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D W Crabb

Publications and source records attributed to D W Crabb.

At least 19 recordsLinked to original sources
PubMed1995

A new family of protein kinases--the mitochondrial protein kinases.

Molecular cloning has provided evidence for a new family of protein kinases in eukaryotic cells. These kinases show no sequence similarity with other eukaryotic protein kinases, but are related by sequence to the histidine protein kinases found in prokaryotes. These protein kinases, responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase complexes, are located exclusively in mitochondrial matrix space and have most likely evolved from genes originally present in respiration-dependent bacteria endocytosed by primitive eukaryotic cells. Long-term regulatory mechanisms involved in the control of the activities of these two kinases are of considerable interest. Dietary protein deficiency increases the activity of branched-chain alpha-ketoacid dehydrogenase kinase associated with the branched-chain alpha-ketoacid dehydrogenase complex. The amount of branched-chain alpha-ketoacid dehydrogenase kinase protein associated with the branched-chain alpha-ketoacid dehydrogenase complex and the message level for branched-chain alpha-ketoacid dehydrogenase kinase are both greatly increased in the liver of rats starved for protein, suggesting increased expression of the gene encoding branched-chain alpha-ketoacid dehydrogenase kinase. The increase in branched-chain alpha-ketoacid dehydrogenase kinase activity results in greater phosphorylation and lower activity of the branched-chain alpha-ketoacid dehydrogenase complex. The metabolic consequence is conservation of branched chain amino acids for protein synthesis during periods of dietary protein deficiency. Two isoforms of pyruvate dehydrogenase kinase have been identified and cloned. Pyruvate dehydrogenase kinase 1, the first isoform cloned, corresponds to the 48 kDa subunit of the pyruvate dehydrogenase kinase isolated from rat heart tissue. Pyruvate dehydrogenase kinase 2, the second isoform cloned, corresponds to the 45 kDa subunit of this enzyme. In addition, it also appears to correspond to a possibly free or soluble form of pyruvate dehydrogenase kinase that was originally named kinase activator protein. Assuming that differences in kinetic and/or regulatory properties of these isoforms exist, tissue specific expression of these enzymes and/or control of their association with the complex will probably prove to be important for the long term regulation of the activity of the pyruvate dehydrogenase complex. Starvation and the diabetic state are known to greatly increase activity of the pyruvate dehydrogenase kinase in the liver, heart and muscle of the rat. This contributes in these states to the phosphorylation and inactivation of the pyruvate dehydrogenase complex and conservation of pyruvate and lactate for gluconeogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1995

The aldehyde dehydrogenase ALDH2*2 allele exhibits dominance over ALDH2*1 in transduced HeLa cells.

Individuals heterozygous or homozygous for the variant aldehyde dehydrogenase (ALDH2) allele (ALDH2*2), which encodes a protein differing only at residue 487 from the normal protein, have decreased ALDH2 activity in liver extracts and experience cutaneous flushing when they drink alcohol. The mechanisms by which this allele exerts its dominant effect is unknown. To study this effect, the human ALDH2*1 cDNA was cloned and the ALDH2*2 allele was generated by site-directed mutagenesis. These cDNAs were transduced using retroviral vectors into HeLa and CV1 cells, which do not express ALDH2. The normal allele directed synthesis of immunoreactive ALDH2 protein (ALDH2E) with the expected isoelectric point. Extracts of these cells contained increased aldehyde dehydrogenase activity with low Km for the aldehyde substrate. The ALDH2*2 allele directed synthesis of mRNA and immunoreactive protein (ALDH2K), but the protein lacked enzymatic activity. When ALDH2*1-expressing cells were transduced with ALDH2*2 vectors, both mRNAs were expressed and immunoreactive proteins with isoelectric points ranging between those of ALDH2E and ALDH2K were present, indicating that the subunits formed heteromers. ALDH2 activity in these cells was reduced below that of the parental ALDH2*1-expressing cells. Thus, the ALDH2*2 allele is sufficient to cause ALDH2 deficiency in vitro.

Aldehyde Dehydrogenase↗
PubMed1994

Seven transmembrane domain receptor subtypes identified in NG108-15 cells by reverse transcription-polymerase chain reaction.

NG108-15 neuroblastoma x glioma cells are widely used for the study of neurotransmitter receptors. We utilized reverse transcription-polymerase chain reaction to amplify members of the seven transmembrane domain class of G-protein linked receptors using RNA isolated from NG108-15 cells. Two complementary DNAs representing receptors were obtained; based upon comparison with the sequence database, they probably represent the murine dopamine D1A receptor and a receptor closely related to the serotonin 5HT1D receptor subtype. The finding of the 5HT receptor subtype is of interest, as only the 5HT3 subtype was previously identified in NG108-15 cells by pharmacological means. Certain responses of NG108-15 cells to serotonin have been described that do not appear to be mediated by known 5HT receptor subtypes. The cDNA we cloned may therefore represent an additional 5HT1D subclass.

Amino Acid Sequence↗
PubMed1992

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.

Aldehyde Oxidoreductases↗
PubMed1992

The first 22 base pairs of the proximal promoter of the rat class I alcohol dehydrogenase gene is bipartite and interacts with multiple DNA-binding proteins.

The rat class I alcohol dehydrogenase (ADH) gene is primarily expressed in the liver. We previously showed that the liver-enriched transcription factor, the CCAAT/enhancer binding protein (C/EBP), binds to the proximal promoter of the rat class I ADH gene between positions -11 and -22 relative to the start site of transcription. We now demonstrate that another transcription factor, the liver activator protein (LAP), also interacts with the same region of the promoter based on the following observations: (1) LAP synthesized by in vitro transcription and translation of cloned cDNA sequence forms complexes with an oligonucleotide containing the C/EBP-binding sequence within the ADH promoter as determined by the electrophoretic mobility shift assay (EMSA), (2) purified LAP interacts with the proximal ADH promoter when analyzed by the DNase I protection assay, and (3) an ADH promoter-reporter gene construct containing the C/EBP-binding site is transactivated by an eukaryotic expression vector containing the LAP sequence. EMSA of an oligonucleotide containing the first 22 base pairs (between positions -1 and -22) of the ADH promoter with rat liver nuclear extracts (RLNE) resulted in the formation of two major complexes. Complex 1 was competed away by a heterologous oligonucleotide containing a C/EBP-binding site within the promoter of the adipocyte 422 (aP2) gene, while complex 2 was not. Additional competition experiments with the ADH or 422 (aP2) oligonucleotide using either RLNE or extracts from 3T3-L1 adipocytes demonstrated that complex 1 contains either C/EBP or LAP, while complex 2 contains a DNA-binding protein that binds to a novel sequence 5'-TGGCCCAGTT-3' between positions -1 and -10 of the ADH promoter. Ultraviolet cross-linking between RLNE and a labeled oligonucleotide containing the above sequence indicates that this protein, designated EDBP (for enhancer-site downstream binding protein), has an estimated molecular weight of 47 kDa, which is larger than that reported for either C/EBP (42 kDa) or LAP (36 kDa).

3T3 Cells↗
PubMed1992

Estradiol regulates class I alcohol dehydrogenase gene expression in renal medulla of male rats by a post-transcriptional mechanism.

Rat kidney contains alcohol dehydrogenase (ADH) activity which appears to be identical to the class I ADH expressed in liver. Treatment of male rats with estradiol for 10 days induced ADH activity and protein in the kidney approximately 3-fold. This was not the result of suppression of testosterone levels by estrogen, as castration did not increase ADH activity. In situ hybridization of kidney sections showed that ADH transcripts were localized to the medulla, that the basal level of mRNA is very low in the male, and that the induction of ADH mRNA by estradiol was approximately 10-fold. As estimated from Northern blot analysis, the induction of the mRNA was approximately 7-fold. Thus, induction of ADH mRNA substantially exceeded the increase of ADH activity and protein. Since the estradiol-treated rats lost weight relative to the oil-injected controls, the effect of starvation on ADH mRNA in kidney was examined. Starvation decreased kidney ADH activity by about 30% but increased mRNA about 2-fold. Time course experiments demonstrated induction of ADH mRNA by estradiol within 1 h with the maximum level achieved by 24 h. The transcription rate of the ADH gene as assessed by nuclear run-on assays performed at 1 and 24 h after treatment with estradiol was unchanged. We conclude that estradiol induces ADH mRNA in kidney by a post-transcriptional mechanism.

Alcohol Dehydrogenase↗
PubMed1992

Corticosterone induces rat liver alcohol dehydrogenase mRNA but not enzyme protein or activity.

Glucocorticoids induced ADH activity and mRNA 2- to 4-fold in rat hepatoma cells (H4IIE and H4IIEC3), but were reported not to alter ADH activity in rat liver. The failure of corticosteroids to induce ADH may have been due to the short-term treatment of the rats or the dose of steroid used. To reevaluate the effect of glucocorticoids in vivo, we studied animals 4.5 weeks after adrenalectomy so that ADH activity and mRNA should have reached a new steady-state level; the dose of glucocorticoid used was estimated to provide physiological replacement. Male Wistar rats were injected with a single daily dose (10 mg/kg/day) of corticosterone-21-acetate or vehicle subcutaneously for 10 days. Liver extracts were assayed for ADH activity, ADH protein, and ADH mRNA. Nuclei were isolated for nuclear run-on assays. Adrenalectomy did not reduce the activity of ADH in liver. Subsequent corticosterone treatment did not alter ADH enzyme activity, nor did it affect ADH protein levels as analyzed on Western blots. However, Northern blot analysis of ADH mRNA indicated a 2-fold increase in ADH mRNA in the treated animals when the data were normalized to the level of the 28S ribosomal RNA or CHO-B mRNA. The rate of transcription of the ADH gene in nuclei isolated at the end of 10 days of treatment from corticosterone-treated adrenalectomized rats was not statistically different from that in the oil-treated adrenalectomized ones. The disparity between ADH activity and protein levels and the mRNA level may have resulted from other effects of corticosterone, e.g., stimulation of protein degradation or effects on translation.

Alcohol Dehydrogenase↗
PubMed1991

Estradiol induces class I alcohol dehydrogenase activity and mRNA in kidney of female rats.

Rat kidney contains alcohol dehydrogenase (ADH) activity which appears to be similar or identical to the class I ADH expressed in liver. Both tissues contain a 1.6-kb transcript which hybridizes with an ADH cDNA under stringent conditions. Kidney ADH activity is responsive to estradiol. The enzyme activity in the kidneys of sham-operated and ovariectomized animals was the same. Treatment of either group of animals by intramuscular injection of estradiol (1 mg/kg body wt/day) for 10 days induced ADH activity in kidney two- to threefold, whether the activity was expressed as U/g tissue, U/g protein, or U/mg DNA. Estradiol induced kidney ADH mRNA in both ovariectomized and sham-operated rats approximately twofold. Thus, induction of ADH mRNA accounts for the increase in ADH activity. In situ hybridization indicated that the ADH mRNA was present in the inner cortex and medulla of the kidney. Methylation patterns of the ADH gene were examined. The gene resides in a methylated region of chromatin without any of the typical features of a HpaII tiny fragment (HTF) island. Two MspI sites flanking the transcription start site are undermethylated in liver compared with kidney and spleen. This suggests that methylation of this gene may play a role in the tissue-specific expression of ADH.

Alcohol Dehydrogenase↗
PubMed1991

Investigation of the role of polymorphisms at the alcohol and aldehyde dehydrogenase loci in genetic predisposition to alcohol-related end-organ damage.

Little is known about factors determining individual susceptibility to the physical complications of alcohol abuse but genetically determined differences in ethanol metabolism may be important. The oxidative metabolism of alcohol is catalyzed by alcohol and aldehyde dehydrogenase. Polymorphisms have been observed at two of the five loci encoding alcohol dehydrogenase subunits: ADH2 (producing three beta subunits) and ADH3 (producing two tau subunits) and also at the locus encoding the metabolically important form of aldehyde dehydrogenase, ALDH2. We have compared ADH2, ADH3 and ALDH2 allele frequencies in patients with alcohol-related cirrhosis (n = 59) and chronic pancreatitis (n = 13) with 79 local healthy control subjects. The different alleles were detected with allele-specific oligonucleotide probes after amplification of leukocyte DNA by the polymerase chain reaction. All patients and all but one control subject were homozygous ADH2*1, encoding the beta 1 subunit. No ADH2*3 alleles were detected. All 34 patients and 39 control subjects tested were homozygous ALDH2*1 encoding the active enzyme. ADH3 allele frequencies were different in patients and control subjects. ADH3*1 frequency: control subjects, 55.1%; cirrhotic patients, 62.7%; chronic pancreatitis patients, 65.4%. The difference between the patient groups combined and the control subjects was significant (p less than 0.05; G-test of Sokal and Rohlf) if it was assumed that the allele frequency in our control population was a reasonable estimate of our local population allele frequency. These results suggest that genetically determined differences in alcohol metabolism may, in part, explain predisposition to alcohol-related end-organ damage.

Alcohol Dehydrogenase↗
PubMed1991

CCAAT/enhancer binding protein binds and activates the promoter of the rat class I alcohol dehydrogenase gene.

CCAAT/enhancer binding protein (C/EBP), a DNA binding protein originally isolated from rat liver, activates the transcription of a number of liver-specific genes. We studied the effect of C/EBP on the transcription of the rat class I alcohol dehydrogenase gene. Purified C/EBP was shown to bind to a segment of the rat class I alcohol dehydrogenase gene promoter between position -10 and -22. Nuclear extracts isolated from rat liver contained a heat stable factor(s) that also bound to this sequence. Cotransfection experiments in HepG2 cells showed transactivation of alcohol dehydrogenase promoter-CAT fusion constructs by an expression vector containing wild-type C/EBP. Expression vector containing a mutated C/EBP that failed to bind DNA also failed to activate the promoter. These experiments demonstrate that C/EBP expression can activate the rat class I alcohol dehydrogenase gene promoter.

Alcohol Dehydrogenase↗
PubMed1991

Polymorphism of the rat liver mitochondrial aldehyde dehydrogenase cDNA.

In humans, a deficiency in mitochondrial aldehyde dehydrogenase (Class 2 ALDH) activity due to a single base-pair exchange in its structural gene serves as a deterrent to excessive alcohol consumption. Differences in Class 2 ALDH isozyme patterns on isoelectric focusing gels have been observed in the selectively bred, alcohol-preferring (P) and alcohol-nonpreferring (NP) lines of rats. To determine whether the differences are the result of sequence variation in the structural gene, we sequenced the cDNAs for Class 2 ALDH from P and NP rats. A synonymous exchange was seen in the codon for amino acid 473 in both lines, when compared with published sequences. Additionally, when the cDNA from P rats was used as reference, a substitution (G for A) was identified in the cDNA of NP rats which changes amino acid 67 from Gln (CAG codon; ALDH2Q allele) to Arg (CGG codon; ALDH2R allele). The Arg for Gln substitution makes the enzyme more basic and could account for the different electrophoretic mobilities. To determine whether the polymorphism was associated with drinking behavior, we genotyped the ALDH2 locus by amplifying rat genomic DNA encompassing the nucleotide exchange followed by probing with allele-specific oligonucleotides. There are highly significant differences in the frequencies of the two alleles in the P and NP rat lines. The frequency of the ALDH2R allele is 63% in the NP line and only 18% in the P line, whereas the frequency of the ALDH2Q allele is 82% in the P line and 37% in the NP line.

Alcoholism↗
PubMed1991

Molecular defects in the E1 alpha subunit of the branched-chain alpha-ketoacid dehydrogenase complex that cause maple syrup urine disease.

Maple syrup urine disease (MSUD) results from an inborn metabolic error caused by a deficiency of the branched-chain alpha-ketoacid dehydrogenase complex (BCKDC). cDNA clones encoding the E1 alpha subunit of BCKDC from rat and human liver have been isolated and characterized. The chromosomal location of E1 alpha on chromosome 19q13.1-13.2 has been determined using complementary methods. The etiology of MSUD has been studied by determining the enzyme activity, protein mass and mRNA level of BCKDC in fibroblasts from a human family and Polled Hereford calves, both with classic MSUD. A TACTyr to AACAsn substitution at residue 394 of the E1 alpha subunit was identified in the human patient by using enzymatic amplification of mRNA followed by DNA sequencing. Amplification of both mRNA and genomic DNA, in combination with allele-specific oligonucleotide hybridization, demonstrated that the patient was a compound heterozygote, inheriting an allele with a structural mutation from the father, and an allele from the mother containing a presumably cis-acting defect in regulation that abolished the expression of one of the E1 alpha alleles. The results revealed for the first time that a case of MSUD was caused by structural and regulatory mutations involving the E1 alpha subunit. Recent studies by others have demonstrated that the same structural mutation as is found in this patient is responsible for the high incidence of MSUD in the Philadelphia Mennonite population.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗