Biosynthesis of an acidic galactan and sulfated glycosaminoglycans during embryonic development of the mollusc Pomacea sp.

The synthesis of acidic polysaccharides by eggs of Pomacea sp. at different stages of development was studied. By day 3 after oviposition an acidic galactan became apparent. This compound reaches its maximum concentration by day 6 and then slowly decreases in concentration, no longer being detectable by day 12. Among the sulfated glycosaminoglycans, chondroitin sulfate is the first to appear (day 10), followed by heparan sulfate and other sulfate glycosaminoglycans, which were synthesized in large amounts until hatching (around day 15). Each one of the compounds was purified and characterized by chemical analysis and enzymatic degradation. The synthesis and characterization of the sulfated glycosaminoglycans was confirmed by the use of radioactive sulfate applied to intact eggs at different stages of development. These studies suggest that the main difference between vertebrate and mollusc development regarding the acidic polysaccharides is that hyaluronic acid seems to be absent during early mollusc development. It is proposed that the acidic galactan may be synthesized from the neutral galactan, already present in the eggs in high amounts, and may replace this glycosaminoglycan in the mollusc embryogenesis.     

Role of glucose metabolism in acid formation by isolated gastric glands

The role played by glucose in providing energy for acid formation was studied in isolated gastric glands from rabbit. The widely-used inhibitors of glycolysis, iodoacetic acid and iodoacetamide were found to inhibit glucose oxidation as well as the indicators of acid formation, respiration and accumulation of aminopyrine. However, the potent inhibition of acid formation was found to involve a nonspecific mechanism other than the simple inhibition of glycolysis. An alternative approach involved use of the glucose transport inhibitor, phloretin. Phloretin blocked glucose oxidation and also inhibited functional responses. Acid formation was restored easily by the addition of pyruvate or various other oxidizable substrates. Measurement of lactate formation in the absence of exogenous glucose showed that the gastric glands contain very little glycogen. Addition of external glucose resulted in a 10-fold increase in lactate formation and this rate was stimulated further by histamine and rotenone. Rotenone also inhibited both respiration and aminopyrine accumulation; however, the inhibition was not complete. Phloretin treatment resulted in total inhibition of the residual aminopyrine accumulation after rotenone treatment. The results are interpreted to indicate that gastric glands are dependent almost totally on external substrate supply to support acid formation; and, that while anaerobic glucose metabolism can sustain a very low level of acid formation, the major role of glucose is to yield pyruvate equivalents for subsequent oxidation.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Control mechanisms in the acceleration of hepatic glycogen degradation during hypoxia

Hepatic glycogen metabolism in aerobic and hypoxic conditions has been assessed with respect to glycogenolysis, phosphorylase a activity and nucleotide content. Insulin did not inhibit glycogen breakdown nor stimulate lipogenesis in the aerobic perfused liver.Partial ischaemia induced glycogen breakdown, release of glucose and changes in nucleotide content in the perfused liver. Phosphorylase a content increased within 2 min in response to total ischaemia, in vivo and in the perfused liver. This change was paralleled by an increase in hepatic AMP. Glycogen synthase a activity decreased, as did the hepatic content of both cyclic AMP and cyclic GMP.     

NAD turnover during early development of Xenopus laevis

The NAD pools of Xenopus laevis oocytes and early embryos can be radioactively labelled by microinjection of [adenine- ³H]NAD. This technique is used to study the metabolism of NAD in oocytes and during early development. The rate at which NAD is degraded in vivo has been monitored by determining the rate of transfer of adenine residues from the NAD pool into other nucleotides and polynucleotides. In oocytes, NAD turnover is extremely slow, with a half-life of about 400 h. NAD turnover increases dramatically after fertilisation, and the half-life of the compound decreases to 37 h in 5-h-old embryos and to 10 h in 40-h-old embryos. 2 mM 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) polymerase, reduces the NAD turnover rate by about 20%, whereas 5 mM isonicotinic acid hydrazide, a specific inhibitor of NAD glycohydrolase, produces no significant inhibition. This indicates that a significant fraction of the considerable NAD turnover observed involves poly(ADP-ribose) polymerase. Our results indicate that poly(ADP-ribose) polymerase is active during early development and suggest that this activity may be involved in one or more aspects of the nuclear metabolism of the embryo.     

Identification and characterization of the adenosine 3′,5′-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3′,5′-[³²P]monophosphate (8-N₃-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5′-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.     

Ligandin concentrations in the steroidogenic tissues of the rat during development

Ligandin, a ubiquitous multifunctional cytoplasmic protein which exhibits glutathione S-transferase, glutathione peroxidase and Δ⁵-3-ketosteroid isomerase activities and binds to cortisol metabolites, is present in relatively high concentrations in gonadal and adrenal tissue. In contrast to hepatic ligandin, little is known about the ontogeny of ligandin in steroid-synthesising tissues. We report here the intracellular concentrations of ligandin as well as the serum concentrations of testosterone and progesterone measured by radioimmunoassay at different stages of development in the rat. Ligandin levels in testis, ovary and adrenal tissue were relatively high soon after birth, decreased by day 9 and increased rapidly during puberty to reach adult levels. These changes appeared to be paralleled by changes in the circulating levels of testosterone and progesterone. In contrast, ligandin levels in non-steroidogenically active tissues, such as liver and kidney, were low at birth and rose progressively to reach adult levels. Whereas hepatic ligandin concentration could be increased at all stages of development by phenobarbital induction, no induction occurred in the endocrine tissues.     

Potential role in development of the major cysteine protease in larvae of the brine shrimp Artemia franciscana

Encysted embryos and larvae of the brine shrimp Artemia franciscana contain a cysteine protease which represents over 90% of the protease activity in these organisms. We have used immunocytochemical methods to determine the localization and potential role of the cysteine protease in development of young larvae. In prenauplius larvae, there is intense staining for the protease on the basal side of the epidermal layer in the posterior region and diffuse staining for the protease throughout the embryo. In first instar larvae, cysteine-protease staining becomes intense in the midgut-forming area where a reticulum-like pattern emerges in cells with an abundance of yolk platelets. Cysteine-protease staining in second instar larvae becomes intense in the apical side of epidermal cells and in the basal and apical zones of midgut cells. Subcellular localization of the protease in the epidermis and midgut of young larvae using immunogold electron microscopy suggests that most is located in the cytosol and extracellular matrix adiacent to these cells. Addition of cysteine-protease inhibitors to the growth medium, especially the fluoromethyl ketone Z-Phe-Ala-CH₂F, inhibits growth and segmentation of the thorax. Collectively, these observations suggest that the major cysteine protease in embryos and larvae functions in yolk utilization, as a hatching enzyme, in apolysis during the molt cycle, and as a digestive enzyme when the swimming larvae begin to feed.     

Ribonucleic acid metabolism during the development and ripening of tomato fruits

The accumulation of RNA in the outer locule tissue of tomato fruits was measured during development and ripening. Labelling studies suggest two peaks of synthesis, the first during early development and the second just before the onset of ripening (colour change). During the second period of increased RNA labelling the amount of total RNA per fruit either remains constant or starts to decline. Synthesis of rRNA and soluble RNA occurred at all stages. Polydisperse RNA containing polyadenylic acid was isolated and shown to direct the synthesis of protein in vitro. No significant changes in the amount of polyadenylic acid, relative to total RNA were detectable during the ripening period.     

Cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus)

The Deleted in Azoospermia (DAZ) family of RNA binding proteins consists of highly conserved genes boule, daz and daz-like (dazl) essential for germ cell development. boule is known for its unisexual meiotic expression in invertebrates and mammals, but meiotic-specific female expression plus meiosis-preferential male expression in trout, and meiosis-preferential bisexual expression in medaka. dazl shows highly conserved bisexual expression throughout gametogenesis in diverse species. Here we report the cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus), an important aquaculture fish. Molecular cloning and sequence analysis led to the identification of tilapia boule and dazl cDNAs. The predicted partial Boule contains a conserved RRM motif and Dazl has the C-terminal sequence. On a phylogenetic tree, tilapia Boule and Dazl are in separate clades of Boule and Dazl homologs from other species, indicating their divergence during early vertebrate evolution. By RT-PCR analysis, boule and dazl showed bisexual gonad-specific expression. By in situ hybridization analysis, both boule and dazl RNAs were restricted to female and male germ cells of adult gonads but absent in gonadal soma. In the ovary, boule and dazl RNAs were abundant in oocytes. In the testis, boule and dazl RNAs were prominent in meiotic spermatocytes but barely detectable in meiotic products. These data show that boule and dazl are expressed bisexually in germ cells and provide useful markers to study gametogenesis in the adult tilapia.     

The formation of bacteriochlorophyll · protein complexes of the photosynthetic apparatus of Rhodopseudomonas capsulata during early stages of development

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg (pO₂). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll.Cells which were incubated at 0.5 mmHg (pO₂) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of ¹⁴C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Purine metabolism in the bloodstream forms of trypanosoma gambiense and Trypanosoma rhodesiense

Bloodstream forms of Trypanosoma brucie gambiense and Trypanosoma brucei rhodesiense are incapable of de novo purine synthesis. Purine bases are converted directly to ribonucleotides and with the exception of guanine, are stable. Guanine is incorporated directly into ribonucleotides and also deaminated to xanthine. Purine ribonucleosides are hydrolyzed rapidly; these reactions may limit their incorporation since purine bases label the nucleotide pools more efficiently than do ribonucleosides. The apparent order of salvage efficiency for ribonucleosides is adenosine>inosine>guanosine>xanthosine for both organisms. T. b. gambiense salvages purine bases in the same order, while T. b. rhodesiense salvages purine bases in the order hypoxanthine>adenine>guanine>xanthine.     

Differential expression of boule and dazl in adult germ cells of the Asian seabass

Fertility genes boule and dazl constitute the evolutionarily conserved DAZ (Deleted in AZoospermia) family of RNA binding proteins essential for germline development across animal phyla. Here we report the cloning and expression analysis of boule and dazl from the Asian seabass (Lates calcarifer), a marine fish that undergoes sequential male-to-female sex reversal. Molecular cloning and sequence comparison led to the identification of boule and dazl cDNAs. RT-PCR analysis showed that both boule and dazl RNAs were restricted to the gonads among adult organs examined. Chromogenic in situ hybridization revealed germ cell-specific expression for both boule and dazl in female and male adults. Importantly, distinct differences were found between boule and dazl in terms of temporospatial expression and subcellular distribution. The boule RNA was abundant in late gametogenic cells except sperm. Interestingly, dazl expression increases in early oocytes and concentrates in a perinuclear speckle that appears to develop ultimately into the Balbiani body in advanced oocytes. The dazl RNA was found to be abundant in spermatocytes but hardly detectable in sperm. These data demonstrate that boule and dazl are germ cell markers in the adult Asian seabass, and that bisexual germline-specific expression has been conserved for boule and dazl in fish.     

Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

Derepression of the high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb⁺ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the low-affinity system has decreased for about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.     

Adaptive character of metabolism in Eothenomys miletus in Hengduan Mountains region during cold acclimation

Environmental factors play an important role in the seasonal adaptation of body mass and thermogenesis in small, wild mammals. The purpose of the present study was to test the hypothesis that ambient temperature was a cue to trigger the seasonal adjustments in body mass, energy intake, uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), and other biochemical characteristics of Eothenomys miletus during 49 days of cold exposure. Our data demonstrated that cold acclimation induced a remarkable decrease in body mass, a significant increase in energy intake and metabolic rate, and high expression of UCP1 in BAT of E. miletus. Biochemical characteristics of BAT and liver respiration were also increased following cold acclimation. These data suggest that E. miletus reduced the body mass and increased energy intake and expenditure under cold acclimation. Increased expression of UCP1 was potentially involved in the regulation of energy metabolism and thermogenic capacity following cold acclimation.     

A fluctuation analysis study of the development of amiloride-sensitive Na+ transport in the skin of larval bullfrogs (Rana catesbeiana)

In the presence of the Na⁺-channel blocker amiloride, the short-circuit current across the skins of bullfrog tadpoles in metamorphic stages XIX–XXIV was subjected to fluctuation analysis. The resulting power spectra contained a Lorentzian component of which the plateau value ($\text{S}{}_0$) decreased while the corner frequency ($\text{f}{}_{\mathrm{<mtext>c</mtext>}}$) increased as the mucosal amiloride concentration was increased from 0.5 to 24 μM. From the linear relationship between the $\text{f}{}_{\mathrm{<mtext>c</mtext>}}$ values and the amiloride concentrations it was possible to determine the binding ($\text{k′}{}_{01}$) and unbinding ($\text{k}{}_{10}$) constants for amiloride to its receptor on the Na⁺ channel. With these parameters as well as short-circuit current and $\text{S}{}_0$ values, the current through the individual Na⁺ channels ($\text{i}$) was calculated (average 0.58 pA). It did not increase significantly during late metamorphosis. The density of Na⁺ channels ($\text{M}$) in the apical membrane, on the other hand, increased significantly. It would appear that the increase in short-circuit current which occurs at this time is due primarily to an increase in amiloride-blockable Na⁺ channels. Unexpectedly, a Lorentzian component could be fitted to power spectra in amiloride-treated skins (stages XIX–XXI) which showed no amiloride-sensitive short-circuit current. Moreover, the typical increase in $\text{f}{}_{\mathrm{<mtext>c</mtext>}}$ with the amiloride concentration did not occur in these animals.     

A study of lactate dehydrogenase levels and turnover rates during postnatal development in the rat

Specific radioimmunoassays for lactate dehydrogenase A and B subunits have been employed to quantify cellular contents of these proteins more precisely than hitherto possible and to monitor changes during postnatal development. Liver, skeletal muscle, heart muscle and kidney cortex all demonstrated alterations in cellular levels of lactate dehydrogenase subunits over the first 56 days of life, the particular pattern being specific to each tissue. Studies on the turnover of lactate dehydrogenase in vivo and in vitro indicated that the developmental changes in total lactate dehydrogenase content in liver and kidney were regulated at some point(s) during both the biosynthesis and the degradation of the proteins.     

Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscle cells in vivo and in vitro

The effect of medium Ca²⁺ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures.There is an easily identifiable class of muscle protein which includes the Ca²⁺-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomyosin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca²⁺ concentration, 0.05–0.3 mM, where fusion was prevented. Similar medium Ca²⁺ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as ‘calcium-modulated proteins’. The increased Ca²⁺ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca²⁺ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca²⁺ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates.The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca²⁺ concentration between 0.06 and 1.8 mM.It is proposed that increase in cytoplasmic free Ca²⁺ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.