Detection and characteristics of membrane form of phenylalanine hydroxylase (EC 1.14.16.1) from the rat liver

The proteins extracted with 0.4% Triton X-100 from the 105000 g homogenate fraction were shown to possess the phenylalanine hydroxylase (EC 1.14.16.1) activity. This phenylalanine hydroxylase fraction was designated as the membrane form of the enzyme. However, immunochemical methods of the antigen analysis performed under non-denaturating conditions and employing monospecific antisera to phenylalanine hydroxylase (double immunodiffusion in agar, racket immunoelectrophoresis, enzyme purification on immunoadsorbents) failed to reveal the antigen among the membrane fraction proteins of the liver. In this fraction the antigen was identified only by immunoblotting performed after electrophoresis of the proteins under denaturating conditions. The molecular mass of the cytoplasmic and membrane forms of the enzyme subunits is identical (52 kD). The Km value of phenylalanine for the cytoplasmic form of phenylalanine hydroxylase is 0.32.10(-3) M, that for the membrane form is 1.66.10(-3) M. Both enzyme forms can bind to phenyl-Sepharose after their activation by the substrate, and they dissociate from the carrier after phenylalanine removal from the incubation mixture, which points to the intactness of the phenylalanine binding allosteric center in the membrane form of the enzyme. This finding allowed for the purification of the membrane form of phenylalanine hydroxylase by affinity chromatography on phenyl-Sepharose.     

The dynamics of the properties of liver phenylalanine hydroxylase in human embryogenesis

Content, subunit composition and activity of phenylalanine hydroxylase's (PH) (EC 1.14.16.1) in cytoplasmic and membrane proteins extract of embryonic liver on week 6-11 of pregnancy were studied. PH enzymatic and antigenic activities were detected starting from the week 6 of pregnancy. Liver cytoplasmic PH antigen content increased gradually during development while its enzymatic activity remained practically unchanged. Concomitantly, relative content of L-subunit increased. Content of liver membrane PH antigen was constant during development. Samples of liver with relatively low specific PH activity were characterized by high content of PH in cytoplasm and vice versa. Since PH activity in extracts prepared from mixture of these samples decreased, an unknown PH inhibitor must be present in cytoplasmic protein extracts with relatively low specific PH activity.     

Immunochemical detection and characteristics of the subunit composition of phenylalanine hydroxylase in the brain of man

Immunochemical properties and subunit structure of an antigen were characterized in autopsy specimens of human liver and brain, using antiserum against human phenylalanine hydroxylase. An identical antigen was revealed in extracts of organs by immunoelectrophoresis. Its content was 1.5-2.0 mg/g tissue in the liver and 20-40 micrograms/g tissue in the brain. One L enzyme subunit and two H subunits were identified in the liver extracts after two-dimensional electrophoresis followed by immunoblotting. Subunit structure of phenylalanine hydroxylase in the brain was similar to that in the liver. The molecular weight of L subunit was 55,000 and it was located in the same area as albumin isoforms. The molecular weight of H subunits was 57,000 and they differed from L subunits in pI. The antigen was purified from crude extracts of biopsy liver by affinity chromatography on immunoadsorbent to phenylalanine hydroxylase and showed phenylalanine hydroxylase activity. An antigen with similar molecular weight was also purified from the brain extract by the same method. These data suggest that phenylalanine hydroxylase can be present in the human brain.     

Phenylalanine hydroxylase-like antibody in human chorionic villi]

An antigen similar by electrophoretic mobility to liver phenylalanine hydroxylase (PH) and cross-reacting with monoclonal antibody PH8 against liver PH was detected in extracts of soluble proteins in 6 from 23 samples of chorionic villi. An antigen with electrophoretic mobility corresponding to 40-41 kDa was detected in extracts of membrane proteins from these 23 samples by immunoblotting with monoclonal antibody PH8. Its molecular weight was similar to that of major chymotryptic peptide of human liver PH. The content of the antigen varied with samples and was less than 20 ng/mg of the extracted protein. Two-dimensional gel electrophoresis revealed only 1 spot of the antigen. The antigen did not react with monoclonal antibodies PH7 and PH9 epitopes of which were located in N-terminal fragment of liver PH. These data suggest that the antigen of membrane fraction could be a PH protein without N-terminal domain.     

Isolation of inactive phenylalanine hydroxylase from autopsy specimens of human liver

An electrophoretically homogeneous protein has been isolated from human liver autoptats, using a procedure employed for the isolation of phenylalanine hydroxylase from rat liver. The procedure includes chromatography of liver extracts on phenyl-Sepharose and subsequent purification on DEAE-Toyopearl. The activity of phenylalanine hydroxylase in the autoptats was markedly decreased in comparison with that in bioptats. The isolated protein possessed no enzymatic activity. However, the subunit composition of the protein, the molecular masses of protein subunits (55 and 57 kD) and the amino acid composition were close to those of the human enzyme. Antibodies to the protein inhibited the phenylalanine hydroxylase activity in human liver bioptats and weakly inhibited the rat enzyme. The experimental results suggest that the structural organization of phenylalanine hydroxylase does not alter as a result of the loss of enzymatic activity in cadaverous human liver.     

Proteolytic modification of the amino-terminal and carboxyl-terminal regions of rat hepatic phenylalanine hydroxylase

Activation of rat liver phenylalanine hydroxylase by limited proteolysis catalyzed by chymotrypsin was investigated with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure gel filtration. Both activation and proteolysis were decreased by the addition of the natural cofactor, (6R)-tetrahydrobiopterin. From chymotryptic digests of the hydroxylase carried out in the presence and absence of (6R)-tetrahydrobiopterin, several different enzyme species were isolated by high pressure gel filtration. One species (subunit Mr = 47,000) with unchanged hydroxylase activity was isolated from the chymotryptic digest in the presence of (6R)-tetrahydrobiopterin; it was derived from the native enzyme (Mr = 52,000) by cleavage of the COOH-terminal Mr = 5,000 portion of the native enzyme. In the absence of (6R)-tetrahydrobiopterin, another species (subunit Mr = 36,000) was isolated. In addition to modification at the COOH-terminal end of the molecule, this species also had lost a Mr = 11,000 fragment from the NH2-terminal end of the hydroxylase. The Mr = 11,000 fragment was shown to include the phosphorylation site of the enzyme. This Mr = 36,000 species was 30-fold more active than the native phenylalanine hydroxylase when assayed in the presence of tetrahydrobiopterin. These results suggest that the regulatory domain that inhibits hydroxylase activity in the basal state may be located at the NH2 terminus of the phenylalanine hydroxylase subunit.     

Isolation and characterization of the Legionella pneumophila outer membrane.

A whole cell lysate of Legionella pneumophila was fractionated into five membrane fractions by sucrose gradient centrifugation. Membranes were characterized by enzymatic, chemical, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Two forms of cytoplasmic membrane (CM-1, CM-2), a band of intermediate density (IM), and two forms of outer membrane (OM-1, OM-2) were detected. The CM-1 fraction was the purest form of cytoplasmic membrane, and fraction CM-2 was primarily cytoplasmic membrane associated with small amounts of peptidoglycan. The IM, CM-1, and CM-2 fractions were enriched in peptidoglycan, and the amount of carbohydrate and 2-keto-3-deoxyoctonic acid was not appreciably greater in outer membrane relative to cytoplasmic membrane. Phosphatidylethanolamine and phosphatidylcholine were found to be the major phospholipids in the membrane fractions. The major outer membrane proteins had molecular sizes of 29,000 and 33,000 daltons and were both modified by heating. The 29,000-dalton protein was tightly associated with the peptidoglycan and was equally distributed in the IM, OM-1, and OM-2.     

Identification of envelope proteins of Candida albicans by vectorial iodination

Intact Candida albicans yeast cells were radiolabelled with 125I, and cell wall, mixed membrane and soluble fractions prepared. The majority (67%) of the 125I was detected in the protein of the cell wall fraction at a specific activity 70-fold higher than that in the membrane or soluble fractions. SDS treatment of the cell wall fraction released 52% of the total protein but only 3% of the wall-bound 125I, and the extract was shown to be severely contaminated with cytosolic and membrane proteins. Zymolyase digestion of SDS treated walls liberated material which contained 93% of the 125I and on electrophoresis migrated as a single diffuse zone (average Mr 260 kD) typical of heterogenous mannoproteins. Protein (1.5%), GlcN (0.08%) and hexose (98.4%) content was measured and amino acid analysis showed enrichment in Ser (15.9%) and Thr (20.2%). These results indicate that the major iodinated protein(s) in the cell envelope is a 260 kD mannoprotein fraction containing both O-linked and Asn-linked oligosaccharides.     

Phenylalanine hydroxylase of human embryos: its homology with the enzyme from the liver of adults

Phenylalanine hydroxylase (PH) activity was discovered in the liver of 7-12 week old human embryos. Embryonic and adult PHs were identical, as shown by immunoelectrophoresis. Unlike the adult liver PH, the PH content of the extract of cytoplasmic proteins of embryonic liver was reduced but the specific activity was increased more than by one order of magnitude. H (57,000 D) and L (55,000 D) subunits were detected by immunoblotting. The L subunit predominates in the extract of membrane proteins of embryonic liver. Hence, the major part of phenylalanine oxidizing activity in the embryonic liver is related to the enzyme immunochemically identical with the PH of adult liver but differing from it in some structural and functional properties.     

Immunochemical identification of the growth hormone and prolactin in the hypophyseal polypeptide spectrum of chickens

Forty-eight fractions of polypeptides including 39 fractions with a molecular weight of 14-95 kD were identified in chick adenohypophysis by sodium dodecyl sulphate electrophoresis in 10-20% gradient polyacrylamide gel slabs. The immunochemical identification of the polypeptides was performed with the aid of the electroblotting of proteins and antisera to human STH, to bovine prolactin, and to the tissue-specific antigen A-1 of chick adenohypophysis. Antisera to human STH and to antigen A-1 reacted with the same major polypeptide fraction, m.w. 26 kD, characteristic of the caudal lobe of the adenohypophysis. Immunoreactive prolactin was present in chick adenohypophysis in the form of a polypeptide fraction with a molecular weight of 25 kD and in the form of two minor fractions of polypeptides with molecular weights of 27 and 28 kD. The data obtained indicate the identity of the adenohypophyseal tissue-specific antigen A-1 to chick STH.     

Two apparent molecular weight forms of human and monkey phenylalanine hydroxylase are due to phosphorylation

Two-dimensional polyacrylamide gel analyses of purified human and monkey liver phenylalanine hydroxylase reveal that the enzyme consists of two different apparent molecular weight forms of polypeptide, designated H (Mr = 50,000) and L (Mr = 49,000), each containing three isoelectric forms. The two apparent molecular weight forms, H and L, represent the phosphorylated and dephosphorylated forms of phenylalanine hydroxylase, respectively. After incubation of purified human and monkey liver enzyme with purified cAMP-dependent protein kinase and [gamma-32P]ATP, only the H forms contained 32P. Treatment with alkaline phosphatase converted the phenylalanine hydroxylase H forms to the L forms. The L forms but not the H forms could be phosphorylated on nitrocellulose paper after electrophoretic transfer from two-dimensional gels. Phosphorylation and dephosphorylation of human liver phenylalanine hydroxylase is not accompanied by significant changes in tetrahydrobiopterin-dependent enzyme activity. Peptide mapping and acid hydrolysis confirm that the apparent molecular weight heterogeneity (and charge shift to a more acidic pI) in human and monkey liver enzyme results from phosphorylation of a single serine residue. However, phosphorylation by the catalytic subunit of cAMP-dependent protein kinase does not account for the multiple charge heterogeneity of human and monkey liver phenylalanine hydroxylase.     

Disposition of glycoproteins in plasma membrane of cultured rat embryonic fibroblasts

Plasma membrane fractions were isolated from untreated and trypsin- or neuraminidase-treated rat embryo fibroblasts and their sialic acids contents per mg membrane protein were determined. The difference represented enzyme releasable sialic acid exposed on the medium side of the cell mambrane. It was 14 to 23% of the total membrane bound sialic acid. Isolated plasma membrane fraction from entreated and enzyme treated cells were then subjected to trypsin or neuraminidase treatment to obtain enzyme-releasable sialic acid from both faces and from the cytoplasmic face of the membrane respectively. Between 30 and 50% of the total membrane bound sialic was released from both the faces and 14 to 30% from the cytoplasmic face. An average of 59% was insusceptible to these enzymes. As an alternative to a cytoplasmic location of sialic acid containing membrane constituents, inaccessibility of enzymes to some of these constituents present on the surface of intact cells is considered.Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of plasma membrane fractions isolated from untreated and trypsin treated cells and of trysinized plasma membrane fraction was carried out to know the number and gel migration of proteins and glycoproteins which are exposed on each of the two faces of the plasma membrane and are sensitive or insensitive to trypsin. The resilts obtained were confirmed by SDS-polyacrylamide gel electrophoresis of untreated and trypsin-treated cells and of isolated plasma membrane fraction after subjecting them to enzymatic radioiodination.     

Analysis of proteins in normal and regenerating rat liver using two-dimensional electrophoresis

The major proteins of homogenate, cytosol, nuclei and nuclear membrane extract from normal and regenerating rat liver were studied by two-dimensional electrophoresis with a view of detecting proteins involved in DNA replication regulation. Essential quantitative differences in three out of 200 polypeptides separated as spots and dyed with Coomassie R-250 on two-dimensional maps were revealed. The content of the p38 nuclear protein (Mr congruent to 38 kD, pI congruent to 4) increases 6-8-fold in the S-phase. The level of another nuclear protein, p50 (Mr congruent to 50 kD, pI congruent to 6.5) decreases 2-3-fold. The cytoplasmic protein p35 (Mr congruent to 35 kD, pI congruent to 8) also decreases 2-3-fold. Moreover, the p40 protein (Mr congruent to 40 kD, pI congruent to 6) whose content in the nuclei sharply rises up to 20 times after sham operation was revealed.     

Reversible inactivation of phenylalanine hydroxylase by catecholamines in cultured hepatoma cells.

Phenylalanine hydroxylase in Reuber H4 hepatoma cell cultures can be rapidly inactivated by the addition of epinephrine, norepinephrine, dopamine, or 3,4-dihydroxyphenylalanine, in order of decreasing effectiveness, to the culture medium. The enzyme was 50% inactivated in 1 hour by 25 muM (R)-epinephrine or 45 muM (R)-norepinephrine in the medium. High concentrations of epinephrine caused a 70% inactivation in 15 min. Phenylalanine hydroxylase appears to be reversibly inactivated by epinephrine within the cells; since washing the compound off the cell cultures resulted in a rapid restoration of enzyme activity (40% in 1 hour), cycloheximide had little effect on the initial rate of recovery of enzyme activity and the same amount of phenylalanine hydroxylase antigen per cell was isolated from treated and normal cultures. Both (S)- and (R)-epinephrine inactivated the enzyme, and 0.1 mM desmethylimipramine, an inhibitor of amine transport, significantly decreased the effect of epinephrine on the hydroxylase activity. The possibility, suggested by the above results, that epinephrine might be directly inactivating phenylalanine hydroxylase within the cells was supported by the finding that purified rat liver phenylalanine hydroxylase would be 50% inactivated by 1.5 muM epinephrine in 10 min.     

The two molecular weight forms of rat phenylalanine hydroxylase are encoded by different messenger RNAs

Immunoprecipitation of the phenylalanine hydroxylase formed by translation of rat liver RNA in a rabbit reticulocyte cell-free protein synthesis system was used to examine the origin of the molecular weight heterogeneity of the enzyme. Sodium dodecyl sulfate-polyacrylamide electrophoresis of the immunoprecipitated products showed that in most cases a single specifically immunoprecipitated polypeptide was produced which corresponded to the higher molecular weight (H) form of phenylalanine hydroxylase (Mr = 50,000). The identity of the product was confirmed by immunological competition and peptide mapping. RNA from other rats, however, coded for both the H-form and the lower molecular weight (L) form of phenylalanine hydroxylase or for only the L-form. The evidence suggests that the L-form derives from a different mRNA, rather than by proteolysis of the H-form, an interpretation which is supported by the isolation of the lower form of phenylalanine hydroxylase from livers of some rats.     

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)     

Isolation and sequence of a cDNA clone which contains the complete coding region of rat phenylalanine hydroxylase. Structural homology with tyrosine hydroxylase, glucocorticoid regulation, and use of alternate polyadenylation sites

Screening of a rat liver cDNA expression library constructed in the vector lambda gt11 with an affinity purified antiserum to rat phenylalanine hydroxylase has resulted in the isolation of two clones which contain the complete coding region (1362 base pairs) of phenylalanine hydroxylase and the entire 3'-untranslated region (562 base pairs). From the nucleotide sequence we deduced the amino acid sequence of the enzyme. The molecular weight is 51,632 (452 amino acids). The rat enzyme is highly homologous to human phenylalanine hydroxylase. The two proteins differ in only 36 amino acids (92% homology), many of which are conservative changes. A dot matrix computer program was used to analyze regions of homology with the amino acid sequence of rat tyrosine hydroxylase. Considerable homology was detected from amino acid 140 in the rat enzyme to the C terminus, but little or no homology was apparent in the N-terminal region. The cDNA clone was used to determine the levels of phenylalanine hydroxylase mRNA in rat tissues using RNA blot hybridization. Two mRNA species were detected, with approximate lengths of 2,000 and 2,400 nucleotides, which appear to derive from use of alternate polyadenylation signals. No difference in mRNA size was found in rats which have different phenylalanine hydroxylase alleles. The kidney was found to contain about 10% of the mRNA found in the liver, and no phenylalanine hydroxylase mRNA was detected in rat brain. Reuber H4 hepatoma cells were also analyzed for phenylalanine hydroxylase mRNA. The parental cells contained mRNA species of the same sizes as in rat liver. Incubation in 10(-6) M hydrocortisone for 24 h resulted in an 18-fold increase in the mRNA level. Mutant hepatoma cells which express very little phenylalanine hydroxylase contained less than 5% of the parental mRNA, but the gene still responded to hydrocortisone.     

Distribution of calmodulin and calmodulin-binding proteins in membranes from bovine epididymal spermatozoa

Reproducible concentrations of calmodulin representing approximately 0.1% of the membrane protein were detected in purified plasma membranes from bovine epididymal spermatozoa. When membranes were isolated in the presence of 1 mM EGTA, the amount of calmodulin associated with the plasma membranes was not reduced. Calmodulin-binding proteins were detected in both purified plasma membranes and in a mixed membrane fraction containing both plasma membranes and cytoplasmic droplet membranes. A calcium-dependent, calmodulin-binding protein of apparent molecular weight 123,000 was detected in both fractions. In the presence of 1 mM EDTA, putative calcium-independent calmodulin-binding proteins of apparent molecular weights 93,000, 32,000, 18,000, and 15,000 were detected in the plasma membrane fraction. The 15,000 Mr polypeptide was also present in the mixed membrane fraction but the three proteins of higher molecular weight were reduced or absent in this fraction.     

5'-Nucleotidase in rat liver lysosomes

5'-Nucleotidase was found in purified rat liver tritosomes. When tritosomes were subfractionated into the membrane and soluble contents fractions, 73% of the total 5'-nucleotidase activity was found in the membrane fraction and 24% in the soluble contents fraction. Immunoblotting using specific polyclonal antibodies against the rat liver plasma membrane 5'-nucleotidase showed that the mobilities on SDS-polyacrylamide gel electrophoresis of both 5'-nucleotidases in the membrane and contents fractions were identical to that of the enzyme in the plasma membranes (Mr = 72,000). 5'-Nucleotidases in the membrane and contents fractions were sensitive to neuraminidase and converted into a form that was 4 kDa smaller after digestion, as observed in the case of plasma membrane enzyme. 5'-Nucleotidases, both from the membrane and contents fractions, were purified using immunoaffinity chromatography, and the isoelectric points, heat stability, and oligomeric structure of the purified enzymes were compared. Isoelectric focusing and the heat stability test indicated the resemblance of the soluble enzyme to the membrane-bound enzyme. However, the membrane-bound enzyme aggregated in the absence of Triton X-100, whereas the soluble enzyme behaved as a dimer. The topography of 5'-nucleotidase in the tritosomal membranes was studied using antibodies against 5'-nucleotidase and neuraminidase treatment. The inhibition of 5'-nucleotidase were not observed in the intact tritosomal fraction until the tritosomes had been disrupted by osmotic shock. These results show that the active sites and the oligosaccharide chains of 5'-nucleotidase are located on the inside surface of the tritosomal membranes.     

Biochemical characterization of recombinant human phenylalanine hydroxylase produced in Escherichia coli

A full-length human phenylalanine hydroxylase cDNA has been recombined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express phenylalanine hydroxylase immunoreactive protein and pterin-dependent conversion of phenylalanine to tyrosine. Recombinant human phenylalanine hydroxylase produced in E. coli has been partially purified, and biochemical studies have been performed comparing the activity and kinetics of the recombinant enzyme with native phenylalanine hydroxylase from human liver. The optimal reaction conditions, kinetic constants, and sensitivity to inhibition by aromatic amino acids are the same for recombinant phenylalanine hydroxylase and native phenylalanine hydroxylase. These data indicate that the recombinant human phenylalanine hydroxylase is an authentic and complete phenylalanine hydroxylase enzyme and that the characteristic aspects of phenylalanine hydroxylase enzymatic activity are determined by a single gene product and can be constituted in the absence of any specific accessory functions of the eukaryotic cell. The availability of recombinant human phenylalanine hydroxylase produced in E. coli will expedite physical and chemical characterization of human phenylalanine hydroxylase which has been hindered in the past by inavailability of the native enzyme for study.