Adenylosuccinate synthetase in rat liver: the existence of two types and their regulatory roles.

Rat liver contains two types of adenylosuccinate synthetase which can be distinguished by isoelectroforcusing or immunochemical analysis. One type is identical with the enzyme in rat skeletal muscle (Type M) and the other is specific for the liver (Type L). Type L was more susceptible to nucleotide inhibition, but less susceptible to inhibition by fructose-1,6-diphosphate than Type M. These differences suggest that these isozymes play different regulatory roles in the liver.     

Thiamine pyrophosphatase and nucleoside diphosphatase in rat brain

Two types of nucleoside diphosphatase were found in rat brain. One (Type L) had similar properties to those of the liver microsomal enzyme with respect to its isoelectric point, substrate specificity, Km values, optimum pH, activation by ATP and molecular weight. The other (Type B), which separated into multiple forms on isoelectric focusing, had lower Km values and a smaller molecular weight than the Type L enzyme, and was inhibited by ATP. The Type B enzyme catalyzed the hydrolysis of thiamine pyrophosphate as well as those of various nucleoside diphosphates at physiological pH, while Type L showed only nucleoside diphosphatase activity at neutral pH. These findings suggest that the two enzymes play different physiological roles in the brain.     

Glycogen debranching enzyme: purification, antibody characterization, and immunoblot analyses of type III glycogen storage disease.

Type III glycogen storage disease is caused by a deficiency of glycogen debranching-enzyme activity. Many patients with this disease have both liver and muscle involvement, whereas others have only liver involvement without clinical or laboratory evidence of myopathy. To improve our understanding of the molecular basis of the disease, debranching enzyme was purified 238-fold from porcine skeletal muscle. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme gave a single band with a relative molecular weight of 160,000 that migrated to the same position as purified rabbit-muscle debranching enzyme. Antiserum against porcine debranching enzyme was prepared in rabbit. The antiserum reacted against porcine debranching enzyme with a single precipitin line and demonstrated a reaction having complete identity to those of both the enzyme present in crude muscle and the enzyme present in liver extracts. Incubation of antiserum with purified porcine debranching enzyme inhibited almost all enzyme activity, whereas such treatment with preimmune serum had little effect. The antiserum also inhibited debranching-enzyme activity in crude liver extracts from both pigs and humans to the same extent as was observed in muscle. Immunoblot analysis probed with anti-porcine-muscle debranching-enzyme antiserum showed that the antiserum can detect debranching enzyme in both human muscle and human liver. The bands detected in human samples by the antiserum were the same size as the one detected in porcine muscle. Five patients with Type III and six patients with other types of glycogen storage disease were subjected to immunoblot analysis. Although anti-porcine antiserum detected specific bands in all liver and muscle samples from patients with other types of glycogen storage disease (Types I, II, and IX), the antiserum detected no cross-reactive material in any of the liver or muscle samples from patients with Type III glycogen storage disease. These data indicate (1) immunochemical similarity of debranching enzyme in liver and muscle and (2) that deficiency of debranching-enzyme activity in Type III glycogen storage disease is due to absence of debrancher protein in the patients that we studied.     

Histochemical and immunohistochemical detection of putative preneoplastic liver foci in women after long-term use of oral contraceptives

Localized areas with altered enzyme patterns were observed in liver tissue surrounding focal nodular hyperplasia in women after long-term use of oral contraceptives. These localized lesions were of three different types. Type I lesions were characterized by glycogen storage, a reduction in ATPase and an increase in gamma-glutamyltranspeptidase (gamma-GT) and UDP-glucuronyltransferase (UDP-GT) detected immunohistochemically. Type II lesions, which were morphologically very similar to small hyperplastic nodules, showed only a decreased ATPase reaction. Type III lesions showed an increase in gamma-GT (detected histochemically) and a slight reduction in ATPase. The results indicated that in human liver from patients given oral contraceptives long-term, localized lesions with altered enzyme patterns may occur which are very similar to those observed in animal models during experimental hepatic carcinogenesis.     

Avian hepatic T3 generation by 5'-monodeiodination: characterization of two enzymatic pathways and the effects of goitrogens

The enzymatic nature of 5'-monodeiodination (5'-D) in avian liver homogenates was demonstrated by abolishment of activity by iopanoic acid (IOP). T3 production from T4 was dependent on enzyme and substrate concentrations, incubation time, incubation temperature, and pH. Two pathways of 5'-D activity were present in avian liver and exhibited characteristics similar to those described in mammalian tissues. Type II activity was identified as propylthiouracil (PTU)-insensitive activity. Type I (PTU-sensitive) was determined by difference between Total and Type II. Km values were 1.58 microM T4 for Total activity and 0.90 nM T4 for Type II, corresponding to the characteristics of the mammalian pathways. The effects of goitrogens on avian hepatic 5'-D were equivalent to those reported for the mammalian enzyme.     

Direct evidence for expression of type II flavoprotein subunit in human complex II (succinate-ubiquinone reductase)

Succinate-ubiquinone reductase (complex II) is an important enzyme complex in aerobic respiration and the tricarboxylic acid cycle. We recently identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual and demonstrated mRNAs of these two isoforms, Type I Fp and Type II Fp, in skeletal muscle, liver, brain, heart, and kidney. Type I Fp was expressed at higher levels than Type II Fp in all cases. In the present study, the biochemical properties of Type II Fp-containing complex II in Raji cells predominantly expressing Type II Fp were investigated. Complex II having Type II Fp was separated from that having Type I Fp by isoelectric focusing in the presence of sucrose monolaurate. Together with the fact that succinate-ubiquinone reductase activity of mitochondria prepared from Raji cell was almost identical to that from human liver, these results clearly indicate the presence of two distinct isoforms of active complex II in human mitochondria.     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.     

The heterogeneity of protein kinase C in various rat tissues

Expression of multiple subspecies of protein kinase C (PKC) was studied in various rat tissues. Three types of the enzyme designated type I, II, and III were analyzed, which have the structures of gamma-, beta- (beta I- and beta II-), and alpha-sequence, respectively. Type I enzyme was found only in the central nervous tissue, whereas type III enzyme appeared to be commonly present in various tissues such as liver, spleen, lung, testis, heart, and kidney. Type II enzyme was also found in these tissues. However, immunoblot and biochemical analysis indicated that type II enzyme of lung and heart was distinct from that of other tissues. The tissue-specific expression of PKC suggests that each subspecies of this enzyme has a defined function in processing and modulating tissue responses to external stimuli.     

Characterization of an analog enzyme to cathepsin L produced by tumor cells

The activity of cathepsin L is examined in the culture supernatants of 38 human, murine and hamster tumor cell lines. It is found that all cell lines secrete the enzyme possessing cathepsin L activity. The supernatant of HPC-YP cell cultures is purified and characterized as the enzyme preparation, because this supernatant shows the highest cathepsin L activity. The results indicate that the enzyme produced in HPC-YP cells is different from cathepsin L of normal liver in the several points. The molecular weight of the enzyme is 68 kd, whereas it is 34 kd for the liver cathepsin L. The enzyme is more stable to heat treatment and at the various pH than the liver cathepsin L. Furthermore, the inhibitors, which inhibit the liver cathepsin L activity, do not inhibit the activity of this enzyme. It is concluded that the enzyme showing cathepsin L activity in the culture supernatants of human tumor cells is different from human normal liver cathepsin L.     

Studies of the residual glycogen branching enzyme activity present in human skin fibroblasts from patients with Type IV glycogen storage disease

Human skin fibroblasts from patients with Type IV glycogen storage disease, in which there is a demonstrable deficiency of glycogen branching enzyme, were shown to be able to synthesize [¹⁴C]glycogen containing [¹⁴C]glucose at branch points when sonicates containing endogenous glycogen synthase $\text{a}$ were incubated with UDP[¹⁴C]glucose. The branch point content of the glycogen synthesized by the Type IV cells was essentially the same as that formed by normal cells, but the total synthetic capacity of the Type IV cells was lower. A new assay for the branching enzyme using glycogen synthase as the indicator enzyme has been developed. Using this assay it has been shown that the residual branching enzyme of affected children and of their heterozygote parents is less easily inhibited by an IgG antibody raised in rabbits against the normal human liver enzyme than is the branching enzyme of normal fibroblasts.     

Activation of rabbit liver high affinity cAMP (type IV) phosphodiesterase by a vanadyl-glutathione complex. Characterization of the role of the sulfhydryl.

Activation of rabbit liver microsomal high affinity cAMP phosphodiesterase (Type IV PDE) by vanadyl-glutathione complexes was studied as a possible model of insulin stimulation of the enzyme in a cell-free system. The effect of VO.2GSH activation of PDE was a 21-fold decrease in the IC50 value for cGMP inhibition and a 2.6-fold increase in the Vmax of the higher affinity cAMP catalytic site. Cyclic AMP and cGMP substrate affinities and cGMP hydrolysis were unaffected by VO.2GSH activation. Selective Type IV PDE inhibitors and cGMP analogs indicated that VO.2GSH complexes activated the cGMP-inhibitable form of the Type IV PDE activities which co-localized in hepatic microsomes. The Type IV PDE activating complex appears to consist minimally of vanadyl ion and 2 oxidized electron donor compounds. The components of the electron donor required to achieve an enzyme activation complex are: 1) a free -SH group as the electron donor for vanadate reduction and 2) a minimum structure of cysteamine (NH2-CH2-CH2-SH). Maximal activation of the enzyme required near 2:1 molar ratios of either glutathione or cysteamine mixed with sodium orthovanadate. Active vanadyl-cysteamine complexes were isolated by reverse- phase high performance liquid chromatography. Tungsten, niobium, and tantalum, but not manganese, chromium, or molybdenum, substituted for vanadium to form enzyme-activating complexes with glutathione. VO.RSH complex activation occurred rapidly upon addition to microsomes and was reversible. We conclude from these studies that VO.RSH complexes and insulin activate the same form of Type IV PDE in rabbit liver microsomes; our findings are discussed with respect to the involvement of a possible electron transfer enzyme oxidation in the activation mechanism.     

Occurrence of two 3-hydroxyacyl-CoA dehydrogenases in rat liver.

3-Hydroxyacyl-CoA dehydrogenase was assayed for acetoacetyl pantetheine-reducing and acetoacetyl-CoA reducing activities in rat liver homegenates. Two isoenzymes of the enzyme, types I and II, were distinguished by the following procedures: trypsin treatment, heat treatment, CM-cellulose chromatography, antibody titration, and sucrose density gradient centrifugation of the light mitochondrial fraction. Type I enzyme was localized in mitochondria, and catalyzed the reduction of both acetoacetyl pantetheine and acetoacetyl-CoA. Type II enzyme was found mainly in peroxisomes, accompanied by a low activity in mitochondria or some other organelles, and was active with acetoacetyl-CoA but not with aceto acetylpantetheine. Both isozymes were induced by the administration to the rats of di-(2-ethylhexyl)phthalate, which enhances the peroxisomal beta-oxidation activity, but the extent of the induction of type II enzyme was much higher than that of type I enzyme. The activity of the former was found only in diethylhexylphthalate-treated rats.     

The processing of human proinsulin and chicken proalbumin by rat hepatic vesicles suggests a convertase specific for X-Y-Arg-Arg or Arg-X-Y-Arg sequences

Vesicles from rat and chicken livers contain very similar Ca2(+)-dependent proteases that respectively cleave (human) proalbumin at an Arg-Arg site and chicken proalbumin at an Arg-Phe-Ala-Arg site. Similar Ca2(+)-dependent proteases are also present in pancreatic secretory granules and cleave proinsulin at two sites, Arg-Arg and Lys-Arg. The mammalian liver processes a large variety of different proproteins and in order to assess its processing site requirements, we investigated the ability of rat hepatic vesicle extracts to cleave purified chicken proalbumin and human proinsulin. Despite having only a monobasic processing site, chicken proalbumin was cleaved faster than human proalbumin which not only contains a dibasic site, but has an identical propeptide to that of the rat's own proalbumin. Human proinsulin was processed by the rat liver extracts; however, no mature insulin was produced. Cleavage occurred in only one place, presumably the Arg-Arg site at the B-C chain junction. This suggests that the mammalian liver might not contain a Type II Lys-Arg-directed convertase, only a Type I Arg-Arg-specific enzyme. The Type I enzyme that cleaves human proalbumin appears to be the same activity that cleaves chicken proalbumin, suggesting a specificity for either X-Y-Arg-Arg or Arg-X-Y-Arg sequences. This proposal is in keeping with the processing site motif of some 16 different proproteins that are known to be processed in the liver and is entirely consistent with the known in vivo specificity of the enzyme defined by naturally occurring variants of human proproteins.     

Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: a biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions

Insertion of Cu2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Cu2+. Ternary complex formation with NAD+ converts the Cu2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.     

Purification and characterization of membrane-bound inositolpolyphosphate 5-phosphatase

Membrane-bound inositolpolyphosphate 5-phosphatase was solubilized and highly purified from a microsomal fraction of rat liver. Its physiochemical and enzymological properties were compared with those of highly purified preparations of two types of soluble enzyme (soluble Type I and Type II) from rat brain. The molecular masses of the membrane-bound and soluble Type I enzymes were 32 kDa, while that of soluble Type II enzyme was 69 kDa, as determined by molecular sieve chromatography. The membrane-bound and soluble Type I enzymes showed similar broad peaks on isoelectric focusing (pI 5.8-6.4), while soluble Type II enzyme showed multiple peaks in the region between pI 4.0-5.8. All three enzymes required divalent cation for activity. Mg2+ was the most effective for both the membrane-bound and soluble Type I enzymes, while Co2+ enhanced soluble Type II enzyme activity about 1.5-fold relative to Mg2+ at 1 mM. The optimal pH of both the membrane-bound and soluble Type I enzymes was 7.8, while that of soluble Type II was 6.8. The Km values for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] of all three enzymes were similar (5-8 microM), but those for inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were quite different, the Km values of membrane-bound and soluble Type I enzymes being 0.8 microM, while that of soluble Type II was 130 microM. These similarities between the membrane-bound and soluble Type I enzymes suggest that these two molecules may be the same protein, and that concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, both of which are considered to play critical roles in the regulation of intracellular Ca2+-concentration, may be differently regulated by two functionally distinct enzymes.     

Thiamine pyrophosphatase (nucleoside diphosphatase) in the Golgi apparatus is distinct from microsomal nucleoside diphosphatase

The properties of thiamine pyrophosphatase in the Golgi apparatus of rat liver were studied. Thiamine pyrophosphatase in an extract of the Golgi apparatus was separated into 6 bands of between pH 5.4 and 6.3 by isoelectric focusing on polyacrylamide gel. On the gels all these subforms catalyzed the hydrolyses of GDP, IDP, UDP, and CDP as well as that of thiamine pyrophosphate. The characteristics resembled those of Type B nucleoside diphosphatase of rat brain, though the enzyme did not have 3 subforms of Type B nucleoside diphosphatase in the higher pH region on isoelectric focusing. Thiamine pyrophosphatase of the Golgi apparatus was separated from microsomal nucleoside diphosphatase by DEAE-cellulose column chromatography. The properties of the enzyme were quite similar to those of Type B nucleoside diphosphatase with respect to its substrate specificity, optimum pH for activity, and inhibition by ATP. These findings suggest that thiamine pyrophosphatase in the Golgi apparatus is different from microsomal nucleoside diphosphatase and that it might be basically the same enzyme as Type B nucleoside diphosphatase except for different extents of modification.     

Unique cathepsin D-type proteases in rat thoracic duct lymphocytes and in rat lymphoid tissues.

Two unique cathepsin D-type proteases apparently present only in rat thoracic duct lymphocytes and in rat lymphoid tissues are described. One, termed H enzyme, has an apparent molecular weight of similar to95,000; the other, termed L enzyme, has an apparent molecular weight of similar to45,000, in common with that of most cathepsins D from other tissues and species. Both enzymes differ from cathepsin D, however, by a considerably greater sensitivity to inhibition by pepstatin and by a smaller degree of inhibition by an antiserum which inhibits rat liver cathepsin D. H enzyme is converted to L enzyme by treatment with beta-mercaptoethanol; the relationship between the two enzymes remains unknown. H and L enzyme have been detected in rat lymphoid tissues and in mouse spleen, but they are not present in other rat tissues (liver, kidney, adrenals), rabbit tissues, calf thymus, bovine spleen, or human tonsils. As measured on acid-denatured bovine hemoglobin as substrate, both enzymes have pH activity curves identical with that of rat liver cathepsin D, with optimal activity at pH 3.6. Activity on human serum albumin is much less and also shows an optimum at pH 3.6; hence, neither enzyme has the properties of cathepsin E. Thiol-reactive inhibitiors have no effect on the activity of H and L enzyme; thus they do not belong to the B group of cathepsins. Additional information, discussed in this paper, leads us to conclude that partially purified H and L enzymes are cathepsin D-type proteases.     

Studies on the interaction between rabbit liver pyruvate kinase and its allosteric effector fructose 1,6-diphosphate

Preparation of the L form of rabbit liver pyruvate kinase (EC 2.7.1.40) in the presence of fructose 1,6-diphosphate yielded an enzyme which was kinetically identical with the M or muscle-type form of pyruvate kinase found in liver. Chromatographic and dialysis studies of this complex showed that most of the fructose 1,6-diphosphate molecules were loosely bound to the enzyme, but dilution-dissociation studies and binding experiments established that there was a high initial affinity between the enzyme and fructose 1,6-diphosphate (K(assoc.)=2.3x10(9)), and that binding of the loosely bound fructose 1,6-diphosphate was concentration-dependent and a necessary condition to overcome the co-operative interaction observed with the homotropic effector phosphoenolpyruvate. Preparation of the liver enzyme in the absence of EDTA did not yield a predominantly M form of the enzyme, and incubation of the M form in the presence of EDTA did not convert it into the L form, but resulted in inhibition of enzyme activity. Immunological studies confirmed that the L and M forms in liver were distinct, and that preparation of the L form in the presence of fructose 1,6-diphosphate did not produce an enzyme antigenically different from the L form prepared in the absence of this heterotropic effector.     

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.