Comparison of D-beta-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria

The properties of D-beta-hydroxybutyrate dehydrogenase (BDH) from rat liver and brain mitochondria were compared to determine if isozymes of this enzyme exist in these tissues. The BDHs from these tissues behaved similarly during the purification process. The enzymes were indistinguishable by sodium dodecyl sulfate-polyacrylamide or acid-urea-polyacrylamide gel electrophoresis and they had identical isoelectric points. The BDHs from rat liver and brain were also quite similar in functional parameters determined by kinetic analysis and phospholipid activation of apo-BDH (i.e., the lipid-free enzyme). Antiserum against rat liver BDH inhibited both enzymes to an equivalent extent in a titration assay. The enzymes had similar patterns of peptide mapping by partial digestion with Staphylococcus aureus V8 protease, followed by immunoblotting using antiserum against the liver enzyme. These results suggest that the BDHs in rat liver and brain are very similar and possibly identical.     

Comparison of ornithine decarboxylase from rat liver, rat hepatoma and mouse kidney.

Comparisons were made of ornithine decarboxylase isolated from Morris hepatoma 7777, thioacetamide-treated rat liver and androgen-stimulated mouse kidney. The enzymes from each source were purified in parallel and their size, isoelectric point, interaction with a monoclonal antibody or a monospecific rabbit antiserum to ornithine decarboxylase, and rates of inactivation in vitro, were studied. Mouse kidney, which is a particularly rich source of ornithine decarboxylase after androgen induction, contained two distinct forms of the enzyme which differed slightly in isoelectric point, but not in Mr. Both forms had a rapid rate of turnover, and virtually all immunoreactive ornithine decarboxylase protein was lost within 4h after protein synthesis was inhibited. Only one form of ornithine decarboxylase was found in thioacetamide-treated rat liver and Morris hepatoma 7777. No differences between the rat liver and hepatoma ornithine decarboxylase protein were found, but the rat ornithine decarboxylase could be separated from the mouse kidney ornithine decarboxylase by two-dimensional gel electrophoresis. The rat protein was slightly smaller and had a slightly more acid isoelectric point. Studies of the inactivation of ornithine decarboxylase in vitro in a microsomal system [Zuretti & Gravela (1983) Biochim. Biophys. Acta 742, 269-277] showed that the enzymes from rat liver and hepatoma 7777 and mouse kidney were inactivated at the same rate. This inactivation was not due to degradation of the enzyme protein, but was probably related to the formation of inactive forms owing to the absence of thiol-reducing agents. Treatment with 1,3-diaminopropane, which is known to cause an increase in the rate of degradation of ornithine decarboxylase in vivo [Seely & Pegg (1983) Biochem. J. 216, 701-717] did not stimulate inactivation by microsomal extracts, indicating that this system does not correspond to the rate-limiting step of enzyme breakdown in vivo.     

Purification of the high-Km aldehyde reductase from rat brain and liver and from ox brain.

A procedure is described that yields an apparently homogeneous preparation of the high-Km aldehyde reductase from rat brain. This procedure is also applicable to the purification of this enzyme from rat liver and ox brain. In the latter case, however, the purified preparation could be resolved into two protein bands, both of which had enzyme activity, by polyacrylamide-gel electrophoresis. Since a sample of the ox brain enzyme from an earlier step in the purification procedure only showed the presence of a single band of activity after electrophoresis, this apparent multiplicity probably results from modification of the enzyme, possibly by oxidation, during the final step of the purification. A number of properties of the rat brain enzyme were determined and these were compared with those of the enzyme from rat liver. The two preparations were similar in their stabilities, behaviour during purification, kinetic properties, electrophoretic mobilities and amino acid compositions. Antibodies to the rat liver enzyme cross-reacted with that from brain and the inhibition of both these preparations by the antiserum was similar, further supporting the view that the enzymes from these two sources were closely similar if not identical.     

Phosphofructokinase from porcine heart, liver and erythrocytes

1. Phosphofructokinase from porcine heart, liver and erythrocytes were purified by affinity chromatographies on Cibacron Blue Sepharose and N6-ATP agarose. 2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the heart and liver enzymes consist of only one kind of subunit, namely, M and L type subunits, respectively, whereas the erythrocyte enzyme comprises of three kinds of subunits, M, L and C types. 3. Some kinetic and regulatory properties of the enzymes were also measured.     

Properties of catechol O-methyltransferases from brain and liver of rat and human.

Kinetic and electrophoretic properties of catechol O-methyltransferases (EC 2.1.1.6) from brain and liver were studied. The enzyme of either rat or human tissues exhibited a single molecular form when subjected to electrophoresis at pH7.9. At pH9 a second, apparently oxidized, form was detected. Isoelectric-focusing experiments also indicated only one enzyme form, which was identical from extracts of brain and liver of each species (pI = 5.2 for rat, 5.5 for human). Similarities between brain and liver catechol O-methyltransferase of a given species were also demonstrated by kinetic parameters, meta/para ratios of products, and inhibitor potencies. Human catechol O-methyltransferase exhibited lower Km values than did the rat enzyme for S-adenosyl-L-methionine, dopamine and dihydroxybenzoic acid. Adrenochrome inhibited both rat and human enzyme. It was concluded (1) that only a single enzyme form could be demonstrated in the physiological pH region; (2) that catechol O-methyltransferase of brain could not be distinguished from the liver enzyme of the same species; and (3) that species differences exist between the enzymes of rat and human tissues.     

Properties of thiamine-binding proteins isolated from rat brain,liver and kidneys

The study of thiamine-binding proteins (ThBP) isolated from liver and kidneys of rats was held in order to find out the peculiarities and physiological role of the ThBP isolated earlier from the rat brain. It was demonstrated that ThBP from liver and kidneys of rats as well as ThBP from rat brain described earlier, were bifunctional: on an equal footing with ability to bind thiamine specifically, they show an ability to hydrolyse the phosphoric esters of thiamine selectively. The ThBP of these tissues (liver, kidneys and brain) didn't differ by the molecular weight, but differed by the enzymatic activities. The molecular weight of ThBP was estimated to be 100 kDa by gel-filtration; 63 kDa and 35 kDa by sodium dodecylsulfate gel electrophoresis. Specific thiamine-binding activity increases as follows: ThBP from rat brain < ThBP from rat liver < ThBP from rat kidneys.     

Comparison of the phosphate-dependent glutaminase obtained from rat brain and kidney.

A phosphate-dependent glutaminase was purified 1200-fold from rat brain. In the absence of a polyvalent anion, the glutaminase exists as an inactive protomer which has an estimated Mr of 126000. The addition of 100mM-phosphate causes maximal activation and a dimerization (Mr 249000) of the glutaminase. The phosphate activation is sigmoidal, with a K0.5 of 25mM and a Hill coefficient (h) of 1.5 Glutamate inhibition is competitive with respect to glutamine and is decreased by increasing the concentration of phosphate. Phosphate also decreases the Km for glutamine. The purified glutaminase contains a predominant peptide (Mr 65000) and a minor peptide (Mr 68000) that are present in an approximate ratio of 4:1 respectively. The glutaminase immunoprecipitated from freshly solubilized brain tissue or from synaptosomal and non-synaptosomal brain mitochondria contains the same distribution of the two peptides. In contrast, the glutaminase purified from rat kidney contains five to seven peptides that range in Mr value from 59000 to 48000, and immunoprecipitates derived from freshly solubilized renal tissue contain only the Mr-65000 peptide. Partial proteolysis and size fractionation of the three immunoprecipitated peptides indicate that they are structurally related. The series of peptides characteristic of the purified renal glutaminase is generated on storage of the solubilized extract of kidney tissue. The glutaminase contained in the solubilized brain extract is not degraded unless a renal extract is added. Thus the difference in the pattern of peptides associated with the two purified enzymes is due to an endogenous renal proteinase that is not present in brain.     

Insulin-degrading neutral protease from plasma membranes of rat liver cells and erythrocytes

Insulin-degrading neutral proteinase with molecular weight of 70 kDa was partly purified from the rat liver and erythrocyte plasma membranes. Incubation of membranes with [gamma-32P]ATP resulted in the enzyme phosphorylation. Intensity of this process greatly increased in the presence of insulin (100 microU/ml), and correlated with the elevation of the insulin-degrading activity in proteinase. Ca2+, Mn2+, dithiothreitol, cysteine were shown to have a stimulatory effect on insulin degradation; p-chloromercuribenzoate significantly repressed this process. Phenylmethylsulphonyl fluoride and soybean trypsin inhibitor did not affect the activity of the proteinase. It was concluded that the investigated enzyme was a calpain and may participate in the mechanism of insulin action.     

Comparison of cytosol retinol binding proteins from bovine retina, dog liver, and rat liver

Cytosol retinol binding proteins (CRBP's) have been purified from rat liver, dog liver, and bovine retina. All had identical molecular weights on sodium dodecyl sulfate electrophoresis. They had different RF values on non-sodium dodecyl sulfate gels at pH 8.9. The three CRBP exhibited similar absorption and fluorescence spectra. The absorbance of the ligand was perturbed after binding, the main band shifting bathochromically and exhibiting a lambda(max) at 350 nm compared with 328 nm for free retinol in hexane. Additionally, subsidiary peaks appeared at 335 and 367 nm. Rabbit antiserum against rat liver CRBP cross-reacted with CRBP's from dog liver and bovine retina. The Ouchterlony immunodiffusion technique indicated that these proteins have molecular structures with identical antigenic determinants. All three CRBP's had amino acid composition that were virtually identical, as judged by our own observations and those of other laboratories. The molecular structure of cytosol retinol binding proteins appears to be highly conserved, irrespective of species or tissue of origin.     

Isolation and structures of xenopsin-related peptides from rat stomach, liver and brain

Using radioimmunoassay for detection, a mammalian counterpart to amphibian xenopsin (XP) was isolated and sequenced from pepsin-treated extracts of three different rat tissues and shown to be H-Phe-His-Pro-Lys-Arg-Pro-Trp-Ile-Leu-OH. This peptide, which shares six of the eight residues in XP, existed primarily in large molecular form(s) in the rat from which it could be liberated by the enzyme, pepsin. The XP-related sequence was differentially distributed through tissues, with concentrations ranging from ca. 80 pmol/g in diaphragm and skeletal muscle to ca. 800 pmol/g in stomach, liver and intestine. Like XP, the mammalian peptide potently crossreacted in a radioreceptor assay for neurotensin. These results prove the existence of radioreceptor-active XP-related sequences in multiple tissues of the rat.     

Purification and immunochemical interrelationship of insulin receptors from rat brain and liver.

Insulin receptors from rat brain and liver were purified. Brain purified receptor exhibited protein bands of apparent Mr = 135,000 and 95,000 molecular weight corresponding to alpha- and beta-subunits, retained a tyrosine specific protein kinase activity and demonstrated phosphorylation that is hormonally sensitive. Antisera were raised against both insulin receptor preparations and enzyme-linked immunosorbent assay was developed. The comparison of two insulin receptors was based on a displacement enzyme-linked immunosorbent assay where antisera were interchanged on predetermined optimal dilutions. This indicated that both insulin receptors possess some unique antigenic determinants thereby implying a structural difference.     

Comparison of the phospholipid requirements and molecular form of CTP : phosphocholine cytidylyltransferase from rat lung, kidney, brain and liver

The cytidylyltransferase activity in fresh cytosol from different tissues of the rat was measured in the absence and presence of phosphatidylglycerol. In all cases addition of this lipid produced large increases in enzyme activity. Agarose gel (A-5.0) filtration profiles of the enzyme activities indicated that the L-form of the enzyme (190 000 molecular weight) predominated in liver, brain, kidney, and fetal lung. However, adult lung cytosol contained 70--80% of the activity in the H-form (molecular weight greater than or equal to 5 x 10(6)). Removal of phospholipid material from the alveolar spaces by lavage produced a significant reduction of the H-form of the enzyme in the cytosol fraction. The L-form of the cytidylyltransferases from fetal lung and adult liver, kidney, and brain all possess the same specificities for activation by phospholipids in vitro. In all cases, phosphatidylglycerol was the most potent activator at 0.2 mM. Lysophosphatidylethanolamine stimulated enzyme activity, whereas lysophosphatidylglycerol was a potent inhibitor. These studies implicate the role of acidic phospholipids in the regulation of cytidylyltransferase activity in vivo and the existence of a common L-form of the enzyme in serveral tissues of the rat.     

Comparison of populations of mRNA coding fumarase in rat brain and liver

The populations of mRNA encoding mitochondrial and cytosolic fumarases in the poly A+ RNA fractions purified from polysomes of rat brain and liver were examined. When the in vitro translation products programmed by the poly A+ RNA fraction obtained from rat brain were purified by immunoprecipitation with anti-fumarase antibody and analyzed by SDS polyacrylamide gel electrophoresis and fluorography, only one polypeptide of 50 KD was detected as a precursor of fumarase. In contrast, by the same method, two polypeptides of 50 KD and 45 KD, which is the same size as mature fumarase, were detected as precursors of rat liver fumarase. These results suggest that rat brain polysomes contain only one population of mRNA coding a 50 KD precursor of mitochondrial fumarase with little or no mRNA of the cytosolic fumarase, whereas rat liver polysomes contain two types of mRNA for mitochondrial and cytosolic fumarases, respectively. These findings are consistent with the fact that the brain is the only organ in rats known not to contain cytosolic fumarase.     

Mutual inhibitory effect of calpastatins on calpains from carp muscle, carp erythrocytes and rat liver

The inhibitory effect of calpastatin (specific inhibitor for calpain) on calpain (Ca2+-dependent cysteine proteinase, EC 3.4.22.17) was examined using carp muscle, carp erythrocytes and rat liver preparations. A mutual inhibitory effect between calpains and calpastatins from different tissues and species was observed. The conservation of the inhibitory effect of calpastatin on calpain among vertebrates suggests that the calpain-calpastatin system may play a biologically fundamental and common role in various cells.     

Phosphofructokinase from the epithelial cells of rat small intestine. Comparison of regulatory properties with those of skeletal muscle, liver and brain phosphofructokinase

The regulatory properties of phosphofructokinase from rat mucosa, liver, brain and muscle were investigated. Mucosal phosphofructokinase displayed cooperativity with respect to fructose 6-phosphate at pH 7.0 and so did the muscle, brain and liver isoenzymes. All these four isoenzymes were inhibited by ATP, the mucosal isoenzyme being the least inhibited. They were also inhibited by citrate and creatine phosphate. AMP, ADP, glucose 1,6-diphosphate, fructose 2,6-bisphosphate and inorganic phosphate were all strong activators for the mucosal, brain, liver and muscle phosphofructokinase, but the mucosal isoenzyme was found to be more activated than the others, accounting for the higher rates of glycolysis observed in mucosa. The results suggest that mucosal phosphofructokinase is unique and different from all the other isoenzymes.     

Bicarbonate measurements in rat liver, brain, heart, and skeletal muscle.

A method is described for measuring total CO₂ and HCO₃^? in tissues rapidly frozen in liquid nitrogen. The method is a modification of the procedure of D. D. Van Slyke and J. M. Neill (1924, J. Biol. Chem.61, 523–573) for use with freeze-clamped tissue where anoxic changes have not occurred. The HCO₃^? content exclusive of tissue CO₂ in fed rats was found to be: liver, 19.4 ± 1.0; brain, 20.2 ± 0.9; thigh, 16.2 ± 0.8; and heart, 15.4 ± 1.4 μmol/g.     

Purification of quinolinic acid phosphoribosyltransferase from rat liver and brain

Quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) was purified 3600-fold from rat liver and 280-fold from rat brain. Kinetic analyses (K_m = 12 μM for the substrate quinolinic acid and K_m 23 μM for the cosubstrate phosphoribosylpyrophosphate), physicochemical properties of the purified enzymes, inhibition by phthalic acid (K_i = 1.4 μM) and molecular weight determination (M_r 160 000 for the holoenzyme, consisting of five identical 32 kDa subunits) indicated the structural identity of quinolinic acid phosphoribosyltransferase from the two rat tissues. This was further confirmed immunologically, using antibodies raised against purified rat liver quinolinic acid phosphoribosyltransferase. Rat quinolinic acid phosphoribosyltransferase differs in several aspects from quinolinic acid phosphoribosyltransferase isolated from other organisms. The purified enzyme will prove a useful tool in the examination of a possible role of quinolinic acid in cellular function and/or dysfunction.     

Quantitation of the efflux of acylcarnitines from rat heart, brain, and liver mitochondria

The efflux of individual short-chain and medium-chain acylcarnitines from rat liver, heart, and brain mitochondria metabolizing several substrates has been measured. The acylcarnitine efflux profiles depend on the substrate, the source of mitochondria, and the incubation conditions. The largest amount of any acylcarnitine effluxing per mg of protein was acetylcarnitine produced by heart mitochondria from pyruvate. This efflux of acetylcarnitine from heart mitochondria is almost 5 times greater with 1 mM than 0.2 mM carnitine. Apparently the acetyl-CoA generated from pyruvate by pyruvate dehydrogenase is very accessible to carnitine acetyltransferase. Very little acetylcarnitine effluxes from heart mitochondria when octanoate is the substrate except in the presence of malonate. Acetylcarnitine production from some substrates peaks and then declines, indicating uptake and utilization. The unequivocal demonstration that considerable amounts of propionylcarnitine or isobutyrylcarnitine efflux from heart mitochondria metabolizing alpha-ketoisovalerate and alpha-keto-beta-methylvalerate provides evidence for a role (via removal of non-metabolizable propionyl-CoA or slowly metabolizable acyl-CoAs) for carnitine in tissues which have limited capacity to metabolize propionyl-CoA. These results also show propionyl-CoA must be formed during the metabolism of alpha-ketoisovalerate and that extra-mitochondrial free carnitine rapidly interacts with matrix short-chain aliphatic acyl-CoA generated from alpha-keto acids of branched-chain amino acids and pyruvate in the presence and absence of malate.