Differential tissue expression of three 35-kDa annexin calcium-dependent phospholipid-binding proteins

We have purified three 35-kDa calcium- and phospholipid-binding proteins from rat liver. These three calcimedins bind to phosphatidylserine in a calcium-dependent manner and have been termed 35 alpha, 35 beta, and 35 gamma based on their relative charge as determined by isoelectric focusing. Purification of the three 35-kDa calcimedins is achieved by phenyl-Sepharose, ion exchange, and gel filtration chromatography. Antibody was produced against the annexin consensus peptide, Lys-Ala-Met-Lys-Gly-Leu-Gly-Thr-Asp-Glu, which was derived from the sequence of several Ca2+/phospholipid-binding proteins including calpactin, lipocortin, endonexin II, 67-kDa calelectrin, lymphocyte 68-kDa protein, and protein II. Recognition of each 35-kDa calcimedin by anticonsensus sequence antibody places them in this protein family. Antibodies against each 35-kDa calcimedin were raised and purified by antigen-affinity chromatography. Each antibody is monospecific for the respective 35-kDa calcimedin. Immunological cross-reactivity defines 35 alpha, 35 beta, and 35 gamma as lipocortins III, IV, and V, respectively. Surveys by immunoblot analysis using these monospecific antibodies demonstrate a markedly different tissue expression pattern for each 35-kDa calcimedin. Furthermore, the levels of 35 alpha, 35 beta, and 35 gamma are differentially regulated in maturing rat ovary and uterus. Each calcimedin has been localized by indirect immunofluorescence within specific cell types. These results support the concept that mediation of the intracellular calcium signal can occur via multiple pathways through several related yet independent mediator proteins.     

An immunological comparison of several novel calcium-binding proteins

Polyclonal antibodies prepared against each of the calcimedins were utilized to determine their tissue distribution. The immunological survey of rat tissues revealed that the levels of the 35-kDa calcimedin varied, while the amount of the 67-kDa calcimedin was relatively constant in the tissues examined. A new immunoreactive species, 52 kDa, was detected with the antibody to the 35-kDa calcimedin; this protein appears to be the predominant immunoreactive species in the tissues examined. Antibodies to the 35-kDa calcimedin were also used to compare many other calcium-binding proteins in order to determine immunological relationships. These comparisons demonstrate that the epidermal growth factor receptor/kinase substrate (p35), the src kinase substrate (pp36), and calregulin are immunologically unrelated to the calcimedins. However, it was found that the 67-kDa calcimedin and the p70 calelectrin are identical, as are the 35-kDa calcimedin and the p32.5 calelectrin. The calimedins are a subset of the chromobindins. In addition, the antibody to the 35-kDa calcimedin also cross-reacts with synexin, which may be related to the new 52-kDa immunoreactive protein identified.     

UM-X7.1 myopathic hamster heart shows altered calcimedin protein levels

Cardiac tissue isolated from the UM- X7 .1 myopathic hamster shows decreasing levels of the calcimedin proteins relative to calmodulin. Liver, an unaffected tissue, does not show this decrease. The ratio of the 67K - calcimedin to calmodulin for several ages of heart tissue clearly indicates a progressive loss of the calcimedins which correlates with the cardiomyopathy progression. The loss of the calcimedins may be related to the development of the muscle myopathy.     

Effect of nifedipine on 67k calcimedin in cultured macrophages and smooth muscle cells

The calcium signal in regulation is mediated by specific calcium-binding proteins. The calcimedins, a newly described set of proteins, bind hydrophobic domains in a calcium-dependent manner and may play a role in calcium signal mediation. We previously showed that mouse peritoneal macrophages contain the 67k calcimedin and that thioglycolate elicited inflammatory macrophages appeared to have elevated levels of the 67k calcimedin. We now report that the cultured macrophage-like cell line J774A.1, as well as the cultured smooth muscle cell line DDT1MF-2, contain the 67k calcimedin as determined by indirect immunofluorescence. We further report that the level of fluorescence is increased in these cell lines after treatment with the calcium channel blocker nifedipine.     

Calcimedins: purification and characterization from chicken gizzard and rat and bovine livers

A procedure for the simultaneous extraction and purification of four calcimedins from chicken gizzard, rat liver, and bovine liver is described. These proteins bind to hydrophobic resins in a calcium-dependent manner similar to calmodulin and troponin C. The four calcimedins purified had molecular weights 67,000 (67K), 35,000 (35K), 33,000 (33K), and 30,000 (30K) as determined by SDS polyacrylamide gel electrophoresis. Their ability to bind calcium was demonstrated using the Hummel-Dreyer method. Their tissue concentration ranged between 1-4 mg/100 g wet weight in the three tissues studied. During gel filtration, calcimedins 67K and 35K, had Rf (Ve-Vo/Vt-Vo) values of 0.46 and 0.74, respectively, indicating monomeric structure. However, the 33K and 30K calcimedins had Rf values of 0.26 (molecular weights greater than 90,000) suggesting that they occur as subunit complexes in their native state. Antibodies raised against the 67K and 35K calcimedins showed cross reactivity suggesting possible common origin. However, peptide mapping studies showed that they are independent proteins with considerable peptide homology. Antibodies to 30/33K calcimedins did not cross-react with either 67K or 35K calcimedins. Moreover, their peptide maps were strikingly different from those of 67K and 35K calcimedins indicating that they are unique. At present, the regulatory function of this group of proteins is not clear. Indirect evidences support the possibility that they are involved in membrane associated events, such as endocytosis and secretion.     

Purification, characterization, and partial sequence analysis of 32-kDa calcimedin from chicken gizzard

Calcimedin is a group of proteins which has a binding ability to several hydrophobic matrices or cellular membrane fractions in the presence of Ca2+. Although the molecular properties were partially clarified, the physiological functions of calcimedins have not been clearly defined. In this study, we describe the isolation and characterization of 32-kDa calcimedin from chicken gizzard. Both structural and functional studies establish that 32-kDa calcimedin is a member of the calpactin/lipocortin family. The 32-kDa calcimedin displays phospholipase A2 inhibitory activity, Ca2(+)-dependent F-actin binding activity, and phospholipid binding activity similar to those of calpactins/lipocortins. Antiendonexin II antibody recognized 32-kDa calcimedin. However, antibodies against calpactin I (lipocortin II), calpactin II (lipocortin I), 35-kDa calcimedin, and 67-kDa calcimedin did not cross-react with 32-kDa calcimedin. One-dimensional peptide maps of the 32-kDa calcimedin and the 35-kDa calcimedin are different, confirming that they are distinct proteins. By comparing the sequence of 32-kDa calcimedin with the predicted sequence of endonexin II, we concluded that the primary structure of the 32-kDa protein is highly conserved. In particular, the sequences AMKGMGTDDEXEIXL, GMGTDEEEIL, VLTEILASR, and ILTSR conform to the endonexin consensus sequence, which is characteristic of the calpactin/lipocortin family.     

Purification, biological properties and partial sequence analysis of 67-kDa calcimedin and its 34-kDa fragment from chicken gizzard

Calcimedin is a group of proteins, originally isolated from chicken gizzard, which are able to bind to several hydrophobic matrices in the presence of Ca2+. Although the molecular properties have been partially discovered, the physiological functions of calcimedins have not yet been clearly defined. In this study, we describe the isolation and characterization of 67-kDa calcimedin and its 34-kDa fragment from chicken gizzard. Both structural and functional studies establish that 67-kDa calcimedin is a member of the calpactin/lipocortin family: it displays phospholipase A2 inhibitory activity, Ca2(+)-dependent F-actin binding and phospholipid binding activity similar to those of calpactins (lipocortins). By comparing the sequence of 67-kDa calcimedin with the predicted sequence of 67-kDa calelectrin, we concluded that the primary structure of these 67-kDa proteins is highly conserved. In particular, the sequences GLGTDEGAIIXVLTQR and EGAGTDESTLIEIMATR conform with the annexin consensus sequence which is characteristic of the calpactin/lipocortin family. A 34-kDa fragment of 67-kDa calcimedin was also purified and their relatedness has been confirmed by antibody cross-reactivity. The sequence data further support that the 34-kDa fragment is derived from the C-terminal portion of 67-kDa calcimedin by limited proteolysis. The 34-kDa fragment, which contains the annexin consensus sequence, preserves the phospholipase A2 inhibitory activity, and binds F-actin and phospholipids.     

67 kDa calcimedin, a new Ca2+-binding protein.

A set of four proteins, termed calcimedins, are isolatable from smooth, cardiac and skeletal muscle by using a fluphenazine-Sepharose affinity column. The calcimedins show apparent Mr values of 67,000, 35,000, 33,000 and 30,000 by SDS/polyacrylamide-gel electrophoresis. The 67,000-Mr calcimedin (67 kDa calcimedin) has now been purified to homogeneity by using DEAE-cellulose chromatography followed by Ca2+-dependent binding to phenyl-Sepharose. The amino acid analysis of the 67 kDa calcimedin shows this protein does not contain trimethyl-lysine but does contain 2 mol of tryptophan/mol of protein. The 67 kDa calcimedin shows positive ellipticity in the near-u.v. range with c.d. Ca2+-binding studies indicate one high-affinity Ca2+-binding site with Kd 0.4 microM. The data show that the 67 kDa calcimedin is distinct from other Ca2+-binding proteins described to date.     

Isolation and purification of an antibody to 67-KD calcimedin

The 67-KD calcimedin is a calcium-binding protein isolated from several muscle tissues. The protein shows apparent Mr of 67,000 by SDS-polyacrylamide gel electrophoresis. An antibody has been prepared by immunizing sheep with the protein purified from chicken gizzard smooth muscle. This antibody recognizes 67-KD calcimedin but not calmodulin, bovine serum albumin, transferrin, or brain p68 calelectrin. The presence of 67-KD calcimedin is demonstrated in the smooth muscle cell lines A10 and DDT1MF-2 as well as in primary cultures of chicken breast and heart muscle, by immunoprecipitation and immunofluorescence. The 67-KD calcimedin, being responsive to calcium, may play a role in calcium-mediated cell regulation. This report identifies several cells that may be useful for further delineation of the cellular role of 67-KD calcimedin.     

Rapid purification of the 30 kDa calcimedin using DNase I affinity chromatography

The 30 kDa calcimedin was found to bind directly to phenyl-Sepharose in a calcium dependent manner similar to calmodulin. The 30 kDa calcimedin was also found to bind to and inhibit DNase I. This calcium-dependent binding was exploited to develop a two-step purification scheme for this calcimedin. In addition, affinity-purified antibodies to the 30 kDa calcimedin were used to examine its tissue distribution. The highest levels were found in lung, trachea and diaphragm while the lowest levels of the 30 kDa calcimedin were found in brain and skeletal muscle.     

67 k calcimedin (67 kDa) is distinct from p67 calelectrin and lymphocyte 68 kDa Ca2+-binding protein.

The 67 k calcimedin is a Ca2+-binding protein present in both muscle cells and peritoneal macrophages. Many tissues, including lymphoid tissues, liver and lymphocytes, have been shown to contain Ca2+-binding proteins of similar molecular size, such as the p67(67 kDa) calelectrin or the 68 kDa lymphocyte protein. We have tested affinity-purified antibodies raised to the smooth-muscle 67 k calcimedin in these several tissues and here report that the 67 k calcimedin is not detectable in liver, thymus, spleen or thymic lymphocytes. These findings support recent biochemical evidence, discussed here, suggesting that the 67 k calcimedin is a protein different from calelectrin and the 68 kDa lymphocyte protein. The more limited tissue distribution of the 67 k calcimedin, which includes muscle and macrophages, suggests that the 67 k calcimedin may function in Ca2+-mediated events special to these cell types. The affinity-purified antibodies to the 67 k calcimedin will be useful in obtaining information concerning the special roles of this Ca2+-binding protein in these cells.     

Calcium-dependent proteolysis of calcium-binding proteins

In several myopathic disorders, the internal muscle cell calcium concentration increases significantly as compared to normal muscle cells. We report that in the presence of elevated calcium levels, the calcium-binding proteins troponin C and calmodulin are protected from digestion by the chymotrypsin-like serine proteinase that co-purifies with isolated myofibrils. Degradation of the 67k calcimedin in the presence of calcium shows altered major cleavage fragments while degradation of myosin is unaffected by the presence of calcium. A role for this serine proteinase in muscle-wasting diseases is suggested.     

Identification and characterization of calmodulin-binding proteins in mammalian sperm flagella

A calcium and calmodulin-regulated cyclic nucleotide phosphodiesterase has been shown to be an integral component of both rat and bovine sperm flagella. The calcium-activated enzyme was inhibited by both trifluoperazine (ID50 = 10 microM) and [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid (EGTA), and the basal activity measured in the presence of EGTA was stimulated by limited proteolysis to that observed in the presence of calcium/calmodulin. 125I-Calmodulin binding to purified rat sperm flagella has been characterized and the flagellar-associated calmodulin-binding proteins identified by a combination of gel and nitrocellulose overlay procedures and by chemical cross-linking experiments using dimethyl suberimidate. 125I-Calmodulin bound to demembranated rat sperm flagella in a time- and concentration-dependent manner. At equilibrium, 30-40% of the bound 125I-calmodulin remains associated with the flagella after treatment with EGTA or trifluoperazine. The majority of the bound 125I-calmodulin, both the Ca2+-dependent and -independent, was displaced by excess calmodulin. A 67-kDa calmodulin-binding protein was identified by both the gel and nitrocellulose overlay procedures. In both cases, binding was dependent on Ca2+ and was totally inhibited by trifluoperazine, EGTA, and excess calmodulin. On nitrocellulose overlays, the concentration of calmodulin required to decrease binding of 125I-calmodulin by 50% was between 10(-10) and 10(-11) M. Limited proteolysis resulted in the total loss of all Ca2+-dependent binding to the 67-kDa polypeptide. Chemical cross-linking experiments identified a major calcium-dependent 125I-calmodulin:polypeptide complex in the 84-90-kDa molecular mass range and a minor complex of approximately 200 kDa. Immunoblot analysis showed that the major 67-kDa calmodulin-binding protein did not cross-react with polyclonal antibodies raised against either the calcium/calmodulin-regulated cyclic nucleotide phosphodiesterase or phosphoprotein phosphatase (calcineurin) from bovine brain.     

Calmodulin-dependent protein kinases purified from rat brain and rabbit liver

A calmodulin-dependent protein kinase was purified from rat brain by the same protocol used previously for a rabbit liver calmodulin-dependent glycogen synthase kinase. The rat brain kinase readily phosphorylated rabbit skeletal muscle glycogen synthase at sites 1b and 2, the same sites phosphorylated by rabbit liver calmodulin-dependent kinase. The two kinases have other similarities: substrate specificity, potent inhibition by sodium fluoride, and nearly equal Ka's (10-20 nM) for calmodulin. Also, both enzymes have similar Stokes radii, 70 A (rabbit liver) and 75 A (rat brain), but quite different sedimentation coefficients, 10.6 S and 17.4 S, respectively. Consequently, the calculated molecular weights are also different: 560,000 for the brain enzyme and 300,000 for the liver enzyme. The major subunit of the rat brain kinase appears to be a single 51-kDa peptide, not a doublet pattern of 51- and 53-kDa subunits that is characteristic of the rabbit liver enzyme. Our findings are consistent with the hypothesis that the rat brain and rabbit liver enzymes belong to a class of closely related calmodulin-dependent protein kinases, possibly isozymes. This class of enzymes may be responsible for regulating several of the known calcium-dependent physiological functions.     

Molecular cloning of cDNA encoding a 55-kDa multifunctional thyroid hormone binding protein of skeletal muscle sarcoplasmic reticulum.

A cDNA clone encoding 55-kDa multifunctional, thyroid hormone binding protein of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 509 amino acids, and a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that an 18-residue NH2-terminal signal sequence was removed during synthesis. The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma beta-subunit of prolyl 4-hydroxylase and hen oviduct glycosylation site binding protein. The protein contains two repeated sequences Trp-Cys-Gly-His-Cys-Lys proposed to be in the active sites of protein disulfide isomerase. Northern blot analysis showed that the mRNA encoding rabbit skeletal muscle form of the protein is present in liver, kidney, brain, fast- and slow-twitch skeletal muscle, and in the myocardium. In all tissues the cDNA reacts with mRNA of 2.7 kilobases in length. The 55-kDa multifunctional thyroid hormone binding protein was identified in isolated sarcoplasmic reticulum vesicles using a monoclonal antibody specific to the 55-kDa thyroid hormone binding protein from rat liver endoplasmic reticulum. The mature protein of Mr 56,681 contains 95 acidic and 61 basic amino acids. The COOH-terminal amino acid sequence of the protein is highly enriched in acidic residues with 17 of the last 29 amino acids being negatively charged. Analysis of hydropathy of the mature protein suggests that there are no potential transmembrane segments. The COOH-terminal sequence of the protein, Arg-Asp-Glu-Leu (RDEL), is similar to but different from that proposed to be an endoplasmic reticulum retention signal; Lys-Asp-Glu-Leu (KDEL) (Munro, S., and Pelham, H.R.B. (1987) Cell 48, 899-907). This variant of the retention signal may function in a similar manner to the KDEL sequence, to localize the protein to the sarcoplasmic or endoplasmic reticulum. The positively charged amino acids Lys and Arg may thus interchange in this retention signal.     

Manipulating thyroid status alters endoplasmic reticulum calcium homeostasis in rat cerebellum

Thyroid-related hormones regulate the efficiency and expression of sarco-endoplasmic reticulum calcium ATPases in cardiac and skeletal muscle. However, little is known about the relationship between thyroid hormones and calcium (Ca2+) homeostasis in the brain. It is hypothesized that manipulating rat thyroid hormone levels would induce significant brain Ca2+ adaptations consistent with clinical findings. Adult male Sprague-Dawley rats were assigned to one of three treatment groups for 28 days: control, hypothyroid (6-n-propyl-2-thiouracil (PTU), an inhibitor of thyroxine (T4) synthesis), and hyperthyroid (T4). Throughout, rats were given weekly behavioral tests. Ca2+ accumulation decreased in the cerebellum in both hyper- and hypothyroid animals. This was specific to different ER pools of calcium with regional heterogeneity in the response to thyroid hormone manipulation. Behavioral tasks demonstrated sensitivity to thyroid manipulation, and corresponded to alterations in calcium homeostasis. Ca2+ accumulation heterogeneity in chronic hyper- and hypothyroid animals potentially explains clinical manifestations of altered thyroid status.     

The modulation of mitochondrial nitric-oxide synthase activity in rat brain development

Different mitochondrial nitric-oxide synthase (mtNOS) isoforms have been described in rat and mouse tissues, such as liver, thymus, skeletal muscle, and more recently, heart and brain. The modulation of these variants by thyroid status, hypoxia, or gene deficiency opens a broad spectrum of mtNOS-dependent tissue-specific functions. In this study, a new NOS variant is described in rat brain with an M(r) of 144 kDa and mainly localized in the inner mitochondrial membrane. During rat brain maturation, the expression and activity of mtNOS were maximal at the late embryonic stages and early postnatal days followed by a decreased expression in the adult stage (100 +/- 9 versus 19 +/- 2 pmol of [(3)H]citrulline/min/mg of protein, respectively). This temporal pattern was opposite to that of the cytosolic 157-kDa nNOS protein. Mitochondrial redox changes followed the variations in mtNOS activity: mtNOS-dependent production of hydrogen peroxide was maximal in newborns and decreased markedly in the adult stage, thus reflecting the production and utilization of mitochondrial matrix nitric oxide. Moreover, the activity of brain Mn-superoxide dismutase followed a developmental pattern similar to that of mtNOS. Cerebellar granular cells isolated from newborn rats and with high mtNOS activity exhibited maximal proliferation rates, which were decreased by modifying the levels of either hydrogen peroxide or nitric oxide. Altogether, these findings support the notion that a coordinated modulation of mtNOS and Mn-superoxide dismutase contributes to establish the rat brain redox status and participate in the normal physiology of brain development.     

Developmental regulation of calmodulin gene expression in rat brain and skeletal muscle.

Three different calmodulin genes that encode the identical protein have been identified in the rat (Nojima, 1989); however, calmodulin gene expression at the various stages of tissue differentiation and maturation has not been previously determined. We have quantitated the content of mRNAs encoding calmodulin in the developing brain and skeletal muscle using RNA blot analysis with three specific cDNA probes. Our results show that five species of calmodulin mRNAs: 4.0 and 1.7 kb for CaM I, 1.4 kb for CaM II, and 2.3 and 0.8 kb for CaM III are detectable at all ages in the brain as well as in skeletal muscle but exhibit a tissue-specific developmental pattern of expression. The comparison of the temporal pattern of calmodulin gene expression with both mitotic activity, as demonstrated by cyclin A mRNA levels, and differentiation and maturation of specific brain or muscle regions is consistent with calmodulin involvement in development.     

Effect of vitamin D deficiency on the composition and in vitro labeling of rat kidney proteins

Densitometric analysis of single-dimension gels consistently demonstrated that, in addition to rat renal calcium binding protein (CaBP) (Mr 28,000), two other kidney proteins of Mr 16,500 and Mr 18,000 were significantly enriched in their contents in the vitamin D-replete rat. Partial characterization of the Mr 18,000 and 16,500 proteins revealed that these proteins were heat-stable and distinct from calmodulin, as determined by their inability to undergo the calcium-dependent mobility shift in sodium dodecyl sulfate gels which is characteristic of calmodulin. The Mr 16,500 and Mr 18,000 kidney proteins did not cross-react with rat renal or rat intestinal CaBP antisera, as assessed by radioimmunoassay and Western blot analysis. A comparison of peptide maps of tryptic digests of these proteins and purified rat renal CaBP, as analyzed by high-pressure liquid chromatography, revealed no apparent homology. Protein synthesis studies using [35S]methionine and short-term tissue culture of kidney cortex fragments indicated that the most pronounced effect of vitamin D or 1,25 dihydroxyvitamin D3 was increased synthesis of the Mr 28,000 protein (3.2- to 4.6-fold increase compared to -D rats, P less than 0.001). Synthesis of a Mr 54,500 protein increased by 1.3- to 1.5-fold (P less than 0.05) and [35S]methionine incorporation into a Mr 66,000 protein decreased by 1.2- to 1.3-fold (P less than 0.05) in +D rats. This study represents the first detailed characterization of the effects of vitamin D on the composition and synthesis of rat kidney proteins. The data indicate that the most significant effect of vitamin D on kidney proteins is increased synthesis of the Mr 28,000 CaBP, suggesting that a major role of vitamin D in renal function is regulation of calcium transport at the distal tubule. However, dietary vitamin D or 1,25(OH)2D3 can influence the expression as well as the suppression of other specific kidney proteins.     

Influence of thyroid hormone status on expression of genes encoding G protein subunits in the rat heart.

We have studied the influence of thyroid hormone status in vivo on expression of the genes encoding guanine nucleotide-binding regulatory protein (G protein) alpha-subunits Gs alpha, Gi alpha(2), Gi alpha(3), and both the 36-kDa form (beta 1) and the 35-kDa form (beta 2) of the beta-subunit in rat ventricle. The relative amounts of immunoactive Gi alpha(2) and Gi alpha(3) were greater in ventricular membranes from hypothyroid animals than from euthyroid animals (1.9- and 2.6-fold, respectively). A corresponding 2.3-fold increase in Gi alpha(2) mRNA was observed as well as a 1.5-fold increase in Gi alpha(3) mRNA. The relative amounts of immunoactive beta 1 and beta 2 polypeptides were also increased (2.8- and 1.8-fold, respectively) in the hypothyroid state and corresponded with comparable increases in the relative levels of beta 1 and beta 2 mRNAs. No difference was seen between the amounts of Gi alpha(2), Gi alpha(3), beta 1, and beta 2 in the euthyroid state and the hyperthyroid state. In contrast to these effects of thyroid hormone status on Gi alpha and beta, the steady-state amounts of Gs alpha protein and mRNA were not altered by thyroid hormone status. Thyroid hormone status did not alter sensitivity of adenylyl cyclase to stimulation by sodium fluoride or guanyl-5'-yl imidodiphosphate (GppNHp), nor did it influence GppNHp-induced inhibition of forskolin-stimulated enzyme activity. These results demonstrate that thyroid hormone status in vivo can regulate expression of specific G protein subunits in rat myocardium. However, the physiological consequences of these changes remain unclear.