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PubMed1998

Identification of novel chicken estrogen receptor-alpha messenger ribonucleic acid isoforms generated by alternative splicing and promoter usage.

Using the rapid amplification of complementary DNA ends (RACE) methodology we have identified three new chicken estrogen receptor-alpha (cER alpha) messenger RNA (mRNA) variants in addition to the previously described form (isoform A). Whereas one of the new variants (isoform B) presents a 5'-extremity contiguous to the 5'-end of isoform A, the two other forms (isoforms C and D) are generated by alternative splicing of upstream exons (C and D) to a common site situated 70 nucleotides upstream of the translation start site in the previously assigned exon 1 (A). The 3'-end of exon 1C has been located at position -1334 upstream of the transcription start site of the A isoform (+1). Whereas the genomic location of exon 1D is unknown, 700 bp 5' to this exon were isolated by genomic walking, and their sequence was determined. The transcription start sites of the cER alpha mRNA isoforms were defined. In transfection experiments, the regions immediately upstream of the A-D cER alpha mRNA isoforms were shown to possess cell-specific promoter activities. Three of these promoters were down-regulated in the presence of estradiol and ER alpha protein. It is concluded, therefore, that the expression of the four different cER alpha mRNA isoforms is under the control of four different promoters. Finally, RT-PCR, S1 nuclease mapping, and primer extension analysis of these different cER alpha mRNA isoforms revealed a differential pattern of expression of the cER alpha gene in chicken tissues. Together, the results suggest that alternative 5'-splicing and promoter usage may be mechanisms used to modulate the levels of expression of the chicken ER alpha gene in a tissue-specific and/or developmental stage-specific manner.

Alternative Splicing↗
PubMed1993

Insulin-responsive human adipocytes express two glucose transporter isoforms and target them to different vesicles.

We have characterized the insulin-dependent increase in glucose transport in human adipocytes using subcellular fractionation and antibodies specific for the two isoforms of the glucose transporter that are expressed in these cells. Plasma membranes isolated from untreated human fat cells contain the erythroid/GLUT1 isoform of the glucose transporter almost exclusively whereas the muscle-fat/GLUT4 transporter isoform is most abundant in intracellular microsomal membranes in resting cells. After exposure of adipocytes to insulin, the muscle-fat isoform is dramatically increased in the plasma membrane whereas the erythroid isoform barely changes in response to insulin. Thus, the total insulin-mediated increase in plasma membrane glucose transporters, confirmed by affinity labeling of both transporter isoforms, must be due to the increase in the muscle-fat/GLUT4 transporter. The two isoforms exist in different vesicle populations as shown by immunoadsorption of the muscle fat isoform-containing vesicles which are essentially devoid of the erythroid transporter. These data indicate that the insulin-mediated increases in glucose transport in human fat cells is a result of the translocation of vesicles uniquely containing the muscle-fat glucose transporter isoform.

Adipose Tissue↗
PubMed1994

Serum gonadotropin isoforms become more basic after an exogenous challenge of gonadotropin-releasing hormone in children undergoing pubertal development.

Recent clinical studies have questioned whether there is a qualitative change in the circulating isoforms of LH and FSH after stimulation by GnRH. The present study investigated the median charge of serum gonadotropin isoforms before and after an exogenous challenge of 100 micrograms GnRH in 10 girls and 10 boys undergoing pubertal development. All of the children had basal serum levels of LH and FSH and responses 30 and 60 min after GnRH treatment that were considered normal for puberty. LH and FSH in serum and eluates after electrophoresis in 0.10% agarose suspension were measured with sandwich fluoroimmunoassays. The increases in serum gonadotropin concentrations were generally similar for both sexes, but girls had significantly (P < 0.05) higher LH levels at 30 and 60 min than boys and a larger (P < 0.05) relative increase in serum FSH levels after GnRH treatment. In terms of the median charge of serum isoforms, the girls had significantly less negative (i.e. more basic) isoforms of LH (P < 0.05) and FSH (P < 0.001) than the boys. There was a change to more basic isoforms of both LH and FSH in all children 30 min after GnRH administration. For LH, the charge had returned to pretreatment values by 90 min after GnRH, but for FSH, the charge was still significantly (P < 0.05) more basic at this time (n = 4/sex). When the LH isoforms were more acidic before GnRH treatment, the change in median charge was larger than when the isoforms were more basic beforehand. A similar relationship was not found for FSH. Conversely, there was for FSH, but not for LH, a significant (P < 0.001) relationship between the relative increase in serum concentrations and the change in charge of the isoforms 60 min after GnRH treatment. These findings show that the circulating forms of LH and FSH become more basic after an exogenous challenge of GnRH in children undergoing pubertal development and suggest that the differences in the responses of LH and FSH isoforms may be due to differing degrees of selective secretion and/or survival.

Adolescent↗
PubMed1997

Circulating non-22-kilodalton growth hormone isoforms in acromegalic men before and after transsphenoidal surgery.

GH represents several molecular isoforms in addition to the main 22-kDa (22K) GH. There have been reports suggesting that circulating non-22K GH isoforms are increased in acromegaly, but the possible implications of such observations in the management of the disease have not been addressed. The aim of this study was to evaluate the proportion of circulating non-22K GH isoforms in acromegaly. In addition, the relationships between the amount of non-22K GH and tumor size, biochemical measurements, and body composition also were investigated. Samples with different GH levels were selected from 24-h GH profiles from 15 acromegalic men evaluated before and 1 yr after transsphenoidal surgery and from 13 healthy men. The serum non-22K GH levels, expressed as percentage of total GH concentration, were determined by the 22K GH exclusion assay, which is based on immunomagnetic extraction of 22K GH from serum and quantitation of non-22K GH using a polyclonal GH assay. The proportion of non-22K GH isoforms was fairly constant in different samples from the same patient, regardless of the GH level. However, a wide variation of values was observed among acromegalics, both before (14-51%) and after surgery (8-62%). The proportion of non-22K GH isoforms was increased in untreated patients, compared with controls (26.6 vs. 17.4%; P < 0.01), and the values correlated significantly to tumor size, mean 24-h GH concentration, serum PRL, and extracellular water. After surgery, patients not truly cured, with mean 24-h GH concentration of 1 microg/L or more, had an increased proportion of non-22K GH, compared with those with levels less than 1 microg/L (P < 0.01). In the former group, the median values were similar than those in untreated acromegalics (34 vs. 26.6%, respectively), whereas in the latter, they were comparable with those in the controls (15.2 vs. 17.4%, respectively). We conclude that acromegalics have an increased proportion of circulating non-22K GH isoforms. The values are fairly constant in different samples from an individual, regardless of GH level, but a large spectrum can be observed among patients. This variability suggests that different pituitary adenomas secrete GH isoforms in variable amounts. Our observation that a higher proportion of non-22K GH isoforms is present in patients not truly cured after surgery suggests that the evaluation of non-22K GH isoforms can be useful in the follow-up of acromegalic patients.

Acromegaly↗
PubMed2003

Expression of two isoforms of the vacuolar-type ATPase subunit B in the zebrafish Danio rerio.

In the present study we tested the hypothesis that two isoforms of the regulatory subunit B of vacuolar-type ATPase (V-ATPase) are expressed in the zebrafish Danio rerio. The complete coding sequences for both isoforms, vatB1 and vatB2, were cloned and sequenced. BLASTX analysis revealed the greatest similarity to amino acid sequences of B subunits from the European eel Anguilla anguilla and rainbow trout Oncorhynchus mykiss. The isoforms were expressed in a bacterial system and the recombinant proteins verified using isoform-specific antibodies directed against vatB isoforms of the eel. The distribution of both isoforms in zebrafish tissues was investigated using reverse transcriptase-polymerase chain reaction and western blot analysis. The results revealed that at the RNA level both isoforms were expressed in all tested organs, i.e. the gills, swimbladder, heart, kidney, liver, spleen, intestine and skeletal muscle. At the protein level, however, there were tissue-specific variations in the levels of the two vatB isoforms expressed. The highest amounts of V-ATPase were detected in total protein preparations from gill, heart and liver tissue. In liver tissue, however, the western blot analysis indicated that vatB1 was not as prominent as vatB2, and immunohistochemistry revealed that antibodies directed against vatB1 yielded a very weak staining in a number of cells, while an antibody directed against vatB1 and vatB2 yielded a strong staining in virtually every cell. Similarly, neurosecretory cells of the small intestine were stained with an antibody directed against vatB1 and vatB2, but not with an antibody specific for vatB1. Therefore we conclude that the differential expression of two isoforms of the V-ATPase subunits, which may serve different functions as in several mammalian species, may also be a common phenomenon in teleost fish.

Amino Acid Sequence↗
PubMed2003

Differential induction of protein kinase C isoforms at the cardiac hypertrophy stage and congestive heart failure stage in Dahl salt-sensitive rats.

Several protein kinase C (PKC) isoforms may play important roles in cellular signaling pathways. Recent reports have suggested that PKC plays critical isoform-specific roles in the development of cardiac hypertrophy and heart failure. The purpose of the present study was to examine the expression profiles of PKC isoforms in models of cardiac hypertrophy and heart failure. We examined the cardiac expression of individual PKC isoforms at the cardiac hypertrophy stage and the heart failure stage in Dahl salt-sensitive rats by Western blot analysis. The levels of all PKC isoforms increased at the cardiac hypertrophy stage and the heart failure stage, but the pattern of increase differed among PKC isoforms at the heart failure stage. The expressions of PKCalpha, beta, and delta increased at the cardiac hypertrophy stage and remained elevated at the heart failure stage. On the other hand, the expression of PKCepsilon and atypical PKCs (aPKCs) increased at the cardiac hypertrophy stage, but this increase tended to decline at the congestive heart failure stage. These results suggest that there are two groups of PKC isoforms. Several reports have shown that PKCalpha, beta, and delta are involved in the development of cardiac hypertrophy and heart failure, and that PKCepsilon plays a role in the physiological hypertrophic responses and cardioprotective actions. These facts suggest that all PKC isoforms (PKCalpha, beta, delta, epsilon, and aPKCs) expressed in the heart may have similar functions at the cardiac hypertrophy stage, but that two groups of PKC isoforms (PKCalpha, beta, delta, and PKCepsilon, aPKCs) have different functions at the congestive heart failure stage.

Animals↗
PubMed1993

Effect of immunization against LHRH on isoforms of LH in the ovine pituitary.

The role of LHRH in modulating intrapituitary LH content as well as the distribution of LH among its isoforms was examined in sheep. Rams (n = 3) and wethers (n = 6) were actively immunized against an LHRH-human serum globulin conjugate. Pituitaries collected from these animals plus pituitaries from corresponding numbers of nonimmunized rams and wethers were extracted with a buffered saline solution containing protease inhibitors. Immunization markedly reduced total amounts of immunoreactive LH in the pituitary. An aliquot of each pituitary extract was desalted by flow dialysis against water and chromatofocused on a pH 10.5-7.0 gradient. Concentrations of LH in chromatofocusing fractions were determined by radioimmunoassay. LH in pituitary extracts resolved into nine peaks during chromatofocusing which were coded with letters beginning with the most basic isoform. The percentage of LH as the two most basic isoforms, A' and B, was similar (P > 0.05) in all treatment groups. Isoform H constituted a higher percentage (P < 0.05) of the LH in both castrate groups. Nonimmunized wethers had higher percentages of isoforms C, D and E (P < 0.05) and lower percentages (P < 0.05) of the acidic isoforms (coded as peak Z herein) than did other treatment groups. Thus, castration shifted the pattern of intrapituitary isohormones towards the more basic forms. Nonimmunized rams had a higher percentage (P < 0.05) of isoform G than did other groups. Isoform F, the most abundant isoform, was present as a higher percentage (P < 0.05) in immunized rams and wethers than in nonimmunized animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Fatty acid and phorbol ester-mediated interference of mitogenic signaling via novel protein kinase C isoforms in pancreatic beta-cells (INS-1).

It is possible that activation of protein kinase C (PKC) isoforms by free fatty acids (FFA) plays a role in the failure of pancreatic beta-cell mass expansion to compensate for peripheral insulin resistance in the pathogenesis of type-2 diabetes. The effect of lipid moieties on activation of conventional (PKC-alpha and -beta1), novel (PKC-delta) and atypical (PKC-zeta) PKC isoforms was evaluated in an in vitro assay, using biotinylated neurogranin as a substrate. Oleoyl-Coenzyme A (CoA) and palmitoyl-CoA, but not unesterified FFA, significantly increased the activity of all PKC isoforms (P< or =0.05), particularly that for PKC-delta. It was found that FFA (0.4 mM oleate/complexed to 0.5% bovine serum albumin) inhibited IGF-I-induced activation of protein kinase B (PKB) in the pancreatic beta-cell line (INS-1), but this was alleviated in the presence of the general PKC inhibitor (Gö6850; 1 microM). To further investigate whether conventional or novel PKC isoforms adversely affect beta-cell proliferation, the effect of phorbol ester (phorbol 12-myristate 13-acetate; PMA)-mediated activation of these PKC isoforms on glucose/IGF-I-induced INS-1 cell mitogenesis, and insulin receptor substrate (IRS)-mediated signal transduction was investigated. PMA-mediated activation of PKC (100 nM; 4 h) reduced glucose/IGF-I mediated beta-cell mitogenesis (>50%; P< or =0.05), which was reversible by the general PKC inhibitor Gö6850 (1 microM), indicating an effect of PKC and not due to a non-specific PMA toxicity. PMA inhibited IGF-I-induced activation of PKB, correlating with inhibition of IGF-I-induced association of IRS-2 with the p85 regulatory subunit of phosphatidylinositol-3 kinase. However, in contrast, PMA activated the mitogen-activated protein kinases, Erk1/2. Titration inhibition analysis using PKC isoform inhibitors indicated that these PMA-induced effects were via novel PKC isoforms. Thus, FFA/PMA-induced activation of novel PKC isoforms can inhibit glucose/IGF-I-mediated beta-cell mitogenesis, in part by decreasing PKB activation, despite an upregulation of Erk1/2. Thus, activation of novel PKC isoforms by long-chain acyl-CoA may well contribute to decreasing beta-cell mass in the pathogenesis of type-2 diabetes, similar to their inhibition of insulin signal transduction which causes insulin resistance.

Acetophenones↗
PubMed2006

Expression pattern and ontogenesis of thyroid hormone receptor isoforms in the mouse heart.

Nuclear thyroid hormone (T3) receptors (TR) play a critical role in mediating the effects of T3 on development, differentiation and normal physiology of many organs. The heart is a major target organ of T3, and recent studies in knockout mice demonstrated distinct effects of the different TR isoforms on cardiac function, but the specific actions of TR isoforms and their specific localization in the heart remain unclear. We therefore studied the expression of TRalpha1, TRalpha2 and TRbeta1 isoforms in the mouse heart at different stages of development, using monoclonal antibodies against TRalpha1, TRalpha2 and TRbeta1. In order to identify distinct components of the embryonic heart, in situ hybridization for cardiac-specific markers was used with the expression pattern of sarcoplasmic reticulum calcium-ATPase 2a as a marker of myocardial structures, while the pattern of expression of connexin40 was used to indicate the developing chamber myocardium and peripheral ventricular conduction system. Here we show that in the ventricles of the adult heart the TRbeta1 isoform is confined to the cells that form the peripheral ventricular conduction system. TRalpha1, on the other hand, is present in working myocardium as well as in the peripheral ventricular conduction system. In the atria and in the proximal conduction system (sinoatrial node, atrio-ventricular node), TRalpha1 and TRbeta1 isoforms are co-expressed. We also found the heterogeneous expression of the TRalpha1, TRalpha2 and TRbeta1 isoforms in the developing mouse heart, which, in the case of the TRbeta1 isoform, gradually revealed a dynamic expression pattern. It was present in all cardiomyocytes at the early stages of cardiogenesis, but from embryonic day 11.5 and into adulthood, TRbeta1 demonstrated a gradual confinement to the peripheral ventricular conduction system (PVCS), suggesting a specific role of this isoform in the formation of PVCS. Detailed knowledge of the distribution of TRalpha1 and TRbeta1 in the heart is of importance for understanding not only their mechanism of action in the heart but also the design and (clinical) use of TR isoform-specific agonists and antagonists.

Animals↗
PubMed1995

Two isoforms of Drosophila dynamin in wild-type and shibire(ts) neural tissue: different subcellular localization and association mechanisms.

The temperature-sensitive mutations of the shibire (shi) gene in Drosophila cause endocytic arrest, resulting in neurotransmission block and paralysis at high temperatures. However, underlying mechanism for the defects is not yet known. We examined the subcellular distribution of dynamin, a product of the shi gene, by immunoblotting and immunocytochemical assays. Two isoforms of dynamin with apparent M(r) of 92 kD and 94 kD have been detected in wild-type and shi(n) adult neural tissue. The two isoforms were reproducibly associated with different subcellular fractions of head homogenates. The 94kD isoform is fractionated in the low speed (2.000 x g) pellet containing plasma membrane fragments, and the 92kD isoform in the high speed (130,000 x g) pellet. In this procedure, very little dynamin remained in the high speed supernatant fraction. The 94 kD isoform represents the majority (65-75%) of total dynamin and appears to be a peripheral membrane protein. It can be extracted from the low speed membrane pellet by high salt, Na2CO3 (pH 11) or Triton X-100 treatments. Extracted 94kD dynamin from both wild-type and mutant homogenates is able to reassociate with artificial phospholipid vesicles at both permissive and restrictive temperatures. Binding of the 94 kD dynamin to liposomes appears to be pH-dependent, varying most significantly within the physiological pH range, which may be functionally important. The 92 kD isoform cannot be released by high salt or Na2CO3 treatments and only a small fraction is released by Triton X-100, suggesting a different mechanism of association with cell structures. The distribution of the two isoforms is not altered by the presence of stabilized microtubules in homogenates. No apparent degradation or subcellular redistribution of mutant dynamin was detected in two shi(n) alleles after heat shock or block of the dynamin GTPase activity, suggesting that intracellular redistribution or degradation of mutant dynamin are not involved in the endocytosis arrest in these mutants. These observations resemble the effect of endocytosis arrest by GTP-gamma-S in rat brain synaptosomes (Takei et al., 1995), in which dynamin is trapped at the neck of invaginated pits but is absent in the clathrin-coated distal end undergoing internalization. Our finding that endocytosis arrest by shi(n) mutations and GTP-gamma-S do not lead to cumulation of dynamin in the low speed pellet fraction further suggests that the 94 kD isoform remains associated with the plasma membrane during coated vesicle pinch-off and that the two isoforms do not appear to correspond to different functional states of dynamin but are likely to be involved in separate cellular compartments within the membrane cycling pathway (e.g., the plasma membrane, endosomes, and endoplasmic reticulum).

Amino Acid Sequence↗
PubMedDate not supplied

Effects of platelet-derived growth factor isoforms on human lung fibroblast proliferation and procollagen gene expression.

Platelet-derived growth factor (PDGF), a potent mitogen for fibroblasts, is a potentially important cytokine in the pathogenesis of fibroproliferative disorders of lung. Different isoforms of PDGF include a heterodimer composed of A and B chains and homodimers composed of A or B chains. The biological significance of the different isoforms is unknown, but they have been shown to differ in their mitogenic potency in some systems. Their effects on other fibroblast functions have not been fully examined. We undertook this study to determine the effect of PDGF isoforms on human lung fibroblast proliferation and procollagen synthesis. Cultured lung fibroblasts (IMR-90, WI-38, GeNA) were incubated in the presence of varying concentrations of highly purified PDGF-AB obtained from platelets or recombinant PDGF-AA or -BB homodimers. Incorporation of [3H]thymidine was determined as a measure of mitogenic activity. Fetal lung fibroblasts (IMR-90, WI-38) and an adult fibroblast strain (GeNA) responded similarly to the different isoforms, with maximum mitogenic activity observed at 5-10 ng/mL. Cell cycle analysis using three additional normal adult lung fibroblast strains indicated that all PDGF isoforms stimulated similar proportions of cells to cycle over a 7-day period. Fibroblast procollagen synthesis, measured after pulse labeling with [3H]proline, was not increased even at concentrations of the PDGF isoforms that were maximally mitogenic. Moreover, steady-state levels of alpha 1(I) and alpha 1(III) procollagen mRNA levels, determined by northern analysis and dot blot hybridization, were not changed after exposure to any of the PDGF isoforms. While all PDGF isoforms are potent mitogens for fetal and adult lung fibroblasts, it was concluded that they do not directly stimulate procollagen gene expression or procollagen synthesis in vitro. The results suggest that PDGF isoforms are potentially important mitogens for lung fibroblasts, but other factors are likely to be involved in the stimulation of fibroblast procollagen synthesis that is observed in fibroproliferative disorders of lung.

Cell Cycle↗
PubMed1994

Catalytic subunit isoforms of mammalian lens Na,K-ATPase.

To identify the Na,K-ATPase isoforms present in the mammalian lens, seven antisera were prepared to selected peptide sequences of the catalytic (alpha) subunit. Three antisera were prepared to peptide sequences at the N-terminus of the three sequenced rat alpha isoforms. There is < 53% sequence homology among the isoforms in this region. Three antisera were prepared to peptide sequences at the ouabain binding site in the extracellular loop between membrane spanning sequences 1 and 2 of the sequenced rat alpha isoforms; sequence homology among the isoforms in this region is < 69%. An antiserum was also prepared to the carboxyl terminal region of the alpha 2 rat isoform. The sequenced isoforms (rat and human) in this region are > 94% homologous. The results from stains of Western blots of SDS-PAGE separations of lens membranes are presented. Alpha 1 is the predominant isoform of the epithelium. It is not found in cells of the central epithelium but is present in cells located more toward the equator. Alpha 3 is the catalytic subunit of the central 43% of the epithelium. The lens fiber cell membranes have a catalytic subunit that is related to the alpha 2 isoform. In the fiber cell a 98-100 kDa band stains with the antiserum to the alpha 2 N-terminus and the antiserum to the alpha 2 ouabain site. The antiserum to the alpha 2 C-terminus does not stain the 98-100 kDa band. (Preliminary reports of these results were presented at the 1992 and 1993 meetings of the Association for Research in Vision and Ophthalmology).

Amino Acid Sequence↗
PubMed2001

Respiratory syncytial virus infection results in activation of multiple protein kinase C isoforms leading to activation of mitogen-activated protein kinase.

Respiratory syncytial virus (RSV) is an important respiratory pathogen that preferentially infects epithelial cells in the airway and causes a local inflammatory response. Very little is known about the second messenger pathways involved in this response. To characterize some of the acute response pathways involved in RSV infection, we used cultured human epithelial cells (A549) and optimal tissue culture-infective doses (TCID(50)) of RSV. We have previously shown that RSV-induced IL-8 release is linked to activation of the extracellular signal-related kinase (ERK) mitogen-activated protein kinase pathway. In this study, we evaluated the upstream events involved in ERK activation by RSV. RSV activated ERK at two time points, an early time point consistent with viral binding and a later sustained activation consistent with viral replication. We next evaluated the role of protein kinase C (PKC) isoforms in RSV-induced ERK kinase activity. We found that A549 cells contain the Ca(2+)-dependent isoforms alpha and beta1, and the Ca(2+)-independent isoforms delta, epsilon, eta, mu, theta, and zeta. Western analysis showed that RSV caused no change in the amounts of these isoforms. However, kinase activity assays demonstrated activation of isoform zeta within 10 min of infection, followed by a sustained activation of isoforms beta1, delta, epsilon, and mu 24-48 h postinfection. A cell-permeable peptide inhibitor specific for the zeta isoform decreased early ERK kinase activation by RSV. Down-regulation of the other PKC isoforms with PMA blocked the late sustained activation of ERK by RSV. These studies suggest that RSV activates multiple PKC isoforms with subsequent downstream activation of ERK kinase.

Enzyme Activation↗
PubMed2001

Tartrate-resistant acid phosphatase isoform 5b as serum marker for osteoclastic activity.

BACKGROUND: Tartrate-resistant acid phosphatase (AcP) 5b is a marker of osteoclastic activity and bone resorption. Immunoassays for serum TRAcP may lack sensitivity and specificity because of the presence of non-bone isoform 5a. The purpose of this study was to isolate the serum isoforms, quantify their disease-related expressions, and test an improved immunoassay for TRAcP 5b. METHODS: We separated TRAcP isoforms chromatographically from pooled sera of healthy, rheumatoid arthritis (RA) and endstage renal disease (ESRD) subjects. TRAcP isoforms were identified by electrophoresis and quantified by biochemical and immunochemical assays. Serum TRAcP activity in healthy, RA, and ESRD cohorts was assessed at pH 5.5 and 6.1, and compared with bone alkaline phosphatase (BAP) and N-telopeptides of type I collagen (NTx). RESULTS: TRAcP isoforms 5a and 5b were present in all sera; 5b was identical to osteoclastic TRAcP. In serum from healthy subjects, 5a accounted for 87% of the enzyme protein but only 55% of the activity. In RA, both isoforms were increased two- to threefold in protein, but their specific activities were subnormal. In ESRD, only 5b was abnormal, being increased fivefold in protein and threefold in activity. In RA sera, TRAcP activity did not correlate with either BAP or NTx. In ESRD sera, TRAcP activity correlated with BAP and NTx only when measured at pH 6.1. CONCLUSIONS: All sera contained both TRAcP isoforms 5a and 5b, but only 5b was present in bone. TRAcP isoform expression was variable in different diseases. Measurement of TRAcP activity at pH 6.1 improves the specificity of immunoassay for isoform 5b.

Acid Phosphatase↗
PubMed1992

Isoforms of the alpha subunit of Na,K-ATPase and their significance.

Recent studies of the Na,K-ATPase have demonstrated that multiple isoforms of both the alpha and beta subunits exist and that these are expressed in a tissue and developmental specific manner. In the case of the alpha subunit, there are three known isoforms, alpha 1, alpha 2 and alpha 3. We have examined adult human heart for the presence of these isoforms and found that all three exist in approximately equal amounts. This is in contrast to the adult rat heart which contains only alpha 1 and alpha 2 isoforms. The difference in abundance of various isoforms in various tissues could result from the necessity to express Na,K-ATPase with different properties at various developmental stages or in specific cell types. For example, enzymes with differences in Na+ or K+ affinity or the ability to respond to various effector molecules may be required. Alternatively, the presence of three isoforms may simply result from the triplication of the alpha subunit gene and the divergence of expression of these genes during evolution. In this case the isozymes would not confer a specific function to the Na,K-ATPase. In order to provide information with respect to these two alternatives, cell lines producing rat alpha 1, alpha 2 and alpha 3 were developed and the enzymatic properties of the resulting enzyme determined. The results indicate that Na,K-ATPase carrying the alpha 1 are alpha 2 isoforms are fairly similar while enzyme with the alpha 3 isoform differs in its apparent affinity for sodium. The K0.5 for Na+ is approximately three fold lower for this isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Apolipoprotein(a) (Apo(a)) glycoprotein isoforms result from size differences in Apo(a) mRNA in baboons.

Like humans, baboons possess serum lipoprotein(a) that contains apolipoprotein(a) (apo(a], and baboon apo(a) also occurs in distinguishable glycoprotein isoforms. To investigate the molecular basis for isoform size variation, we isolated hepatic RNA from baboons possessing different apo(a) isoforms for Northern blot analyses with rhesus apo(a) cDNA probes. In a survey of 22 baboons, we detected a variety of sizes of apo(a) transcripts (5.2-11.2 kilobases) that corresponded with relative mobilities and numbers of serum apo(a) isoforms. In unrelated baboons possessing apo(a) isoforms of similar mobilities, we detected apo(a) transcripts with similar mobilities. In related baboons possessing apo(a) isoforms that were identical by descent, apo(a) transcripts were also identical in size. Using data from 22 baboons included in this study, we compared the sizes of 20 apo(a) isoforms and corresponding transcripts. Linear regression analysis established a highly significant correlation (p less than 0.001) between estimated apo(a) transcript size and glycoprotein mass (r2 = 0.996). From these results, we conclude that apo(a) glycoprotein isoforms are due to structural differences in apo(a) transcripts. However, we also noted exceptions to correspondence between apo(a) transcripts and isoforms that suggest the action of additional post-transcriptional control of serum apo(a) levels.

Alleles↗
PubMed1989

Human serum amyloid A protein. The assignment of the six major isoforms to three published gene sequences and evidence for two genetic loci.

Serum amyloid A protein (apo-SAA) is an acute-phase reactant and an apolipoprotein of high density lipoproteins (HDL). Six major isoforms of apo-SAA occur in humans (pI 6.0, 6.4, 7.0, 7.4, 7.5, 8.0). In this report we have rationalized the phenotypic expression of apo-SAA isoforms with published apo-SAA structures predicted from apo-SAA cDNA's pA1 and pSAA82 and the genomic DNA SAAg9. The six apo-SAA isoforms fall into three pairs, pI 6.0/6.4, 7.0/7.5, and 7.4/8.0, which are products of cDNA pA1, cDNA pSAA82, and genomic DNA SAAg9, respectively. The second of each isoform pair (i.e. pI 6.4, 7.5, and 8.0) is the "primary" product: a 104-residue peptide with the NH2-terminal sequence Arg-Ser-Phe-Phe. Each primary product is processed either to a major 103-residue peptide with the NH2-terminal sequence Ser-Phe-Phe or processed to a minor 102-residue product which results from the loss of both an Arg and a Ser residue from the NH2 termini. These "secondary" products have the lower pI values of 6.0, 7.0, and 7.4, respectively. The isoelectric points of the SAAg9 products were confirmed by expression of SAAg9 in transfected mouse L-cells. Both the pI 8.0 and 7.4 isoforms were present in cellular extracts, suggesting that post-translational modification of apo-SAA may occur intracellularly. However, the greater relative abundance of the pI 7.4 isoform extracellularly suggests that the major conversion may occur after secretion. Whereas the gene corresponding to the pA1 cDNA sequence does not show allelic variation, the segregation characteristics of the pI 7.0/7.5 and 7.4/8.0 isoform pairs amongst individuals suggests that these isoforms are the products of genes (with sequences corresponding to pSAA82 and SAAg9, respectively) which are allelic variants at a single locus distinct from that for the pI 6.0/6.4 isoform pair.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of two isoforms of the plasma membrane Ca2+-transporting ATPase from rat brain. Structural and functional domains exhibit similarity to Na+,K+- and other cation transport ATPases.

Complementary DNAs for two isoforms of the plasma membrane Ca2+-ATPase from rat brain have been isolated. The cDNAs were identified using an oligonucleotide probe derived from a conserved amino acid sequence of the ATP binding site of the aspartylphosphate family of transport ATPases. The complete nucleotide sequences have been determined, and the primary structures for both isoforms have been deduced. The Mr of isoform 1, which has 1,176 amino acids, is 129,500 and that of isoform 2, which has 1,198 amino acids, is 132,605. A region of isoform 1 exhibits perfect amino acid identity with a fragment consisting of 17 amino acids from the phosphorylation domain of the human erythrocyte calmodulin-sensitive Ca2+-ATPase (James, P., Zvaritch, E.I., Shakhparonov, M.I., Penniston, J.T., and Carafoli, E. (1987) Biochem. Biophys. Res. Commun. 149, 7-12). A comparison of transport ATPases from diverse species has allowed the identification of a sequence that may form part of a second ATP binding site. Amino acid similarity and hydropathy profile comparisons suggest that the transmembrane organization of the plasma membrane Ca2+-ATPase is the same as that of the Na+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase; the data indicate that each of these enzymes has an even number of transmembrane domains and that their C termini are located on the cytoplasmic side of the membrane. An arginine-rich sequence that is highly characteristic of the "A" domain of a calmodulin binding site is located near the C termini of both isoforms. This putative A domain is identical in isoforms 1 and 2 but their "B" domains, and the remaining C-terminal sequences, are very different. However, 3'-untranslated sequences of the isoform 1 transcript have the potential to encode a calmodulin binding B domain and a C terminus that is very similar to that of isoform 2. This suggests the possibility that additional diversity may occur via alternative processing of the primary transcript.

Adenosine Triphosphatases↗