Analysis of the tissue-specific expression, developmental regulation, and linkage relationships of a rodent gene encoding heart fatty acid binding protein

The rat contains at least three homologous cytosolic proteins that bind long chain fatty acids, termed liver (L-), intestinal (I-), and heart (H-) fatty acid binding protein (FABP). I-FABP mRNA is confined to the gastrointestinal tract while L-FABP mRNA is abundantly represented in hepatocytes as well as enterocytes. We have isolated a rat heart FABP cDNA clone and determined the pattern of H-FABP mRNA accumulation in a wide variety of tissues harvested from late fetal, suckling, weaning, and adult rats. RNA blot hybridizations and primer extension analysis disclosed that the distribution of H-FABP mRNA in adult rat tissues is different from that of I- or L-FABP mRNA. H-FABP mRNA is most abundant in adult heart. This mRNA was also present in an adult slow twitch (type I) skeletal muscle (soleus, 63% of the concentration in heart), testes (28%), a fast twitch skeletal muscle (psoas, 17%), brain (10%), kidney (5%), and adrenal gland (5%). H-FABP mRNA was not detected in adult small intestine, colon, spleen, lung, or liver RNA. Distinct patterns of developmental change in H-FABP mRNA accumulation were documented in heart, placenta, brain, kidney, and testes. Myocardial H-FABP mRNA levels rise rapidly during the 48 h prior to and after birth, reaching peak levels by the early weaning period. The postnatal increase in myocardial H-FABP mRNA concentration and its relative distribution in adult fast and slow twitch skeletal muscle are consistent with its previously proposed function in facilitating mitochondrial beta-oxidation of fatty acids. However, the presence of H-FABP mRNA in brain, a tissue which does not normally significantly oxidize fatty acids in late postnatal life, suggests that H-FABP may play a wider role in fatty acid metabolism than previously realized. Mouse-hamster somatic cell hybrids were utilized to map H-FABP. Using stringencies which did not produce cross-hybridization between L-, I-, and H-FABP DNA sequences, we found at least three loci in the mouse genome, each located on different chromosomes, which reacted with our cloned H-FABP cDNA. None of these H-FABP-related loci were linked to the gene which specifies a highly homologous adipocyte-specific protein termed aP2 or to genes encoding two other members of this protein family, cellular retinol binding protein and cellular retinol binding protein II.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of the cDNA encoding rat skeletal muscle myosin light chain kinase. Sequence and tissue distribution

A cDNA clone encoding skeletal muscle myosin light chain kinase (MLCK) was isolated from a rat skeletal muscle library using oligonucleotide probes. The total length of the rat skeletal muscle MLCK cDNA was 2823 base pairs with an open reading frame of 1830 base pairs. The deduced sequence of the 610-amino acid protein exhibited 96% amino acid identity to rabbit skeletal muscle MLCK in the carboxyl-terminal portion of the molecule, which contains the catalytic and the calmodulin-binding domains, and 58% identity in the amino-terminal region. Analysis of total rat mRNA revealed a single mRNA species of 3.4 kilobases that was unique to skeletal muscle. Further analysis of skeletal muscle tissue using fast-twitch glycolytic, fast-twitch oxidative glycolytic, and slow-twitch oxidative fibers isolated from rat leg revealed that the mRNA level for MLCK varied among the three fiber types. The results of kinase assays performed on the fibers showed that MLCK activity levels paralleled the MLCK mRNA levels found in each of the three types of skeletal muscle fibers studied. Fast-twitch oxidative glycolytic (gastrocnemius red) and slow-twitch oxidative (soleus) exhibited 60 and 13%, respectively, of the enzymatic activity present in fast-twitch glycolytic (gastrocnemius white) fibers.     

Characterization and expression of the rat heart sarcoplasmic reticulum Ca2+-ATPase mRNA

Sarcoplasmic reticulum Ca2+-ATPase cDNA clones have been isolated from an adult rat heart cDNA library and the nucleotide sequence of the Ca2+-ATPase mRNA determined. The sequence has an open reading frame of 997 codons. It is identical to a cDNA isolated from a rat stomach cDNA library and 90% isologous to the rabbit and human slow/cardiac cDNAs. Nuclease S1 mapping analysis indicates that this sequence corresponds to the main Ca2+-ATPase mRNA present in heart and in slow skeletal muscle and that it is expressed in various proportions in smooth and non-muscle tissues, together with another isoform which differs from the cardiac form in the sequence of its 3'-end.     

Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the first step in formation of AMP from IMP. At least two isozymes exist in vertebrate tissue. An acidic form, present in most tissues, has been suggested to be involved in de novo biosynthesis while a basic isozyme, which predominates in muscle, appears to function in the purine nucleotide cycle. Antibodies specific for the basic isozyme detect a single protein in mouse tissues with highest levels in skeletal muscle, tongue, esophagus, and heart tissue consistent with a role for the enzyme in muscle metabolism. A series of degenerate oligonucleotides were constructed based on peptide sequences from purified rat muscle enzyme and then used to clone a mouse muscle cDNA encoding the basic isozyme. The clone contains a open reading frame of 1356 bases with 452 amino acids. Northern analysis of RNA from mouse tissues showed a tissue distribution similar to that of the protein, indicating a high level of gene expression in muscle. Transfection of COS cells with the mouse muscle cDNA allows expression of a functional protein with a molecular mass of approximately 50 kDa, consistent with the open reading frame and the size of the isolated rat enzyme. The deduced amino acid sequence of the mouse synthetase is 47 and 37% identical to the synthetase sequences from Dictyostelium discoideum and Escherichia coli, respectively. The availability of antibodies and cDNA clones specific for the basic isozyme of adenylosuccinate synthetase from muscle will facilitate future genetic and biochemical analysis of this protein and its role in muscle physiology.     

Fatty acid binding protein 4 in human skeletal muscle

The mechanisms that regulate intramyocellular triglycerol (IMTG) storage and mobilization are largely unknown. However, during the last decades several intracellular fatty acid binding proteins (FABPs) have been identified. FABP3 is the dominating FABP in skeletal muscle. Expression of additional FABPs is suggested from findings in FABP3-null mutated mice. In the present study, our aims were to investigate if FABP4 is expressed within skeletal muscle fibers and if FABP3 and FABP4 are more abundant in skeletal muscle fibers in endurance-trained than in control subjects. We show that FABP4 protein is expressed within the skeletal muscle fibers and that FABP4 mRNA and protein are more abundant in the endurance trained subjects. Still, FABP4 is markedly less expressed than FABP3, which is the generally accepted dominating FABP in skeletal muscle tissue.     

Cloning, expression, and mapping of PDCD9, the human homolog of Gallus gallus pro-apoptotic protein p52

We report the sequence, tissue distribution, and chromosome location of a novel gene, PDCD9 (programmed cell death 9). PDCD9 is the mammalian counterpart of the Gallus gallus pro-apoptotic protein p52. The human cDNA has an open reading frame of 1,314 nucleotides and was predicted to encode a protein of 438 amino acids with a calculated mass of 50 kDa. The protein sequences of chicken, mouse, and human PDCD9 are remarkably conserved. PDCD9 mRNA is expressed ubiquitously in adult tissues, displaying a stronger signal in heart, skeletal muscle, kidney, and liver. PDCD9 was mapped to chromosome 5q11.     

Intracellular transport of lipids

Summary Translocation of lipids inside mammalian cells is considered to be facilitated by a number of low-molecular weight lipid binding proteins. An overview of these proteins is given, with particular reference to the heart. Three distinct phospholipid transfer proteins specifically stimulate the net transfer of individual phospholipid classes between membrane structures. In rat cardiac muscle their content is 15–140 pmol/g ww. Fatty acid-binding proteins (FABP) are abundantly present in tissues actively involved in the uptake or utilization of long-chain fatty acids, such as intestine, liver and heart. The four distinct FABP types now identified show a complex tissue distribution with some tissues containing more than one type. Heart (H-) FABP comprises about 5% of the cytosolic protein mass; its content in rat heart is 100 nmol/g ww. Immunochemical evidence has been obtained for the presence of H-FABP in several other tissues, including red skeletal muscle, mammary gland and kidney. Beside long-chain fatty acids FABP binds with similar affinity also fatty acyl-CoA and acyl-L-carnitines. In heart the latter compound may be the primary ligand, since normoxic acyl-L-carnitine levels are several fold higher than those of fatty acids. In addition, H-FABP was found to modulate cardiac energy production by controlling the transfer of acyl-L-carnitine to the mitochondrial -oxidative system. H-FABP may also protect the heart against the toxic effects of high intracellular levels of fatty acid intermediates that arise during ischemia.     

CoA-dependent methylmalonate-semialdehyde dehydrogenase, a unique member of the aldehyde dehydrogenase superfamily. cDNA cloning, evolutionary relationships, and tissue distribution.

Three overlapping cDNA clones encoding methylmalonate-semialdehyde dehydrogenase (MMSDH; 2-methyl-3-oxopropanoate:NAD+ oxidoreductase (CoA-propanoylating); EC 1.2.1.27) have been isolated by screening a rat liver lambda gt 11 library with nondegenerate oligonucleotide probes synthesized according to polymerase chain reaction-amplified portions coding for the N-terminal amino acid sequence of rat liver MMSDH. The three clones cover a total of 1942 base pairs of cDNA, with an open reading frame of 1569 base pairs. The authenticity of the composite cDNA was confirmed by a perfect match of 43 amino acids known from protein sequencing. The composite cDNA predicts a 503 amino acid mature protein with M(r) = 55,330, consistent with previous estimates. Polymerase chain reaction was used to obtain the sequence of the 32 amino acids corresponding to the mitochondrial entry peptide. Northern blot analysis of total RNA from several rat tissues showed a single mRNA band of 3.8 kilobases. Relative mRNA levels were: kidney greater than liver greater than heart greater than muscle greater than brain, which differed somewhat from relative MMSDH protein levels determined by Western blot analysis: liver = kidney greater than heart greater than muscle greater than brain. A 1423-base pair cDNA clone encoding human MMSDH was isolated from a human liver lambda gt 11 library. The human MMSDH cDNA contains an open reading frame of 1293 base pairs that encodes the protein from Leu-74 to the C terminus. Human and rat MMSDH share 89.6 and 97.7% identity in nucleotide and protein sequence, respectively. MMSDH clearly belongs to a superfamily of aldehyde dehydrogenases and is closely related to betaine aldehyde dehydrogenase, 2-hydroxymuconic semialdehyde dehydrogenase, and class 1 and 2 aldehyde dehydrogenases.     

Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol

Cellular retinol-binding protein, type I (CRBP-I) and type II (CRBP-II) are the only members of the fatty acid-binding protein (FABP) family that process intracellular retinol. Heart and skeletal muscle take up postprandial retinol but express little or no CRBP-I or CRBP-II. We have identified an intracellular retinol-binding protein in these tissues. The 134-amino acid protein is encoded by a cDNA that is expressed primarily in heart, muscle and adipose tissue. It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the FABP family. In situ hybridization demonstrates that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse. Fluorescence titrations of purified recombinant protein with retinol isomers indicates binding to all-trans-, 13-cis-, and 9-cis-retinol, with respective K(d) values of 109, 83, and 130 nm. Retinoic acids (all-trans-, 13-cis-, and 9-cis-), retinals (all-trans-, 13-cis-, and 9-cis-), fatty acids (laurate, myristate, palmitate, oleate, linoleate, arachidonate, and docosahexanoate), or fatty alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind. The distinct tissue expression pattern and binding specificity suggest that we have identified a novel FABP family member, cellular retinol-binding protein, type III.     

Rat RI beta isoform of type I regulatory subunit of cyclic adenosine monophosphate-dependent protein kinase: cDNA sequence analysis, mRNA tissue specificity, and rat/mouse difference in expression in testis

A rat complementary DNA (cDNA) for the RI beta isoform of type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase regulatory subunit was cloned and sequenced and was found to contain the entire protein coding and 3'-untranslated regions, with a single (ATTAAA) poly-adenylation site. The largest open reading frame was preceded by a short out-of-phase open reading frame, which is not seen in the corresponding mouse RI beta cDNA due to a single base substitution. The rat RI beta cDNA clone was 2,374 bases long and detected a rat mRNA of approximately 2.8 kilobases. Rat RI beta mRNA was abundant in adult rat brain and testis but was undetectable in other rat tissues. The rat RI beta cDNA also detected RI beta mRNA in mouse brain, but not mouse testis, from 10-week-old BALB/c or 10- and 6-week-old Swiss Webster mice. Thus, despite a 96% nucleotide identity in the coding region of RI beta in rat vs. mouse, there are at least two differences in these closely related species. First, there is a short open reading frame, which precedes the coding region in the rat but not the mouse. Second, unlike the mouse testis, the rat testis contains abundant levels of RI beta mRNA.     

Fatty acid oxidation capacity and fatty acid-binding protein content of different cell types isolated from rat heart

Summary Heart tissue contains appreciable amounts of fatty acid-binding protein (FABP). FABP is thought to play a crucial role in the transport of fatty acids from the cellular membrane to the intracellular site of oxidation and also, in case of endothelial cells, in the transfer of fatty acids from the vascular to the interstitial compartment through the endothelial cytoplasm. The present study was designed to delineate a possible quantitative relationship between the capacity of different cell types in the heart to oxidize fatty acids and the presence of FABP. Palmitate oxidation capacity, measured in homogenates of cells isolated from adult rat hearts, was 2 nmol/min per mg tissue protein in freshly isolated cardiomyocytes (CMC), but only 0.09 and 0.31 nmol/min per mg tissue protein in cultivated endothelial (CEC) and fibroblast-like cells (CFLC), respectively. Palmitate oxidation rates were closely related to the cytochrome C oxidase activity and, hence, to the mitochondrial density in the cells under investigation. In CMC the content of cytosolic H-FABP (H-FABP_c) was about 4.51 µg/mg tissue protein. However, in CEC and CFLC the FABP content was less than 0.01 and 0.004 µg/mg tissue protein, respectively, corresponding to at maximum 0.2% of the FABP content of CMC. These findings indicate a marked difference between CMC and non-myocytal cells in the heart regarding their capacity to oxidize fatty acids, and a marked disproportion between the fatty acid oxidation capacity and immunochemically determined FABP content in both CEC and CFLC. The functional implication of these observations remains to be elucidated.     

Fatty acid binding proteins from heart

Heart contains a fatty acid binding protein (FABP) concentration comparable to liver, when it is determined with a fatty acid-binding assay. The low concentration detected with anti-liver FABP antibodies is related to the different chemical forms and physiochemical properties of liver and heart FABP. The ratio of fatty acid bound per purified protein molecule is one or lower. Rat heart mitochondria oxidize FABP-bound fatty acids. The FABP content of rat heart is dependent on sex and diurnal cycle, but is not influenced by starvation or clofibrate feeding. It is also not different in the newborn rat. FABP was obtained from human heart in a yield of 11%. It shows similar binding characteristics to palmitic, oleic and arachidonic acid. The functional significance of the specific heart FABP is discussed in relation to myocardial fatty acid metabolism in normal and pathological conditions.     

Fatty acid-binding protein and its relation to fatty acid oxidation

A relation between fatty acid oxidation capacity and cytosolic FABP content was found in heart and various muscles of the rat. Other tissues do not show such a relation, since they are involved in more or other pathways of fatty acid metabolism. At postnatal development FABP content and fatty acid oxidation capacity rise concomitantly in heart and quadriceps muscle in contrast to in liver and kidney. A dietary fat content of 40 en. % increased only the FABP content of liver and adipose tissue. Peroxisomal proliferators increased fatty acid oxidation in both liver and kidney, but only the FABP content of liver, and had no effect on heart and skeletal muscle. The FABP content of muscle did not show adaptation to various conditions. Only it increased in fast-twitch muscles upon chronic electrostimulation and endurance training.     

The fatty acid-binding protein from human skeletal muscle

Fatty acid-binding protein (FABP) was isolated from human skeletal muscle by gel filtration and anion- and cation-exchange chromatography. The isolation procedure, however, with rat and pig skeletal muscle gave mostly inactive preparations. Rat muscle FABP preparations contained parvalbumin as a contaminant. FABP from human muscle had a Mr of about 15 kDa, a pI value of 5.2, and a Kd value with oleic acid of 0.50 microM. Skeletal muscle and heart FABPs and their antisera showed a strong cross-reactivity on Western blots and in enzyme-linked immunosorbent assays (ELISA). No cross-reactivity was observed with liver FABP and its antiserum. On the basis of amino acid composition, electrophoretic behavior, fatty acid binding, and immunochemical properties, human skeletal muscle FABP must be similar or closely related to human heart FABP. The FABP content determined by ELISA was comparable in various human muscles and cultured muscle cells, but lower than that in rat muscles.     

Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development

A full-length cDNA clone that codes for glucose transporter protein was isolated from a rabbit brain cDNA library by using synthetic oligonucleotide probe derived from the sequence of human glucose transporter cDNA. The coding region shared 93.2% nucleotide and 97.0% amino-acid similarities with those of human glucose transporter and 89.4% nucleotide and 97.4% amino-acid similarities with those of rat transporter. Northern blot analysis revealed that glucose transporter mRNA is most abundant in the placenta and that it is also abundant in the brain. The fat tissue, heart, liver, and skeletal muscle of adult rats contained a very small amount of mRNA, while heart, liver, skeletal muscle and kidney of fetal rats contained a very high amount of glucose transporter mRNA. These results suggest that this type of glucose transporter might be closely related with cell proliferation and tissue development.