Changes in relative concentrations of the hemoglobin components in Paramecium caused by cell growth and temperature of the culture

The ciliated protozoan Paramecium contains a heterogeneous hemoglobin (Hb), which is resolvable electrophor-etically into several definite components (Davis and Steers, 1976; Steers and Davis, 1979). The Hb components show a species-specificity, as has been pointed out for the species complex of P. aurelia (Irie and Usuki, 1980; Usuki and Irie, 1983a), and for P. multimicronucleatum, P. caudatum and P. jenningsi (Steers et al. 1981; Usuki and Irie, 19836; Usuki et al. 1989). This finding may indicate that the expression of these Hb components is controlled by different loci on the genes.

Paramecium Hb is a monomeric heme protein with a smaller size than any other Hbs reported from a variety of organisms (Prosser, 1973; Linzen, 1986). The primary structure of the major Hb component from P. caudatum has been determined, indicating a unique sequence of 116 amino acid residues with a molecular mass of 12 565 Da (Iwaasa et al. 1989). However, few attempts have been made to find a role for Hbs in this animal group.

On the other hand, the Hb content in some invertebrates is known to vary considerably according to some environmental factors, especially the ambient oxygen tension (Riggs and Van Holde, 1973; Weber, 1980; Kobayashi and Hoshi, 1984). Moreover, certain organisms have been reported to change their Hb components dramatically during the process of ontogenetic growth (Moens and Kondo, 1976; Sehin et al. 1979). In Paramecium, however, it has been shown that the Hb level in a given stock does not show significant change even when the organism is subjected to different conditions, although the Hb content hows a remarkable variation among species in this organism (Usuki and Hino, 1987).

In this paper we report the finding that Paramecium accommodates changes in cell growth and ambient temperature by a change in the relative amounts of Hb components, like higher invertebrates, which vary their Hb according to their physiological state.

The ciliates used were two stocks (Sh 23 and YC) of Paramecium caudatum syngen 3, two stocks (NE 25 and CH 312) of Paramecium multimicronucleatum syngen 2, and two stocks (ATCC 30997 and ATCC 30998) of Paramecium jenningsi. As the culture medium, a mixture of fresh lettuce infusion and pea broth was used throughout this study, and it was supplemented with 0.5 mg I^-1 of stigmasterol (Merck) and inoculated with Entero-bacter aerogenes one day before use. Each stock of the paramecia cells was grown previously in flasks at 27 °C, to the extent of 20-301 by addition of an equal volume of fresh medium when the cells reached stationary phase. The grown cells were concentrated and washed with the Dryls solution under a light centrifugal force (150g). Then the concentrated cells were divided in two: they were cultured again with fresh media in tanks at different room temperatures maintained to 18.0 (±0.1)ºC and 27.0 (±O.I)°C, respectively. These cultures were increased several times in volume (amounting to 60–1201 in total) by addition of fresh medium after the cells reached stationary phase. These cells were harvested at logarithmic phase or stationary phase. Hb from the respective cells was partially purified as described (Usuki and Irie, 1983a; Usuki et al. 1989), and then stored in a deep freeze at -80°C until use.

Polyacrylamide gel electrophoresis (PAGE) was performed with a 15% mini-slab gel (lmm×85mm×85mm) in a discontinuous buffer system. Isoelectric focusing (IEF) was carried out using a Pharmacia flat-bed apparatus FBE-3000 (Uppsala, Sweden) and an LKB Ampholine PAG plate (pH 3.5–9.5) (Bromma, Sweden). PAGE and IEF were performed at 2 °C. Densitometric analysis was done using the Shimadzu dual-wavelength chromatoscanner CS-910 (Kyoto, Japan), with a 420nm sample beam and a 650nm reference beam on unstained gels.

Paramecium cells in logarithmic phase were harvested after about 24 h at 27 °C and after 3–4 days at 18 °C after the final addition of freshly prepared culture medium. At this time the culture medium retained considerable turbidity, reflecting the presence of an excess of food bacteria. The cells in stationary phase were generally obtained after 3–4 days culture at 27°C and after 7–14 days culture at I8°C; in this time the media had become less turbid, as surging crowds of paramecia came into sight.

The Hb of P. caudatum was resolved by IEF into six components: basic component (bHb), Hb₂, Hb3, Hbθ, Hb10 and a trace amount of HbII. The Hbs of P. multimicronuc-leatum and P. jenningsi were analyzed by PAGE: the former species contained Hb₂, Hb₃, Hb₆, Hb₁₀, Hb₁₁and Hb₁₂, and the latter species had similar components to the former, except for bHb instead of Hb10. These Hb compositions were not modified by cell growth or culture temperature, showing a species specificity as reported (Usuki and Irie, l983a,t>; Usuki et al. 1989).

The effects of cell growth from logarithmic phase to stationary phase were examined by comparison of the relative amounts of the Hb components in each of the stocks, which were cultured at a constant temperature.

In 27 °C culture, the Hb of stock Sh 23 of P. caudatum contained 29 % of bHb and 65 % of Hb10 at logarithmic phase, while at stationary phase it contained 10 % of bHb and 81% of Hb10, resulting in a 19% decrease in the former and a 16 % increase in the latter during cell growth (Fig. IB). Similar changes were observed for both of the Hbs from stocks Sh 23 and YC at I8°C in culture, although the effects were slightly less at this temperature (Fig. 1A). The variation in other minor Hb components was negligible in this species. In the same way, the Hb of stock NE 25 of P. multimicronucleatum at logarithmic phase in 27°C culture was composed of 63% of Hb₁₁ and 25% of Hb₂, and at stationary phase it yielded 80% of Hb₁₁ and 12% of Hb₂ (Fig. ID): namely, the stock caused an increase in Hb?i of approximately 17 % during cell growth at 27°C, accompanied by a decrease in Hb₂ as well as other minor Hbs. The cells at I8°C in culture also showed a similar change, although the change in concentration was less than that at 27°C in culture (Fig. IC).

During the transition from logarithmic phase to stationary phase, stock CH 312 of P. multimicronucleatum in 27°C culture showed an approx. 23% increase in Hb₂ together with a comparable decrease in Hbn, opposite to the response to those of stock NE 25 of the same species (Fig. IF). In culture at I8°C stock CH 312 underwent a drastic change in the concentration of the Hb components: Hb₂ increased from 17 % to 90 % and Hb₁₁ decreased from 76% to 8% (Fig. IE). Such a change in concentration between Hb₂ and Hbn caused a dramatic alteration in the major Hb component that this stock contained before and after the cell growth.

The growth-dependent changes in the Hb in stock ATCC 30997 of P. jenningsi in culture at I8°C resemble those for stock CH 312. Growth of the cell caused a noticeable increase in Hb₂, from 22% to 60%, in association with a decrease from 54 % to 26 % in Hbn ^aHd from 21 % to 5 % in Hb₁₁ (Fig. 1G). As a consequence, the major Hb component in this stock varied between Hb₁₁ and Hb₂ before and after cell growth, as found for stock CH 312. In 27°C culture, however, cell growth caused a 20 % increase in Hb₁₁ from 49 % to 69 % and a 25 % decrease in bHb from 29 % to 4 %, while Hb₂ was practically unchanged, remaining at 19 % and 16%, respectively (Fig. 1H). A similar change was observed in the Hb of stock ATCC 30998 in culture at 27 °C, and these phenomena were similar to those for stock NE 25 of P. multimicronucleatum and the stocks of P. caudatum cultured at this temperature.

The effects of environmental temperature were investigated by comparison of the relative amounts of the Hb components in each of the stocks that were cultured to a homologous growth phase at different temperatures.

At logarithmic phase, stock Sh 23 of P. caudatum produced Hb containing 79 % of Hb?o and 18 % of bHb in culture at 18 °C, while at 27 °C Hb?o decreased to 65 % and bHb increased to 29 % (Fig. 2A). At stationary phase the effect of temperature was rather small, showing a small percentage of variation with a similarity to those at logarithmic phase (Fig. 2B). Stock YC at stationary phase also showed essentially the same variation between 18 and 27 °C.

The Hb of stock NE 25 of P. multimicronucleatum contained 40 % of Hbn and 50 % of Hb₂ at logarithmic phase in culture at 18 °C. Cells at 27 °C in culture contained 63 % of Hb₁₁ and 25 % of Hb₂, resulting in a 23 % increase in Hb₁₁ and a 25 % decrease in Hb₂ by increasing the temperature (Fig. 2C). At stationary phase, the Hb in culture at I8°C contained 49% of Hb₁₁ and 44% of Hb₂. At 27 °C the cells changed, to contain 80 % of Hb₁₁ and 12 % of Hb₂ (Fig. 2D). Thus, the shift of temperature from 18 to 27 °C causes a noticeable increase in Hb₁₁ and a decrease by a comparable amount of Hb₂, especially when the cells have become full grown.

Temperature-dependent change in stock CH 312 was abrupt at stationary phase. The cells grown in culture at 18 °C produced Hb containing 90 % of Hb₂ and 8 % of Hb₁₁, whereas the cells in culture at 27 °C generated 30 % of Hb₂ and 59% of Hb₁₁, reversing the quantitative relation between the two components (Fig. 2F). At logarithmic phase, the Hb composition in the cell was scarcely affected by temperature (Fig. 2E). The effects of temperature on P. jenningsi were similar to those on stock CH 312. When the cultivation temperature was shifted from 18 to 27 °C, the cells in stationary phase increased their concentration of Hbn from 26 % to 69 % and decreased their Hb₂ from 60 % to 16 %, resulting in an inverse relation of the quantities of the two components (Fig. 2H). At logarithmic phase, however, the effects of temperature remained only small, as in stock CH 312 (Fig. 2G).

The data obtained from the four stocks of different species of Paramecium are compiled three-dimensionally in Fig. 3. As seen in this figure, cell growth and lowering the temperature bring about an increase in Hb₁₀ in association with a decrease in bHb in P. caudatum, although the concentration change in this species is moderate and smaller than in other species. Stock NE 25 of P. multimicronucleatum also exhibits a consistent change, giving an obvious increase in Hb₁₁ and a compensatory decrease in Hb2 on increasing the temperature and on cell growth. On the other hand, the changes in stocks CH 312 and ATCC 30997 are curious, reflecting the result of an anomalous reduction in Hb₁₁ at the time of growth phase at 18 °C.

In Paramecium a method for reliable synchronous culture has not yet been established. In this study, therefore, the organism was cultured by the usual method and the cells were harvested at logarithmic phase or stationary phase: the former phase was expected to be full of growing and proliferating young cells and the latter should contain mature cells. The difference in growth phase may affect intracellular conditions greatly. For example, many authors have remarked that the susceptibility of respiration to cyanide varies considerably during the growth of a variety of eukaryotic cells (Pace, 1945; Edwards and Lloyd, 1977; Lloyd et al. 1980; Palmer, 1981). These findings have been studied in connection with the function of an alternative respiratory system that produces a large excess of electrons at the time of active biosynthesis (Doussière et al. 1979; Young, 1983). In addition to this, paramecia in this study were cultured at two different temperatures, 18 and 27°C. In ciliates temperaturedependent changes have been reported in ciliary activity or swimming behaviour (Tawada and Oosawa, 1972; Connolly et al. 1985a), in lipid composition and membrane fluidity (Nozawa et al. 1974; Connolly et al. 1985b), and in surface antigens (Preer, 1986; Bannon et al. 1986; Love et al. 1988), etc. We ascertained that each of the paramecia stocks that are transplanted into fresh culture medium proliferates at a rate of 0.3-0.8 division every day at 18°C and 2.5–3 divisions at 27°C.

This study made it clear that the cell growth of Paramecium from logarithmic, young phase to stationary growth phase is accompanied by an obvious change in the relative concentrations of the Hb components, and that the cultivation temperature also affects the equilibria among them. Both growth-dependent and temperature-dependent changes differ fundamentally from each other: the former occurs in association with the cell cycle even under constant temperature, whereas the latter is found in the cells only when the cultivation temperature is shifted.

At this point, we must note that both growth-dependent and temperature-dependent changes in Paramecium Hb depend upon a concentration variation of two or three major components: i.e. Hb₁₀ and bHb in P. caudatum, Hb₁₁ and Hb₂ in P. multimicronucleatum, and Hb₁₁ and Hb₂ or bHb in P. jenningsi, respectively. These components increase or decrease in opposite directions to each other according to the growth of the cell and the ambient temperature, although the physiological meaning of these changes remains obscure; whereas, other Hb components in these Paramecium species are too small in quantity and are generally less effective, even if their amounts vary.

On the other hand, the Hb components from various stocks in different species of Paramecium have been divided into two groups by their molecular masses: estimation by SDS–PAGE shows 1.1 kDa for Hb10 and Hb₁₁, and 1.3 kDa for Hb₂ and bHb (Usuki and Irie, 1983a,b; Usuki et al. 1989). However, the isoelectric point measured in our laboratory varied greatly among the Paramecium species: the pl value was 3.9 for Hb?₁₀ and 9.8–10.5 for bHbs from P. caudatum, 4.0–4.2 for Hbi?s and 6.2–6.5 for Hb₂s from P. multimicronucleatum and P. jenningsi, and 8.8 for bHb from P. jenningsi (Usuki et al. 1989; unpublished data). Therefore, we can state that both the growth-dependent and temperature-dependent changes observed in this study are realized by participation of two different types of Hb components: one of them shows far faster migration by PAGE, has a smaller molecular mass and a significantly lower pl value than those of the other. This may permit a reverse control for the two Hb components in a cell.

These results were unexpected and showed that stocks CH 312 of P. multimicronucleatum and ATCC 30997 of P. jenningsi exhibited an abnormal decrease in Hb?? at stationary phase, which was reached after a prolonged period at 18 °C. P. jenningsi is known to be a species of tropical origin (Diller and Earl, 1958). According to Dr Y. Takagi, stock CH 312 is a descendant of artificially induced conjugants, which have been gained through chemical treatments by Dr A. Miyake (personal communication). We have little information about the physiological, biochemical and ecological characteristics of these two stocks. However, we have found that the isoelectric points of Hbn from these stocks are lower by approximately 0.2 pH unit than that of stock NE 25 (unpublished data). This finding may indicate that Hb₁₁ possesses some difference in its molecular structure. So, we put forward the hypothesis that, during a prolonged cultivation period at 18°C, the stability of the Hb₁₁ molecule in these stocks breaks down and/or some process necessary for Hb₁₁ synthesis in these cells is susceptible to temperature. In connection with this, an interesting finding has been reported that the expression of a temperature-dependent surface antigen of Tetrahymena is directly correlated with mRNA abundance, which is controlled by a dramatic temperature-dependent change in mRNA stability (Love et al. 1988).

We thank Dr T. Watanabe, College of General Education, Tohoku University, Sendai, and Dr K. Mikami, Miyagi College of Education, Sendai, for the supply of stocks of P. caudatum. The stocks of P. multimicronucleatum were provided by Dr Y. Takagi, Faculty of Science, Nara Women’s University, Nara. We are also indebted to Dr M. Fujishima, Faculty of Science, Yamaguchi University, Yamaguchi, for supply of the seed cells ofP. jenningsi. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.