Molecular properties of multiple forms of acid phosphatase from horse liver.

1. Horse liver acid phosphatase was separated into two partially purified fractions differing in molecular weight (enzyme I about 100 00, enzyme II about 25 000). 2. Enzyme I was separated into several subfractions by DEAE-cellulose chromatography and isoelectric focusing. 3. Molecular weight, sedimentation coefficient and effective molecular radii were determined for acid phosphatases I and II by gel filtration and density-gradient centrifugation.     

Characterization of multiple forms of carbonyl reductase from chicken liver.

Three enzyme forms (CR1, CR2 and CR3) of carbonyl reductase were purified from chicken liver with using 4-benzoylpyridine as a substrate. CR1 was a dimeric enzyme composed of two identical 25-kD subunits. CR2 and CR3 were monomeric enzymes whose molecular weights were both 32 kD. CR1 exhibited 17 beta-hydroxysteroid dehydrogenase activity as well as carbonyl reductase activity in the presence of both NADP(H) and NAD(H). CR2 and CR3 had similar properties with regard to substrate specificity and inhibitor sensitivity. They could exhibit the activity only with NADPH and had no hydroxysteroid dehydrogenase activity. CR2 and CR3 cross-reacted with anti-chicken kidney carbonyl reductase antibody, though CR1 did not. The results suggest that CR1 is a hydroxysteroid dehydrogenase, and CR2 and CR3 are similar to each other and to the kidney enzymes.     

Detection of multiple forms of rat liver ornithine decarboxylase.

Rat liver ornithine decarboxylase induced by injection of thioacetamide has been separated into at least two fractions by covalent chromatography on an activated thiol-Sepharose 4B column. The two major fractions could be distinguished by ion exchange chromatography and electrophoresis on acrylamide gels. In addition, the two forms displayed different Km values for ornithine. Although the two forms are separable, they display identical antigenic properties, pH optima, and they appear to be the same molecular size. The biological significance or the relationship between multiple forms of ornithine decarboxylase is not understood.     

Characterization of dolichyl diphosphate phosphatase from rat liver

Dolichyl diphosphate phosphatase (DolPPase) has been characterized in rat liver. Subcellular distribution studies indicate that the enzyme is localized in the endoplasmic reticulum. The in vitro enzymatic activity is stimulated by EDTA, due to release of inhibition by trivalent cations found in the assay tubes. All di- and trivalent cations tested were inhibitory, with the trivalent ions Al3+ and Fe3+ showing greater than 70% inhibition at a concentration of 10 microM. The assay requires the presence of a detergent for optimal activity, with Triton X-100 giving maximum activity at 0.1%. The substrate specificity of DolPPase toward polyprenyl diphosphates has been determined and indicates that there is little preference of the enzyme for substrates of different chain length, and either stereochemical orientation or degree of saturation of the alpha-isoprene unit. Km values of 11-14 microM were obtained for all substrates tested. Preliminary studies on the transmembrane topology of the DolPPase using latency assays, indicate that the active site of the enzyme may reside on the cytoplasmic face of the endoplasmic reticulum.     

Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase.

UDP-glucuronosyltransferase (EC 2.4.1.17) activity was solubilized from male Wistar rat liver microsomal fraction in Emulgen 911, and six fractions with the transferase activity were separated by chromatofocusing on PBE 94 (pH 9.4 to 6.0). Fraction I was further separated into Isoforms Ia, Ib and Ic by affinity chromatography on UDP-hexanolamine-Sepharose 4B. UDP-glucuronosyltransferase in Fraction III was further purified by rechromatofocusing (pH 8.7 to 7.5). UDP-glucuronosyltransferases in Fractions IV and V were purified by UDP-hexanolamine-Sepharose chromatography. The transferase isoforms in Fractions II, III, IV and V were finally purified by h.p.l.c. on a TSK G 3000 SW column. Purified UDP-glucuronosyltransferase Isoforms Ia (Mr 51,000), Ib (Mr 52,000), Ic (Mr 56,000), II (Mr 52,000), IV (Mr 53,000) and V (Mr 53,000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoform III enzyme showed two bands of Mr 52,000 and 53,000. Comparison of the amino acid compositions by the method of Cornish-Bowden [(1980) Anal. Biochem. 105, 233-238] suggested that all UDP-glucuronosyltransferase isoforms are structurally related. Reverse-phase h.p.l.c. of tryptic peptides of individual isoforms revealed distinct 'maps', indicating differences in primary protein structure. The two bands of Isoform III revealed distinct electrophoretic peptide maps after limited enzymic proteolysis. After reconstitution with phosphatidylcholine liposomes, the purified isoforms exhibited distinct but overlapping substrate specificities. Isoform V was specific for bilirubin glucuronidation, which was not inhibited by other aglycone substrates. Each isoform, except Ia, was identified as a glycoprotein by periodic acid/Schiff staining.     

Yeast contains multiple forms of histone acetyltransferase

We have assayed several methods to quantitatively recover yeast histone acetyltransferases in an attempt to study the multiplicity of enzymatic activities. Two methods, namely (NH4)2SO4 precipitation and salt dissociation of chromatin in 0.5 M NaCl, yielded convenient preparations of total histone acetyltransferases. DEAE-Sepharose chromatography of the crude extracts resulted in the separation of three peaks of activity when total yeast histones were used as substrate. However, the scanning of the enzymatic activity toward individual histones along the chromatography, achieved by determining the specific activity of the individual histones after incubating whole histones and [14C]acetyl-CoA with the chromatographic fractions, yielded four peaks. The first two peaks showed specificity toward H2B and H3, respectively. Although they partially overlapped, rechromatography on cation exchangers allowed us to resolve the two activities, and several criteria were used to prove that they correspond to different enzyme molecules. The last two peaks were H4-specific, but the present data suggest that one of the activities is chromatin-bound, whereas the other surely corresponds to the cytoplasmic B-form of the enzyme. The enzyme specific for yeast H2B acetylates chicken erythrocyte H2A, rather than H2B. The detected multiplicity of yeast histone acetyltransferases may correspond to the multiplicity of roles proposed for histone acetylation.     

Characterization of D-myo-inositol 1,4,5-trisphosphate phosphatase in rat liver plasma membranes

D-myo-Inositol 1,4,5-trisphosphate has been previously demonstrated to act as a second messenger for the hormonal mobilization of intracellular calcium in rat liver. In this study, the breakdown of D-myo-inositol 1,4,5-trisphosphate by a phosphatase activity was characterized. Using partially purified subcellular fractions, it was found that D-myo-inositol 1,4,5-trisphosphate phosphatase (I-P3ase) specific activity was highest in the plasma membrane fraction, while D-myo-inositol 1,4-bisphosphate phosphatase specific activity was highest in the cytosolic and microsomal fractions. The plasma membrane I-P3ase was Mg2+-dependent with optimal activity observed at 0.5-1.5 mM free Mg2+. The enzyme had a neutral pH optimum, suggesting that it was neither an acid nor alkaline phosphatase. Neither LiCl nor NaF inhibited the I-P3ase activity. However, both L-cysteine and dithiothreitol stimulated the activity 2-fold. Spermine (2.0 mM) inhibited the I-P3ase activity by 50%, while putrescine and spermidine had little or no effect.     

Studies on the conversion of multiple forms of tyrosine aminotransferase in rat liver.

Studies from several laboratories have demonstrated the existence of at least three separable forms of the hepatic enzyme, tyrosine aminotransferase. The significance of these separable forms of the enzyme isolated in vitro for the nature and regulation of the enzyme in vivo has been the subject of some controversy. The studies reported in this paper demonstrate the existence of a heat-labile, pH- and temperature-dependent, nondialyzable component associated predominantly with the lysosomal and mitochondrial fraction of rat liver which catalyzes the conversion of form II to forms III and IV of the enzyme. The activity of this conversion factor is not significantly affected by F^?, molybdate ions, or two inhibitors of proteases. On the other hand, the cyanate ion completely inhibits the conversion of form II to forms III and IV of tyrosine aminotransferase, as do iodoacetate and oxidized glutathione. p-Chloromercuribenzoate also markedly inhibits the conversion. Kinetic studies suggest that the shift from one form to another follows the pathway: II to III to IV. Titration of the available sulfhydryl groups of the three forms of the enzyme demonstrates that form II possesses between 16 and 17 titratable SH groups per mole, while forms III and IV possess 15 and 13 or 14, respectively. The possible catalytic mechanism by which the conversion of the multiple forms of tyrosine aminotransferase is accomplished is discussed.     

Regulation of synthase phosphatase and phosphorylase phosphatase in rat liver.

Using substrates purified from liver, the apparent Km values of synthase phosphatase ([UDPglucose--glycogen glucosyltransferase-D]phosphohydrolase, EC 3.1.3.42) and phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) were found to be 0.7 and 60 units/ml respectively. The maximal velocity of phosphorylase phosphatase was more than a 100 times that of synthase phosphatase. In adrenalectomized, fasted animals there was a complete loss of synthase phosphatase but only a slight decrease in phosphorylase phosphatase when activity was measured using endogenous substrates in a concentrated liver extract. When assayed under optimal conditions with purified substrates, both activities were present but had decreased to very low levels. Mixing experiments indicated that synthase D present in the extract of adrenalectomized fasted animals was altered such that it was no longer a substrate for synthase phosphatase from normal rats. Phosphorylase a substrate on the other hand was unaltered and readily converted. When glucose was given in vivo, no change in percent of synthase in the I form was seen in adrenalectomized rats but the percent of phosphorylase in the a form was reduced. Precipitation of protein from an extract of normal fed rats with ethanol produced a large activation of phosphorylase phosphatase activity with no corresponding increase in synthase phosphatase activity. Despite the low phosphorylase phosphatase present in extracts of adrenalectomized fasted animals, ethanol precipitation increased activity to the same high level as obtained in the normal fed rats. Synthase phosphatase and phosphorylase phosphatase activities were also decreased in normal fasted, diabetic fed and fasted, and adrenalectomized fed rats. Both enzymes recovered in the same manner temporally after oral glucose administration to adrenalectomized, fasted rats. These results suggest an integrated regulatory mechanism for the two phosphatase.     

A lysosomal factor that interconverts multiple forms of rat liver tyrosine aminotransferase.

A particulate factor of rat liver is described which interconverts three forms of rat liver cytosolic tyrosine aminotransferase $\text{in}$$\text{vitro}$ with no alteration of enzyme activity. The factor appears to be a heat- and pH-sensitive lysosomal protein. The interconversion process is stimulated $\text{in}$$\text{vitro}$ by 2.5 mM MgCl₂ and 2.5 mM ATP. Asparate aminotransferase multiple forms are also susceptible to $\text{in}$$\text{vitro}$ interconversion by the lysosomal factor. The properties of the factor explain several anomalous effects of $\text{in}$$\text{vitro}$ manipulation on the tyrosine aminotransferase forms which have been reported in the literature and implicate the form interconversion in the degradation of tyrosine aminotransferase.     

Characterization of multiple forms of cholesteryl ester hydrolase in the rat testis

Cholesterol ester hydrolase (EC 3.1.1.13) activity from the 104,000 X g supernatant of rat testis was fractionated into 28-kDa, 72-kDa, and 420-kDa molecular mass forms by high performance size exclusion chromatography. The 72-kDa and 420-kDa forms (temperature-labile) were completely inactivated by elevation of temperature from 32 to 37 degrees C. Apparent disaggregation of the 420-kDa form suggested that the 72-kDa and 420-kDa enzymes are monomeric and multimeric forms of the same enzyme. The 28-kDa form was shown to be a different enzyme (temperature-stable) which retained activity at 37 degrees C. In contrast, cholesteryl ester hydrolase activities from 104,000 X g supernatants of liver or adrenal gland were unaffected and increased 4-fold, respectively, by elevation of temperature from 32 to 37 degrees C. Both testicular enzymes exhibited pH optima at about 7.3, and were activated by sodium cholate at concentrations near the critical micellar concentration (0.03-0.07%), but inhibited by higher concentrations. The temperature-labile cholesteryl ester hydrolase exhibited a high specificity for cholesteryl esters of monoenoic fatty acids of 18-24 carbons, especially nervonate (24:1), whereas the temperature-stable cholesteryl ester hydrolase exhibited highest specificity for cholesteryl oleate and arachidonate. Neither enzyme hydrolyzed cholesteryl acetate, myristate, palmitate, linoleate, or docosahexaenoate . Both enzymes reached maximum rates of hydrolysis at 150 microM substrates, with each substrate and at both reaction temperatures. Substrate inhibition was observed at higher concentrations (200 microM). The temperature-labile cholesteryl ester hydrolase was induced 20-fold in hypophysectomized rats by injection of follicle-stimulating hormone (FSH) and was localized in Sertoli cells, the target cells for FSH, but was not induced by luteinizing hormone. The temperature-stable cholesteryl ester hydrolase was induced by both FSH and LH and was found in both Sertoli cells and Leydig cells, the respective target cells for FSH and luteinizing hormone. Neither form of the enzyme was present at detectable levels in the germinal cells. The unique properties, localization, and hormonal regulation of both temperature-labile and temperature-stable cholesterol ester hydrolases suggest important roles for these enzymes in the testis.     

Multiple molecular forms of phosphoprotein phosphatase. III. Phosphorylase phosphatase and phosphohistone phosphatase of rabbit liver.

1. Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) in the soluble fraction of rabbit liver which catalyzes the dephosphorylation of muscle phosphorylase a and phosphohistone (P-histone) was resolved into three active fractions by NaCl gradient elution from a DEAE-cellulose column (Fraction I, 11 and III in order of elution). They have different relative reaction rates for the two substrates and different degrees of stimulation by Mn-2+. Apparent Km values of Fraction I, II and III were 15, 20 and 16 muM for phosphorylase a, and 6.9, 5.3 and 4.4 muM for P-histone, respectively (with Mn-2+ in the assay mixture). 2. On sucrose density gradient centrifugation Fraction I and II were revealed to contain a major peak (7.0 S and 7.8 S, respectively) and a minor peak (4.0 S) of activity, while Fraction III contained only one peak (5.8 S). Freezing and thawing in the presence of 0.2 M mercaptoethanol dissociated all three fractions into subunits of similar molecular size (3.4 S), with concomitant enhancement of phosphorylase phosphatase activity. The Km values all became essentially the same (20 muM for phosphorylase a and 16 muM for P-histone). 3. The phosphorylase phosphatase and P-histone phosphatase activities could not be separated with any of the procedures described. Competition between the two phosphoprotein substrates was observed with some of the fractions.?     

Differences in thiol groups and multiple forms of rat liver alcohol dehydrogenase

Rat liver alcohol dehydrogenase was analyzed for multiple forms by gel-electrophoresis and for protein thiol groups by differential car?ymethylation before and after reduction. Differences in protein SH-groups were associated with different electrophoretic forms of the enzyme. In the form with highest mobility all cysteine residues were free to alkylation but in forms with lower mobilities some residues were unreactive. Three cysteine residues likely to be affected are reported. Different combinations of a few alternative SS-bridges, superficially located and of no importance in catalytic activity, are suggested to constitute one explanation to the multiplicity of the protein.     

Raman and infrared studies of homogeneous forms of acid phosphatase from rat liver

Raman spectra of acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) forms from rat liver in water solution, and infrared spectra of the same forms as thin films, have been investigated. The spectra show strong bands belonging to phosphodiester or phosphomonoester residues. These groups are modified during the postsynthetic modification of acid phosphatase and are probably connected with the process of bonding and splitting of mannose 6-phosphate and N-acetylglucosamine, in agreement with previous biochemical models for the intracellular transport of newly synthesized lysosomal hydrolases to lysosomes. Some other bands in the infrared spectra are assigned to vibrations of N-H groups which may belong to N-acetylglucosamine.