European Journal of Phycology

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A comparative study of alkaline phosphatase

activity in two species of Gelidium (Gelidiales,
Rhodophyta)

I. Hernández , J.A. Fernández & F.X. Niell

To cite this article: I. Hernández , J.A. Fernández & F.X. Niell (1995) A comparative study of
alkaline phosphatase activity in two species of Gelidium (Gelidiales, Rhodophyta), European
Journal of Phycology, 30:1, 69-77, DOI: 10.1080/09670269500650811

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Published online: 23 Feb 2007.

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 Eur. J. Phycol. (1995),30: 69-77. Printedin GreatBritain 69

A comparative study of alkaline phosphatase activity

in two species of Gelidium (Gelidiales, Rhodophyta)

I. H E R N A N D E Z 1, J. A. F E R N A N D E Z 2 A N D F. X. NIELL 3

i Departamento de Ecolog&, Facultad de Ciencias del Mar, Universidad de Cadiz, E-II510 Puerto Real, Cddiz, Spain
2 Departamento de Fisiologfa Vegetal, Universidad de Mdlaga, Campus Universitario de Teatinos, E-29071 Mdlaga, Spain
3 Departamento de Ecologfa, Universidad de Mdlaga, Campus Universitario de Teatinos, E-29071 Mdlaga, Spain

(Received I1 October 1993; revised and accepted 20 July 1994)

A comparative study of alkaline phosphatase activity (APA) was carried out on repeat samples of representativepopulations of two
species of Gelidium (Rhodophyta), G. latifoliurn and G. sesquipedale, using p-nitrophenylphosphate (pNPP) as substrate. The APA kinetics
of both species exhibited an apparent negative cooperativity for the hydrolysis of pNPP. Differenceswere found in phosphatase activities
of the two species: G. latifolium showed higher APA per unit biomass and a higher proportion of the phosphate cleaved from the model
substrate than G. sesquipedale. Temperature,pH, salinity and external phosphate all had significanteffectson the APA of both species. The
influence of salinity on APA was due in part to the effectsof specificions (Na+ and Mg2+). No correlationbetween photon irradiance
and APA was observed in G. latifolium, but in G. sesquipedale maximum APA was observed at a relatively low photon [Link] of
the two species is discussed with respect to a possible relationship with ecophysiologicalconditions in nature.

Key words: Alkaline phosphatase, Gelidium latifolium, Gelidium sesquipedale, light, osmotic effects,phosphorus.

Introduction of two species of Gelidium, the most important raw

material for the extraction of agar in Spain. The main
Phosphorus is generally not considered to be a limiting
species growing in southern Spain were selected: G.
nutrient in the marine environment (Goldman et al., 1979;
latifolium in the Mediterranean and G. sesquipedale in the
Lobban et al., 1985). Increasingly, however, evidence has
Atlantic. Along our coasts these two species live under
been reported for the role of phosphate as a regulator of
different and well-defined conditions, with differing
primary production in a diversity of marine environments,
environmental factors such as wave action, water tem-
particularly coastal waters (Rivkin & Swift, 1985;
perature, phosphate concentration and photon irradiance.
Sakshaug & Olsen, 1986; Hern~ndez et aI., 1993a).
Therefore, APA was compared in the two species, giving
Many algae respond to conditions of limiting inorganic
particular attention to the influence of environmental
phosphorus by producing non-specific alkaline phospha-
factors important in the field.
rases (Kuenzler & Perras, 1965; Siuda, 1984). These
enzymes cleave inorganic phosphate groups from phos-
phomonoesters (PME), making phosphorus available to Materials and methods
the algae (Cembella et al., 1983). The origins of PME,
Seaweeds and sampling sites
which contribute some of the dissolved organic phos-
phorus compounds (DOP), are diverse (van Boekel, 1991), Gelidium sesquipedale (Clemente) Bornet et Thuret, a
so the reported concentrations of PME and DOP in species of Atlantic distribution, was collected from the
natural waters are variable (Chr6st et al., 1984; Atkin- low intertidal at Punta Carnero, a cape in the area of the
son, 1987, Veldhuis et al., 1987; Herndmdez et al., 1993b). Straits of Gibraltar. We found the specimens throughout
Moreover, short-term pulses of DOP are probably quite the year in limestone crevices of the low intertidal zone,
common in many natural environments (Islam & Whitton, where photon irrafliance was low (maximum = 140-}-
1992) and may be quickly utilised by the algae. 68 #mol photon m -2 s-l; Torres et al., 1991). Gelidium
The alkaline phosphatase activity (APA) of species from latifolium (Greville) Thuret et Bornet was collected at
different groups of macroalgae has received increasing Maro, a non-tidal rocky shore situated on the eastern
attention (Lapointe & O'Connell, 1989; Weich & Gran61i, coast of M~laga. This species was found from January to
1989; Hem~dez et al., 1993b) since Walther & Fries (1976) October. It forms dense fringes on rocks, between the
first identified this enzyme from macroalgae. Recently, high lower limit of the littoral zone and the upper limit of the
APA in Porphyra umbilicalis (L.) Kfitzing has been related to sublittoral zone. Maro is characterised by clearer waters
phosphorus-limited growth (Hern~dez et al., 1993a). and lower algal production than Punta Carnero (Aranfla
The aim of the present study was to estimate the APA et al., 1984; C. Jim6nez, unpublished data).

Published online 23 Feb 2007

I. Herndndez, ]. A. Ferndndez and F. X. Niell 70

After collection, plants were kept cool in darkness and maximum velocity by extrapolation of the high-substrate
transported to the laboratory in an icebox. Macrophytes portion of the plot. The apparent half-saturation constants
were generally free of visible epiphytes and, if necessary, (Ks) were obtained from the Eadie-Hofstee transforma-
the epiphytes were removed carefully with forceps. tion (v vs v/S), since in this plot the distribution of errors
Finally, to minimise any possible acclimation to labora- is more uniform (Price & Stevens, 1982). pNPP concen-
tory conditions, plants were maintained for a short time trations ranged from 7 x I 0 - 6 M to 4"9 x 10-3M.
(24 h maximum, except in the experiments on phosphorus Apparent cooperativity was tested from the slope of a
enrichment) in frequently changed, aerated, filtered nat- Hill plot [log v / ( V m a x -v)/log S].
ural seawater (pH 8"2) at a constant temperature (15°C) The temperature response of APA in both species was
and a photon irradiance of 150#mol photon m -I s-I determined at pH8"3 from 10 to 30°C. Temperature-
(Sylvania F18W/GRO tube). dependent changes in pH from 8"3 were compensated for
by the addition of Tris or HC1. Parameters concerning the
influence of temperature on the enzymatic reaction were
Alkaline phosphatase activity assay
calculated from the Arrhenius equation (Price & Stevens,
Alkaline phosphatase is evident in the outer cortical cells or 1982). The effect of pH on enzymatic activity was tested
the external cell walls of these species, as was demonstrated using different Tris-HCl buffers at 0"25 pH unit intervals
by a histochemical stain (Hernfindez et al., 1994a). Surface between 7 and 9. Different salinities of the assay medium
alkaline phosphatase was assayed colorimetrically in three were obtained by making up solutions of artificial sea-
replicate samples by the method of Reichardt et al. (1967), water from 0 salinity (distilled water) to 52"5, varying
which uses p-nitrophenyl phosphate (pNPP) as substrate, as quantities of all reagents in the artificial seawater. The
modified by Hern~indez et al. (1992). Plants were incubated effects of C1-, Na + and Mg 2+ on APA were determined
in 100 ml of a reaction mixture consisting of 700 #M pNPP by replacing some ions/molecules of the artificial sea-
(Sigma), 50raM Tris-HC1 buffer, pH 8"3. The initial sub- water (NaC1, MgC12 and MgSO4) with mannitoL adding
strafe concentration was sufficiently high to ensure that no the necessary quantity to keep the osmotic pressure
more than 10% was hydrolysed during assay. Reagents constant. The equivalence between concentration and
were dissolved in filtered (0"2#m) artificial seawater of 35 osmotic pressure was taken from Wolf et al. (1982) and
salinity (practical salinity units) (Kalle, 1945). Air was checked with a cryoscopic osmometer (Gonotec Osmo-
bubbled continuously during the assay to oxygenate and mat 030).
supply CO2, and to stir the medium. After 45 min of To test the relationship between photon irradiance and
incubation at 25°C the absorbance was read at 410nm APA, the plants were assayed in the incubation chamber
against a blank (buffer and substrate solution without under illumination with a light source (a 150W solar
plants) in a Beckman DU-7 spectrophotometer. Activity lamp) in which different light intensity selective filters
is reported as #mol paranitrophenol (pNP) released g dry were inserted. The mean values obtained in the following
wt -~ h -z, formed by the hydrolysis of pNPP to phos- subranges of light intensities (in #mol photon m -2 s-I)
phorus (P) and pNP. were considered: 0, 0-10, 10-20, 20-50, 50-100, 100-
To estimate the proportion of the phosphate liberated 200, 200-500, 500-1000, 1000-2000. The effect of
enzymatically that was taken up by the algae in relation external phosphate concentration on APA was studied
to the pNP liberated, at different times (15, 30, 45, by pre-incubating the plants for 5 days in 400ml of
60min) 2"5 ml of the assay medium was sampled to natural seawater enriched with different quantities of
determine P and APA. P was quantified as soluble Na2HPO 4 as a source of orthophosphate. The final
reactive phosphorus (SRP) by the molybdenum blue concentration of P added ranged from 0 to 20 #M.
method (Murphy & Riley, 1962). We have assumed
that all the pNP from pNPP hydrolysis is released into
Statistical analyses
the medium. According to previous research with
glucose-6-phosphate as a substrate for alkaline phospha- For each treatment, differences among means were tested
tase (Hern~indez et aI., unpublished data), any possible by a single factor analysis of variance (ANOVA). Multi-
retention of pNP by the algae can be considered ple post hoc comparisons were performed by the Tukey-
negligible. The non-enzymatic hydrolysis of pNPP by Kramer test (Zar, 1984). Differences of the pNP and SRP
acid conditions can also be assumed to be negligible concentrations during the enzymatic assays were tested
(McComb et al., 1979). Finally the absence of a third for significance by a test of equality of slopes (Zar, 1984).
product, acceptor phosphate (phosphoethanolamine; In all cases the null hypothesis was rejected at the 5%
Wilson et al., 1964), was proved by the absence of any significance level.
differences between two sets of P standards, one using
distilled water and the other using buffered artificial
Results
seawater as a solvent.
Apparent kinetic parameters were studied from two
pNP and P concentrations during the assay
transformations of the Michaelis-Menten equation. The
Lineweaver-Burk transformation (1/v vs 1/S) provided Fig. 1 shows the pNP and SRP concentrations (in #M g
Phosphatase activity in Gelidium 71

300" (a) . ~
pNP = 25.2+ 4
"7,
200

~0.04 ];
100"

P=16+2t r2= 0.98

0 i • i • i • i • i • i

0 10 20 30 40 50 60
t (rain)
~ 0.02~ ~>o~112!6~7 ~ . _2

4O (b)
0 10 20
30 pNP = - 0.03 + 0.58t r 2 0.99 J=
-1
1/[pNPP] ( r a m )

2" (t,)
10

,.=
0 10 20 30 40 50 60
t (min)

Fig. 1. Concentrationsof the two products of the pNPP ~0

hydrolysis - pNP (open circles)and SRP (filledcircles) (in/zM g
dry wt-~) - measuredduring the assay of alkalinephosphatase
activity of Gelidium latifolium (a) and Gelidium sesquipedale (b).
Values are means of three replicates and SD was negligible.

dry wt -I) measured in the medium during the assay of

APA. The slopes of the regression lines for both products -5.5 -4~5 -3.5 -Z5
log [pNPP]
of the enzyme reaction were significantly different. The 0 i i i

difference between both hydrolysis products was consid- 0 50 100 150

ered to be the percentage of P taken up by the algae after -1
pNPP cleavage, assuming that all the pNP is released into 1/[pNPP] (raM)
the medium. This proportion was higher in Gelidium Fig. 2. Double reciprocal plot showing the dependenceof the
latifolium (56%) than in Gelidium sesquipedale (35%). velocity of hydrolysisof pNPP on substrate [Link]
apparent negative cooperativitywas suggestedby the two
straight lines drawn in the [Link], replot of the data
Kinetic parameters of APA according to the Hill [Link] point represents the
mean of three experimentson (a) Gelidium latifolium and (b)
The Lineweaver-Burk transformation of a Michaelis-
Gelidium [Link] is expressed as #tool
Menten plot shows the dependence of APA on substrate pNP g dry wt-~ h-I and substrate concentrationas mM pNPP.
concentration (Fig. 2). This non-linear reciprocal plot
suggested the existence of a negative cooperativity for
the hydrolysis of pNPP. Two apparent Ks values for centrations, similar to those observed in the sampling
pNPP can be deduced from the Eadie-Hofstee plot (Table sites (Herndmdez et al., I993b), the apparent Ks value of
1) and can be interpreted in term of apparent negative higher affinity showed a V max/Ks ratio 2-fold higher in
cooperativity as deduced from the slope of the Hill plot, G. latifolium than in G. sesquipedale (Table 1).
the Hill coefficient nH. This coefficient was nH = 0"72
(r2 : 0"97, p < 0"001) in G. latifolium and nH = 0"47
Temperature and pH effects
(r2 = 0"91, p < 0"02) in G. sesquipedale (Fig. 2).
The ratio V m a x / K s may be a better index of Temperature showed a significant effect on A P A in both
competition in PME use than is Km alone, as Healey species of Gelidium (Fig. 3). APA increased from 5 to
(1982) stated for phosphate uptake. At low PME con- 25°C, but decreased slightly with temperature above
L Herndndez, ]. A. Ferndndez and F. X. Niell 72

Table 1. Apparent semi-saturation constants (Ks in/zM) 50 (a)

'7,
exhibited by alkaline phosphatase from Gelidium latifolium and
Gelidium sesquipedale "7, 40-

30
Species KsI Ksz V max V max ~Ks1
20
Gelidiumlatifolium 118 1748 110"8 0"93
Gelidiumsesquipedale 9 385 3"8 0"43
10"

Vmax (#mol pNP g d r y w t - I h-I) was calculatedfrom the double

1 0 . . . . ~ . . . . ! . . . . ,! . . . . ,! . . . . .! . . . . .~

reciprocalplot (see text). Ks~ and Ks2 refer to the "high" and "low" 6.5 7.0 7.5 8.0 8.5 9.0 9.5
affinityphase of the [Link] ratio V max/Kin pH
was calculatedusing the apparenthigh affinityconstant (Ks~).

25°C. For this reason, data were fitted to the second-order

polynomial, with optimum temperatures for A P A of 30°C
in G. latifoliurn and 25°C in G. sesquipedale. O n the other
hand, over the range in which a linear increase of A P A 12 (b)
with temperature was found (I0-25°C), the Arrhenius
plot (log V m a x = Ea/R x 1 / T + constant) revealed an '7, 10

6"

4"

2"

0 . . . . i . . . . i . . . . ! . . . . | . . . . i . . . . |

6.5 7".0 7.5 8.0 8.5 9.0 9.5

1
pH

1o
Fig. 4. pH profiles of alkaline phosphatase activity of Gelidium
0 i i i i
latifolium (a) and Gelidiumsesquipedale(b) (temperature 25°C).
0 10 20 30 40 Enzymatic activity is expressed as #tool pNP g dry wt - I h -1.
Temperature (°C) Values are means of three replicates and bars denote SD.

activation energy (Ea) of 41"6 kJ mo1-1 for G. latifolium

and 35'8 kJ mol - I for G. sesquipedale, giving, respectively,
Qi0 values of 1'7 and 1"6.
The rate at which G. latifolium and G, sesquipedale
25- Co) hydrolysed pNPP was significantly dependent on pH
(Fig. 4). In the range assayed, maximum activity for G.
~, 20-
.
latifolium (46"4 ± 8"8#mol pNP g dry wt -1 h -1) was
.~ 15- found at the highest pH tested (pH9) whereas in G.
~ - sesquipedale maximum A P A (84"4 q- 2"3 # m o l pNP g dry
~ 10- wt -~ h -s) was achieved at p H 8'8.

5-
Salinity and light effects
0 i i i i
The effects of salinity on A P A showed a similar pattern in
0 10 20 30 4O
both species (Fig. 5). Plants assayed in distilled water and
Temperature (°C) low salinities exhibited very low activities. The activity
Fig. 3. Alkaline phosphatase activity as a function of temperature increased with salinity from distilled water to a maximum
(pH 83). Data were fitted to second-order polynomials: A P A at 43"8 salinity, which resulted in 37-4 # m o l pNP g
A P A = -5-910 -z T2 + 3"6T - 12"9 (rz ----0"56,p < 0'005) in dry wt - I h - I for G. latifolium and 7'2 # m o l pNP g dry
Gelidium latifoliurn(a) and APA = - 4 ' 0 I 0 - 2 T2 + 2"03T - 7"5
wt -1 h -~ for G, sesquipedale. Higher salinities caused a
(r2 = 0"68, p < 0"025) in Getidium sesqnipedale(b). Equations are
illustrated by the curves. Enzymatic activity is expressed as #mol decrease in APA.
pNP g dry wt -I h -~. Values are means of three replicates and However, increased salinity has an osmotic effect as
bars denote SD. well as an increase in the specific ion concentrations. The
Phosphatase activity in Gelidium 73

45' (a) Table 2. Percentage of alkaline phosphatase activity observed in

'7, Gelidium latifolium and Gelidium sesquipedale after the replacement
'7, of NaC1, MgCl2 and MgSO4 by mannitol in the artificial
seawater used in the assay medium
30

-NaCI, -MgC[2, -IgIgC12,-MgSO4,

15' Species + mannitol + mannitol + mannitol

Getidium latifolium 54-4* 103"5 55"6*

Gelidiumsesquipedale 54"9* 100 62"3~
0 I i | i i i

0 10 20 30 40 50 60
Salinity Values are referred to 100%of activity in the control (plants assayedin
standard conditions). Values are means of three replicates.
*Decrease significantat the 5% level.

Effect of added phosphate on A P A

8"

'7, When phosphate was added to the cultures up to a

concentration of 20 #M, a gradual decline in A P A was
'7,
6- observed (Fig. 8). Maximum A P A occurred when plants
were incubated for 5 days in seawater without added
4¸

~, 70 (a)
"~ 60
0 i i ! i i

0 10 20 30 40 50 60 50
Salinity 40
~ 30
Fig. $. Influence of salinity on alkaline phosphatase activity of
~ 20
Gelidium lah'folium (a) and Gelidium sesquipedale (b) (pH 8"3;
temperature 25°C). Enzymatic activity is expressed as/zmol pNP lO
g dry wt -1 h -I. Values are means of three replicates and bars 0 i i i i ~ ! i

denote SD. 0 200 400 600 800 1000 1200 1400

Photon irradiance (~mol photon m -2 8 -1)
use of a non-electrolytic molecule (mannitol) permitted
the two effects to be separated. A significant decrease in
A P A was observed in both species after the substitution
of mannitol for NaCI (Table 2) compared with the control
(plant assayed in standard conditions). The replacement of
MgC12 did not cause any significant effects. Nonetheless, '7, 30 (b)
the replacement of all the magnesium compounds by
mannitol led to a significant decline in A P A (Table 2). '7
The effect of photon irradiance on A P A differed in the ~ 20
two species (Fig. 6). In G. latifolium, neither a significant
correlation nor any particular pattern was found between
A P A and photon irradiance. Thus, it seems that in this lO
species A P A is not influenced by photon irradiance level,
at least in a short-term incubation. However, G. sesquipe-
dale showed a more definite pattern. A P A was low in the 0 l - 1 - ! • 1 i ~

200 400 600 800 1000 1200 1400

dark but showed a significant maximum (25"4 #mol pNP
g dry wt - I h -2) at a relatively low photon irradiance Photon irradiance (~tmol photon m -2 s -1 )
(74 #mol photon m -2 s-l). A time course of the differ-
ence between light (50-100 #mol photon m -2 s -s) and Fig. 6. Alkaline phosphatase activity as a function of photon
irradiance (pH 8"3; temperature 25°C). In Gelidium latifolium (a) no
dark showed that 15rain were enough to detect a
relationship was found between the two variables, whereas in
difference in enzymatic activity (Fig. 7). Photon irra- Gelidium sesquipedale (b) a significant maximum APA (25'4 #tool
diances higher than 150 #mot photon m -z s - I led to a pNP g dry wt -~ h -1) was found at 74 #tool photon m-2 s-~.
smooth decline in phosphatase activity. Values are means of three replicates and bars denote SD.
I. Herndndez, ]. A. Ferndndez and F. X. Niell 74

30- 7O (a)
"7,
60
"7,
.~20 50
t~o
40
~0
%30 %.---
+
20
0 w • i • , • i i i
10 • i • • , , • i • , i - , , • • ! • • i

0 10 20 30 40 50 60 0 3 6 9 12 15 18 21
t (rain) P added (~tM)

Fig. 7. Gelidium sesquipedale. Time course of the difference

between pNP released (in #mol g dry wt -I) through alkaline
phosphatase activity in light (50-100/zmol photon m -2 s -1)
(open circles) and dark (filled circles). SD is shown as a bar when
it exceeds the symbol size.
'7.
"7
phosphate. The data shown in the figure were fitted to a
7-
"single exponential decay plus offset" model:

APA = APA c + APA~m~ x e -bP (1)

5~
where APA c is the APA not affected by orthophosphate
addition, APA~max is t h e apparent maximum phosphatase 4- i I
• I I
activity (i.e. the difference between the absolute APAmax 3 • . i • • i • • i • • i • • i . • i • • i

and APAc), which was achieved with no addition of 0 3 6 9 12 15 18 21

phosphate, b is the decay coefficient and P is the P added (IxM)
amount of orthophosphate added. The values of these
parameters are shown in Table 3.
Fig. 8. Rate of hydrolysis of pNPP by alkaline phosphatase in
In both cases, a strong inhibition (greater than 50%) Gelidium latifolium and Gelidium sesquipedalein media of
was recorded with low levels of added phosphate (less increasing inorganic phosphate concentration (pH 8"3;
than 6 # M phosphate). However, lower phosphate addi- temperature 25°C). Algae were incubated for 5 days in different
tions were necessary in G. latifolium than in G. sesquipedale regimes of external orthophosphate. In G. latifoliurn (a), final APA
to repress APA (Table 3). was 4Z% of the maximum rate, whereas in G. sesquipedale(b) this
percentage was 49%. Enzymatic activity is expressed as #tool
pNP g dry wt -~ h -~. Values are means of three replicates and
Discussion bars denote SD.

Both Gelidium latifolium and Gelidium sesquipedale showed

obvious alkaline phosphatase activity. The activities were In most cases, APA of the two species of Gelidium varied
always higher in G. latifoliurn than in G. sesquipedale, but similarity in response to changes in environmental vari-
were similar to values reported by Lapointe & O'ConneU ables. However, in some cases a different response was
(1989) for species from inshore waters of Bermuda. observed. Such responses may be explained by the
However, higher phosphatase activities have been ecological characteristics of the species.
reported in other Rhodophyta (Hem~adez et al., 1992). Although these differences between the two species

Table 3. Values of the parameters of the "single exponential decay plus offset" model (equation 1) fitted to the effect of added
phosphorus on alkaline phosphatase activity (APA)

APA c APA~, b FI PFI Ps0%

Species (#mol pNP g dry wt-' h -z) (#M -z) (%) (#M) (#M)

Gelidium latifolium 26.7 (1"4) 36"4 (3.4) 2"6 (0"60) 57'7 1"7 0"75
Gelidium sesquipedale 4"01 (0"41) 4"2 (0"64) 0'91 (0"30) 51"04 5"07 4"3

Values are the mean (SE).

APA,, APA not affected by orthophosphate addition; A P A ~ , apparent maximum phosphatase activity (difference between the absolute APAm~ and
APA~); b, decay coefficient; FI, percentage of final repression of APA; PFI, added phosphate required to achieve 99% of the final inhibition; Ps0o/., added
phosphate sufficient to induce 50% inhibition.
Phosphatase activity in Gelidium 75

could be genetic or environmental, we have convincing species of Gelidium (30°C in G. latifolium and 25°C in G.
reasons to suspect that they are essentially genetic in sesquipedale) were related to the temperature conditions in
nature. Sosa & Garcfa-Reina (1992) found the lowest the field. In Maro, mean temperatures fluctuate between
genetic variability described for seaweeds in subpopula- 13°C (winter) and 26"5°C (summer), whereas in Punta
tions of Gelidium arbuscula Bory (either gametophytic or Camero these fluctuations are smaller (14 to 22°C). Thus,
sporophytic) from the Canary Islands. Other data suggest the data suggest that temperature in the field probably
a relatively stable genetic composition in Gelidium reaches the optimum for A P A and could even limit
(Freshwater & Rueness, 1994; Gorostiaga, personal com- phosphatase activity, especially if G. latifolium can be
munication), G. sesquipedale being one of the least variable exposed for short but stressing periods of time to the
species of this genus. atmosphere. The Q,0 found for the two species is similar
Non-linear reciprocal plots for APA have been to other values observed (Pettersson & Jansson, 1978;
reported previously in the dinoflagellate Pyrocystis nocti- Hem~indez et al., 1992). G. sesquipedale showed a lower
luca (Murray) Schuett (Rivkin & Swift, 1980). The Q10 than G. latifolium, which suggests a greater regulation
behaviour of APA in Gelidium may result from two or of A P A with temperature. This is in agreement with the
more reactive sites on the same enzyme showing negative position of the alga in the intertidal: G. sesquipedale lives in
cooperativity (Cadenas, 1978), although further work is limestone crevices immersed in colder waters, which may
needed to demonstrate our hypothesis. In Escherichia coli enforce the development of enzymes optimally adapted
(Migula) Castellani et Chalmers, a dimeric alkaline phos- for a narrow temperature range (Lfining, 1990).
phatase was found to bind the substrate in a negatively pH is known to have a marked influence on algal A P A
cooperative fashion (Lazdunski, 1972). Similar results (Siuda, 1984). A higher pH optimum was found for A P A
have been found in the mycelial alkaline phosphatase in G. latifolium (pH ~,9) than in G. sesquipedale (pH 8"8). In
from Neurospora crassa Shear et Dodge (Palma et at., 1989). general these values are quite close to the values reported
This kinetic behaviour is also possible in monomeric for many cyanobacteria (Healey, 1982; Islam & Whitton,
enzymes (Comish-Bowden & Cardenas, 1987). Sosa & 1992), phytoplankters (Kuenzler & Perras, 1965; Petters-
Garcla-Reina (1992, 1993) showed by electrophoresis in son & Jansson, 1978; Siuda, 1984) and macroalgae
Gelidium arbuscula and Gelidium canariensis (Grunow) (Hemfindez et aL, 1992), although an optimum of up to
Seoane-Camba that alkaline phosphatase presented pH 12 has been reported (Mahasneh et al., 1990). The
typical banding patterns that conformed to its interpreta- optimum pH levels for both species of Gelidium are
tion as a diallelic, monomeric enzyme, with two bands unlikely to be encountered in their natural environment,
occurring in heterozygotic sporophytes. ThaUi of the two as they are uncovered only for brief periods of time. The
species of Gelidium assayed in the present work were water probably remains at around pH 8"3, at which value
diploid (tetrasporophytic) plants, as fertile (gametophytic) A P A is about 70% maximum activity. A possible expla-
material of these species is rarely found in southern Spain nation for the weak relationship between the pH optimum
(Seoane-Camba, 1965; F. Conde, personal communica- of APA and the typical pH of the natural environment
tion). On the contrary, the foliose, haploid phase of the from which the plants were harvested is given by Islam
red alga Porphyra umbilicalis showed evident Michaelis- & Whitton (1992). These authors, on the basis of the
Menten type kinetics (Hemdmdez et aL, 1992). study by Fedde & Whyte (1990) on human fibroblasts,
The lower of the two apparent Ks values observed in suggest that the substrate concentration used in the
the two species of Gelidium was in the range of other Ks assays (0-7mM) can induce a higher pH optimum than
values calculated with pNPP as a substrate [from 8"7 #M if the assays were performed at ambient concentration
for Nostoc commune Vaucher (Whitton et al., 1990) to (<2 #M).
320 #M for Aphanizomenon dos-aquae (Bomet et Flahault) The significant influence of salinity on APA in the two
Ralfs (Heath & Cooke, 1975)]. The ecological significance species of Gelidium has been found in other marine
of the apparent negative cooperativity lies in the linkage macrophytes growing in sites where natural fluctuations
of enzymatic kinetic parameters to external substrate in salinity are common (Hem~dez et al., 1994b). How-
concentrations (Levitsky & Koshland, 1976; Cembella ever, it is unlikely that Gelidium is subjected to fluctua-
et aL, 1983). Gelidium would achieve a constant P tions of salinity. The effect of salinity on APA is partly
supply regardless of the PME concentration in the due to the increase of the ionic strength in the assay
environment, as Bums & Beever (1977) hypothesised medium (Wilson et al., 1964). Nevertheless, our results
for a dual P uptake mechanism in Neurospora crassa. showed that this effect is attributable not only to the ionic
Short-term pulses of DOP are probably quite common strength itself, but that there was also a specific effect of
in many natural environments (Rivkin & Swift, 1980; particular ions, such as Na + and Mg z+. Similar results
Islam & Whitton, 1992), including coastal waters. Poten- were found by Mahasneh et al. (1990) in Calothrix viguieri
tially available substrates of the enzyme (PME) have been Fremy. In this species the influence of NaC1 on APA was
recorded from a nearby shore, and it was found that on not just an osmotic effect. The enhancement of A P A in
some days the PME concentration was higher than that of the presence of Mg 2+ has been found in many alkaline
SRP (Hem~indez et al., 1993b). phosphatases from different organisms (McComb et al.,
The temperature optima of A P A estimated for the two 1979; Siuda, 1984; Hino, 1988), possibly because, as in
L Herndndez, J. A. Ferndndez and F. X. Niell 76

mammals (Anderson et al., 1975), magnesium participates previous studies (e.g. Veldhuis et al., 1987) and may reflect
in the structure of some alkaline phosphatases. The an inverse relationship between APA and internal phos-
stimulant effect of NaC1 on APA may lie in the phate (Wynne, 1981), especially when there is a long-term
pronounced effect that both Na + and C1- could have effect of external phosphate upon APA. Moreover,
on the membrane potential of these species, and, thus, natural populations of Porphyra umbilicalis exhibited a
hydrolysis and phosphate uptake (Ullrich-Eberius & regulation of APA dependent on the internal phosphate
Yingchol, 1974; Cembella et al., 1983). status (Hermlndez et al., 1993b), assessed by the cell quota
APA showed a different response to photon irradiance in the sense of Droop (1983), and other results demon-
in the two species of Gelidium (Fig. 6). The lack of strated that phosphatases are controlled by several
relationship found in G. latifolium was also noticed for compartments involved in phosphorus metabolism, such
Porphyra umbilicalis (Hemfindez et al., 1992). These two as particulate internal phosphorus (Gage & Gorham,
species are exposed in their natural environment to a broad 1985). Further experiments need to be done to clarify
range of photon irradiances, which could be a selection the fine control of APA by an internal compartment of
factor for the enzyme to be independent of light intensity. phosphorus.
However, a long period of incubation in the dark (14 h)
before the assay causes higher APA in darkness compared
with an assay at 100 #mol photon m - 2 s - 1 (Hemimdez, Acknowledgements
unpublished data). As APA is inversely related to internal
We wish to thank Professor Dr I. Nfifiez de Castro for his
phosphate (Wynne, 1981), a long pre-incubation in dark-
helpful advice about enzyme kinetics and also Dr F. F. de
ness may stimulate alkaline phosphatase production due to
Castillejo and members of the Spanish Institute of
a decrease in phosphate uptake, which is an energy-
Oceanography in Fuengirola for providing the informa-
dependent reaction (Healey, 1973).
tion about water temperature at the sampling sites. We
Maximum APA in G. sesquipedale coincided with the
are very grateful to two anonymous referees whose
compensation point for photosynthesis (Torres et al.,
critical review greatly improved our manuscript. This
1991), in a pattern similar to that observed for other
work was supported financially by the research group
variables such as photosynthetic rates, chlorophyll, phy-
no. 4062 of the "Junta de Andalucla" and is a contribution
cobilins and C:N ratios. This common pattern, showing a
to the CICYT research project AMB 93-1211. I.H. held a
slight photoinhibition at high irradiances, reflects the
FPI fellowship from the Spanish Ministry of Education
intolerance of G. sesquipedale to high irradiances, which
and Science.
is greater than that of other intertidal species in the same
area (Torres et al., 1991). Other studies have found
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