a linear function of incubation time and enzyme concentration The enzyme reaction was terminated by the addition of I.0 ml of I.5 M HCIO,. After the removal of the precipitated protein by centrifugation, an ahquot of the incubation medium was assayed for inorganic phosphate by the method of Fiske and SubbaRow (1925) with correction by means of appropriate blanks for non-enzymatic hydrolysis. Enzymatic activity was expressed as Atmoles of inorganic phosphate (P,) liberated per mg protein in 1 hr. The enzyme preparations were assayed for protein content according to the method of Lowry et al. (1951). The succinate dehydrogenase (SDH) activities of the NaI-treated and untreated microsomes were measured relative to the mitochondrial (14,000 g) fraction in a Gilford recording spectrophotometer using an indophenol method of Green, Mii and Kahout (1955). The SDH activity of the NaI-treated microsomes was less than 1 per cent of the mitochondrial fraction, In contrast, the SDH activity of untreated microsomes was 90 per cent of that found in the 14,000 g fraction.

A calcium-stimulated ATPase has been found in various tissues in which active calcium (Ca’+) transport occurs. These tissues include red blood cells (Schatzmann and Vincenzi, 1969), intestinal mucosa (Martin, Melancon and DeLuca, 1969) and sarcoplasmic reticulum (Hasselbach and Makinose, 1963). In addition, ATP-dependent Ca2 + uptake occurs in microsomes prepared from skeletal muscle (Ebashi and Lipmann, 1962), cardiac muscle (Katz and Repke, 1967) and nervous tissue (Otsuka, Ohtsuki and Ebashi, 1965). Such uptake is generally believed to represent an active transport mechanism for Ca’+. A similar uptake process has recently been reported for submandibular gland microsomes (Alonso et al., 1971). Although a Ca’+-stimulated ATPase activity has been reported for other microsomal calcium pumps (Katz and Repke, 1967; Hasselbach, 1964). such activity has not been previously demonstrated for salivary gland microsomes. In this study, we report the existence of a Ca’+stimulated ATPase in dog submandibular microsomes. METHODS

The microsomal fraction was prepared according to methods described in detail by Hall, Siegel and Izutsu (1972). The minced glands were first homogenized in a Waring blender for 60 set with 9 volumes (w/v) of an ice-cold solution containing 0.1 per cent desoxycholate (DOC), 250 mM sucrose and 1 mM EDTA (pH 7.3). This homogenate was centrifuged at 14,000 g for 20 min. The supernatant was then centrifuged at 105,000 g for I hr. The resulting pellet was resuspended in 6 volumes (w/v) of 0.05 per cent DOC, 250 mM sucrose and 1 mM EDTA (pH 7.3). The centrifugation procedures were then repeated and the final pellet was suspended in a volume of I mM EDTA (pH 7.2) solution equal to the original tissue weight (ml/g). Half of this suspension was treated with 2 M NaI (Usegi et al.. 1969) and both halves were then washed three times and resuspended in X0 mM sucrose

RESULTS

Properties

of the calcium-stimulated

The effects of varying the calcium the ATPase activity of both untreated 13

All’asr concentration on and Nal-treated

14

Eileen L. Watson.

K. T. lrutsu

microsomrs are shon n in Table I. As the calcium concentration is increased from 0 to 3 mM, calcium activation of the untreated preparation rises. Further elevation of the calcium concentration to 10 M has no significant effect on the activity of this preparation. The maximal activity of the NaI-treated preparation was reached at a calcium concentration of 1 mM. Incrcasing the calcium concentration to 10 mM either had no effect or perhaps resulted in a slight decrease in ATPasc activity. The maximal activities of the untreated and treated prepat-ations were similar.

Figure 1A shows the rates of inorganic phosphate production from Nal-treated microsomes when both magnesium and calcium are present in the incubation medium. The upper curve shows the effect of calcium alone. Increasing the concentration of magnesium (Fig. 1A, lower 3 curves) resulted in a decreased ATPase activity. At a magnesium concentration of 3 mM, the activity approached that determined in the presence of magnesium alone. & ‘51

0

2 4 6 8 [CO’+] mM

B

1

A

IO

0

2

4

[Mg2’]

6

8

and I. A. Siegel

magnesium concentration of 3 mM the ATPase activities of the untreated and treated preparations were 7.5 and 13.X,nM phosphate x mg protein ’ x hr- II respectimely

The calcium-stimulated treated and NaI-treated

ATPase activity of both unmicrosomes did not require

the prcsencc of other ions. Similar specific activities were obtained Mhen the reaction was performed in the presence of sodium but not potassium, complete replacement of both sodium and potassium by choline, and in the presence of sodium, potassium and 10m4 M ouabain (Table 2).

The increments of ATPase activity brought about by addition of 80 mM Na+ and 15 mM K’ to incubation media containing 0.3 and 3 mM Mg*+ are shown in Table 3. In contrast. the same concentrations of Ca’* are relatively ineffective in stimulating the (Na+ + K+)-ATPase. Addition ofCa*+ (2 x 1O--5 M) to incubation media containing Mg’_ Na’ and K+ resulted in a 40 per cent inhibition of the Mg*+-dependent (Na+ + K * )-ATPase activity.

IO

mM

Fig. I. The effects of C‘a’ _ on ATP hydrolysis in the presence ofvarying concentrationsof Mg2+ (A). Results are expressed as /~moles Pi liberated per mg protein in 1 hr. Stimulation by Ca’+ was measured in the presence of: 0 mM Mg’+ (-0). 0.3 mM Mg’ ’ (+--0). 1 mM Mg’+ (m---m) and 3 mM Mg’+ (U-0). The effects of Mg’+ on ATP hydrolysis (B). Results are cxpressed as pmoles P, per mg protein in I hr.

The effects of magnesium on the untreated preparation were similar to those on the NaI-treated preparation. Although the calcium stimulation ofthe treated and untreated preparations were similar, the stimulation of the untreated preparation by magnesium exceeded that of the Nal-treated preparation. At a

The effects of ouabain (10 4 M) and ethacrynic acid (5 x lo- 3 M) were tested on the stimulation brought about by Ca*+ alone and by MgZ + alone. The divalent ion concentrations added were 10m3 M, 2 x 10e5 M and 6 x lo-” M. At each concentration ouabain did

Ca’ --ATPase

of salivary

not affect either the Ca” or the Mg’+ stimulation. In contrast, ethacrynic acid reduced the Ca2+ stimulation by about 50 per cent at each of the three calcium concentrations and decreased the Mg2+ stimulation by about 90 per cent at each of the three concentrations.

glands

rogenase uptake.

activity,

15 provide

an enzymatic

bases

for this

Ackno~oledye/nmt~This work was supported by PHS grant number DE02600 from the National Institute of Dental Research.

DISCUSSION

Previous studies indicate that rat parotid and submandibular glands demonstrate ATP-dependent calcium uptake (Selinger, Naim and Lasser. 1970). Although a Ca” -dependent ATPase activity has been described for the microsomal pump of various tissues (Hasselbach, 1964). such ATPase activity has not been observed in salivary microsomal preparations (Alonso et ul., 1971). The present study describes an ATPase activity in dog submandibular microsomes that is stimulated by Ca 2+ ions. The Ca’+ stimulation is consistently greater than Mg* + stimulation. Recently, Shami and Radde (1971) described an enzyme in placental plasma membranes that is also preferentially stimulated by Ca’ ’ ions. In both studies, increasing concentrations of Mg’- ions decreased the activity of Ca”-ATPase. In our study Ca” did not substitute for Mg ‘+ in activating (Na* + K+)-ATPase. The (Na+ + K+)ATPase activitv when Ca’+ was employed in the incubation medium was less than 5 per cent of the (Na+ + K+)-ATPase activity found when Mg’+ was employed. This was true of both NaI-treated and untreated preparations. Our observations do not permit one to state whether there are different sites for Ca*’ and Mg’ + ions or whether these ions are acting on a similar site. Shami and Radde (1972) suggest the latter possibility for placental ATPase activity. The Ca’+ activity was essentially equal in both Nal-treated and untreated microsomes. However, Ca’+ stimulation of untreated microsomes was consistently less than Mg’+ stimulation in untreated preparations, whereas Ca’ ’ stimulation was greater than Mg’+ stimulation in NaI-treated preparations. Treatment of microsomes prepared from dog submandibular gland with NaI has previously been shown to alter the activity of Mg’+-ATPase (Hall et al.. 1972) and HCOY-ATPase (Izutsu and Siegel, 1972). Presumably the NaI treatment affects mitochondrial contamination of the preparation because this treatment drastically reduces succinate dehydrogenase activity. We suggest that the reason the untreated preparation displays greater Mg’& activity than Ca” activity when compared to the NaI-treated preparation is because of mitochondrial activity in the untreated microsomes. At pi-escnt the physiological significance of this enzyme is obscure. Presumably the enzyme is involved with calcium uptake by microsomes. It has been suggested that microsomal uptake of Ca’+ from the cytoplasm is involved in returning a secreting salivary gland to its resting state (Alonso rf al.. 1971). Our results, which demonstrate the presence of a Ca2+-stimulated ATPase in a preparation with little mitochon-

drial contamination

as measured

by succinate

dehyd-

REFERENCES

Alonso G. L.. Bazerque P. M., Arrigo D. M. and Tumilasci 0. R. 1971. Adenosine triphosphate-dependent calcium uptake by rat submaxillary gland microsomes. J. yen. Physiof. 58, 340-350. Ebashi S. and Lipmann F. 1962. Adenosine triphosphatelinked concentration of calcium ions in a particulate fraction of rabbit muscle. J. Cell Biol. 14, 389-400. Fiske C. H. and SubbaRow Y. 1925. The calorimetric determination of phosphorus. Biol. Cheln. 66, 375-401. Green D. E., Mii S. and Kahout P. M. 1955. Studies on the terminal electron transport system. J. biol. Chern. 217, 55 I-567. Hall S. H.. Siegel 1. A. and Izutsu K. T. 1972. (Na+ + K+)ATPase activity in the dog submandibular gland. Archs oral Biol. 17, 1737-1744. Hasselbach W. and Makinose M. 1963. Uber den Mechanismus des Calciumtransportes durch die Membranen dcs sarkoplasmatischen Reticulums. Bioc,horl. Z. 339,94-l I I. Hasselbach W. 1964. Relaxing factor and the relaxation of muscle. Mol. Biol. 14, 169-222. Izutsu K. T. and Siege1 I. A. 1972. A microsomal HCOYstimulated ATPase from the dog submandibular gland. Biochim

hiophys.

Acta 284, 478-484.

Katz A. M. and Repke D. I. 1967. Quantitative

aspects of dog cardiac microsomal calcium binding and calcium uptake. Circulation Rrs. 21, 153-162. Lowry 0. H.. Rosebrough N. J., Farr A. L. and Randall R. J. 1951. Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. Makinose M. 1969. The phosphorylation of the membranal protein of the sarcoplasmic vesicles during active calcium transport. Europ. J. Biochem. 10, 74-82. Martin D. L.. Melancon M. J., Jr. and DeLuca H. F. 1969. Vitamin D-stimulated, calcium-dependent, adenosine triphosphatase from brush borders of rat small intestine. Biochrm. Biophys. Rex Corntnun. 35, 819-823. Otsuka M.. Ohtsuki 1. and Ebashi S. 1965. ATP-dependent calcium binding of brain microsomes. J. Biochenz. 58, 107-188. Schatzmann H. J. and Vincenzi F. F. 1969. Calcium movements across the membranes of human red cells. J. PhyGo/., Land. 201, 369--395. Selinger Z.. Naim E. and Lasser M. 1970. Adenosine triphosphate-dependent calcium uptake by microsomal preparations from rat parotid and submaxillary . _ &nds. Bio&itn.

hiophy.$. Ac,rct’203, 326334.

Shami Y. and Radde ATPase of guinea-pig

1. C. 1971. Calcium-stimulated placenta. Biochim hiophys. Acta

249,345S352.

Shami Y. and Radde I. C. 1972. Effect of the Ca’+iMg’+ concentration ratio on placental (Ca’+ + Mg”)-ATPase activity. Biochim. biophys. Actu 255, 675.~679. Usegi S.. Kahlenberg A., Medzihradskv F. and Hokin L. E. 1969. Studies on the characteriration of the sodiumpotassium transport adenosine triphosphatase. Archs. hiochirn.

Biophys.

130, 156-163.

16

Eileen L. Watson.

K. T. Ii-u&u and 1. A. Siegel

prtsente dans la fraction microsomiale de glandes sousRizumkGUnr ATPase stimult-e par le Ca”, maxilla& de chiens, a 6tC 6tudiCe. La concentration de Ca’- nCcessaire pour obtenir une activation contenant maximale de moitie de I’enryme cst de 0,3 mM. L’adjonction de Mg’+ au milieu d’incubation le Ca” diminuc l’activitt- ATPasique. La prksence de Na+ ou dc K+ n’est pas nCcessaire pour l’activation Ca” de l’enzyme. Cal+ nc se substitue pas $ Mg’+ dans la r&action MgZi-dt-pendante (Na* + K+)affect&e par l’ouabainc (10e4M). mais est inhibCe ATpase. L’actlvation Ca’+ n’est pas spkcialement Ces etudes suggkrent une explication d’environ 50 pour cent par 5 x 10 ’ M d’acide ithacrynique. enrqmatique d I’incorporation de calcium par les microsome, des glandes salivaires notie par d’autres auteurs. ATPase untersucht. die in einer aus SubZusammenfassung-Es wurde eine durch Ca’ A aktivierte mandibulardrtisen vom Hund prgparierten mikrosomalen Fraktion vorhanden war. Die zur halbmaxiwar etwa 0,3 mM. Der Zusatr von malen Aktivierung des Enzyms erforderliche Ca”- Konzentration cnthaltenden Medien senkte die ATPase-Aktivitlt, Zur Aktivierung des Enzyms mit Mg2- IU Ca” sein. Auch kann Ca” nicht Mg’+ bei der Mg’*Cal+ mu0 cntweder Na+ oder K’ vorhanden wurde anscheinend nicht abhangigen (Na’ + K +)-ATPase-Reaktion ersetzen. Die Ca’ ’ -Aktivierung durch Ouabain (g-Strophanthin) beeinfluflt. im Gegensab zu einer etwa 50 Prozentigen Hemmung durch 5 x IO-’ M Ethacrylssure. Diese Untersuchungen schaffen eine Vorstellung iiber den enzymatischen Mechanismus der Kaiziumaufnahme durch Mikrosomen der Speicheldriise, wie sie von anderen Untersuchern beschrieben wurde.