Manduca sexta prophenoloxidase (proPO) activation requires proPO-activating proteinase (PAP) and serine proteinase homologs (SPHs) simultaneously

In the tobacco hornworm Manduca sexta, proteolytic activation of prophenoloxidase (proPO) is mediated by three proPO-activating proteinases (PAPs) and two serine proteinase homologs (SPHs) (Proceedings of the National Academy of Sciences, USA 95 (1998) 12220-12225; J. Biol. Chem. 278 (2003a) 3552-3561; Insect Biochem. Mol. Biol. 33 (2003b) 1049-1060). While our current data are consistent with the hypothesis that the SPHs serve as a cofactor/anchor for PAPs (Insect Biochemistry and Molecular Biology 33 (2003) 197-208; Insect Biochemistry and Molecular Biology 34 (2004) 731-742), roles of these clip-domain proteins (i.e. PAPs and SPHs) in proPO activation are poorly defined. To better understand this process, we further characterized the activation reaction using proPO, PAP-1 and SPHs. PAP-1 itself cleaved nearly 1/3 of proPO at Arg51 without generating much phenoloxidase (PO) activity. In the presence of SPHs, the cleavage of proPO became more complete while the increase in PO activity was over 20-fold, indicating that the extent of cleavage does not directly correlate with PO activity. Since SPHs and p-amidinophenyl methanesulfonyl fluoride (APMSF)-treated PAP-1 did not generate active PO by interacting with proPO, proteolytic cleavage is critical for proPO activation. After 1/5 of proPO was processed by PAP-1 alone which was then inactivated by M. sexta serpin-1J or APMSF, further incubation of the reaction mixture with SPHs failed to generate active PO either. Thus, SPHs cannot generate PO activity by simply binding to cleaved proPO. M. sexta proPO activation requires active PAP-1 and SPHs at the same time-one for limited proteolysis and the other as a cofactor, perhaps. Gel filtration chromatography and native gel electrophoresis revealed the PAP-SPH, proPO-PAP, and SPH-proPO associations, essential for generating high Mr, active PO at the site of infection.     

Regulation of the pentose phosphate cycle. Cofactor that controls the inhibition of glucose-6-phosphate dehydrogenase by NADPH in rat liver.

A cofactor of Mr 10(4), characterized as a polypeptide, was found to co-operate with GSSG to prevent the inhibition of glucose-6-phosphate dehydrogenase by NADPH, in order to ensure the operation of the oxidative phase of the pentose phosphate pathway, in rat liver [Eggleston & Krebs (1974) Biochem. J. 138, 425-435; Rodriguez-Segade, Carrion & Freire (1979) Biochem. Biophys. Res. Commun. 89, 148-154]. This cofactor has now been partially purified by ion-exchange chromatography and molecular gel filtration, and characterized as a protein of Mr 10(5). The lighter cofactor reported previously was apparently the result of proteolytic activity generated during the tissue homogenization. The heavier cofactor was unstable, and its amount increased in livers of rats fed on carbohydrate-rich diet. Since the purified cofactor contained no glutathione reductase activity, the involvement of this enzyme in the deinhibitory mechanism of glucose-6-phosphate dehydrogenase by NADPH should be ruled out.     

Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei.

Dihydroorotate dehydrogenase (DHODase) has been purified 400-fold from the rodent malaria parasite Plasmodium berghei to apparent homogeneity by Triton X-100 solubilization followed by anion-exchange, Cibacron Blue F3GA-agarose affinity, and gel filtration chromatography. The purified enzyme has a molecular mass of 52 +/- 2 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of 55 +/- 6 kDa by gel filtration chromatography, and it has a pI of 8.2. It is active in monomeric form, contains 2.022 mol of iron and 1.602 acid-labile sulfurs per mole of enzyme, and does not contain a flavin cofactor. The purified DHODase exhibits optimal activity at pH 8.0 in the presence of the ubiquinone coenzyme CoQ6, CoQ7, CoQ9, or CoQ10. The Km values for L-DHO and CoQ6 are 7.9 +/- 2.5 microM and 21.6 +/- 5.5 microM, respectively. The kcat values for both substrates are 11.44 min-1 and 11.70 min-1, respectively. The reaction product orotate and an orotate analogue, 5-fluoroorotate, are competitive inhibitors of the enzyme-catalyzed reaction with Ki values of 30.5 microM and 34.9 microM, respectively. The requirement of the long-chain ubiquinones for activity supports the hypothesis of the linkage of pyrimidine biosynthesis to the electron transport system and oxygen utilization in malaria by DHODase via ubiquinones [Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].     

Glycogen phosphorylase from Neurospora crassa: purification of a high-specific-activity, non-phosphorylated form.

A highly active glycogen phosphorylase was purified from Neurospora crassa by polyethylene glycol fractionation at pH 6.16 combined with standard techniques (chromatography and salt fractionation). The final preparation had a specific activity of 65 +/- 5 U/mg of protein (synthetic direction, pH 6.1, 30 degrees C) and was homogeneous by the criteria of gel electrophoresis, amino-terminal analysis, gel filtration, and double immunodiffusion in two dimensions. The enzyme had a native molecular weight of 180,000 +/- 10,000 (by calibrated gel filtration and gel electrophoresis) and a subunit molecular weight of 90,000 +/- 5,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each subunit contained one molecule of pyridoxal phosphate. No phosphoserine or phosphothreonine was detected by amino acid analysis optimized for phosphoamino acid detection. The enzyme isolated from cells grown on high-specific-activity 32Pi (as sole source of phosphorus) contained one atom of 32P per subunit. All the radioactivity was removed by procedures that removed pyridoxal phosphate. Thus, the enzyme could not be classified as an a type (phosphorylated, active in the absence of a cofactor) or as a b type (non-phosphorylated, inactive in the absence of a cofactor). The level of phosphorylase was markedly increased in mycelium taken from older cultures in which the carbon source (glucose or sucrose) had been depleted. The polyethylene glycol fractionation scheme applied at pH 7.5 to mycelial extracts of younger cultures (taken before depletion of the sugar) resulted in co-purification of glycogen phosphorylase and glycogen synthetase.     

Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp.

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg²⁺, Co²⁺, and Mn²⁺ were required for maximum activity of the enzyme. The K_m values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while K_cat values were 2.3 × 10² s^-1 and 0.5 × 10² s^-1, respectively.     

Chemical cleavage of bovine beta-lactoglobulin by BNPS-skatole for preparative purposes: comparative study of hydrolytic procedures and peptide characterization

A comparative study of various procedures for tryptophanyl peptide bond cleavage by BNPS-skatole [2-(2-nitrophenyl)-3-methyl-3-bromoindolenine] was carried out on native and on reduced and alkylated bovine -lactoglobulin (BLG). The reaction yield and the composition of the derived products were studied in acetic acid, trifluoroacetic acid (TFA), and ethanol/TFA. For BNPS-skatole removal, extraction by water or ethyl ether was compared with dialysis and gel filtration. The three expected peptides (1–19, 20–61, 62–162) and incomplete cleaved fragments (1–61, 20–162) were separated and characterized by electrophoresis, reverse-phase high-performance liquid chromatography, and mass spectrometry. The highest hydrolysis yield (67.4%) occurred with native BLG cleaved in 88% acetic acid at 47°C for 60 min. Subsequent water extraction and gel filtration led to total recovery of the material, but reagent elimination was only quantitative after gel filtration. Cleavage specificity was ensured by mass spectrometry and the amino acid composition of peptides 1–19 and 62–162. The chemical side reactions identified are discussed.     

Prophenoloxidase (proPO) activation in Manduca sexta: an analysis of molecular interactions among proPO, proPO-activating proteinase-3, and a cofactor

Proteolytic activation of prophenoloxidase (proPO) is an integral part of the insect immune system against pathogen and parasite infection. This reaction is mediated by a proPO-activating proteinase (PAP) and its cofactor in the tobacco hornworm, Manduca sexta (Proc. Natl. Acad. Sci. USA 95 (1998) 12220; J. Biol. Chem. 278 (2003) 3552; Insect Biochem. Mol. Biol. 33 (2003) 1049). The cofactor consists of two serine proteinase homologs (SPHs), which associate with immulectin-2, a calcium-dependent lectin that binds to lipopolysaccharide (Insect Biochem. Mol. Biol. 33 (2003) 197). In order to understand the auxiliary effect of SPH-1 and SPH-2 in proPO activation, we started to investigate the molecular interactions among proPO, PAP-3, and the proteinase-like proteins. M. sexta SPH-1 and SPH-2 were purified from hemolymph of prepupae by hydroxylapatite, gel filtration, lectin-affinity, and ion exchange chromatography. They existed as non-covalent oligomers with an average molecular mass of about 790 kDa. MALDI-TOF mass fingerprint analysis revealed a new cleavage site in SPH-1 before Asp85. The PAP cofactor did not significantly alter Michaelis constant (KM) or kcat of PAP-3 towards a synthetic substrate, acetyl-Ile-Glu-Ala-Arg-p-nitroanilide, but greatly enhanced proPO activation by PAP-3. The apparent KM for proPO was determined to be about 9.4 microg/ml, close to its estimated concentration in larval hemolymph. In the presence of excess proPO and a set amount of PAP-3, increasing levels of phenoloxidase (PO) activity were detected as more SPHs were added. Half of the maximum proPO activation occurred when the molar ratio of PAP-3 to SPH was 1:1.4. Gel filtration experiments suggested that proPO, PAP-3, and the cofactor formed a ternary complex.     

Biosynthesis of Tetrapyrrole Pigment Precursors : Pyridoxal Requirement of the Aminotransferase Step in the Formation of delta-Aminolevulinate from Glutamate in Extracts of Chlorella vulgaris

The aminotransferase that catalyzes the formation of δ-aminolevulinic acid from glutamate-1-semialdehyde or from glutamate in a reconstituted enzyme system was isolated and partially purified from Chlorella vulgaris. The apparent molecular weight of the aminotransferase was determined by Sephadex G-100 and Ultrogel AcA 54 gel filtration to be 60,000 ± 5,000. Catalytic activity of the aminotransferase required pyrixodal phosphate (PALP). The cofactor could not be removed by gel filtration after exposure of the enzyme to PALP. Aminotransferase was inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). The concentration of gabaculine required for half maximal inhibition was about 0.05 micromolar. Aminotransferase activity could be regained upon the removal of gabaculine by gel filtration and supplementing the assay medium with PALP. Neither the inhibitory action of gabaculine nor its reversibility was affected by preincubation of the enzyme with the keto acids levulinate and δ-aminolevulinic acid.     

Partial purification and some properties of an alkaline cellulase from an alkalophilic Bacillus sp.

A newly isolated Bacillus species, which grew optimally at 30°C and pH 10, produced a carboxymethylcellulase in a medium containing 10 g CM-cellulose/l. The enzyme, when partially purified by gel filtration, had a mass of about 29 kDa as determined by both SDS-PAGE and gel filtration chromatography. It was optimally active at pH 9.5 and 40°C, and was stable from pH 7 to 11 at 4°C for 24 h. The enzyme was stimulated by Ca²⁺ (1mm) but was completely inhibited by Hg²⁺ (1mm). Neither EDTA nor EGTA (10mm) affected the activity.The author is with the Department of Biological Sciences, University of Jordan. PO Box 2686, Amman 11181, Jordan     

Reversible inactivation of bovine plasma amine oxidase by cysteamine and related analogs

Cysteamine (1) was reported many years ago to reversibly inhibit lentil seedling amine oxidase, through the formation of a complex with thioacetaldehyde, the turnover product of 1. Herein, cysteamine (1) and its analogs 2-(methylamino)ethanethiol (3) and 3-aminopropanethiol (6) were found to be reversible inhibitors of bovine plasma amine oxidase (BPAO), but 2-(methylthio)ethylamine (7) was determined to be a weak irreversible inhibitor of BPAO. Based on our results, indicating the necessity of a sulfhydryl-amine for reversible inactivation of BPAO, the failure of inhibited BPAO to recover activity after gel filtration, the first-order kinetics of activity recovery upon dialysis, and 2,4,6-trihydroxyphenylalanine quinine (TPQ) cofactor transformation which indicated from the results of phenylhydrazine titration and substrate protection, we propose a mechanism for the reversible inactivation of BPAO by 1 involving the formation of a cofactor adduct, thiazolidine, between BPAO and 1.     

A cofactor protein required for actin activation of myosin Mg2+ATPase activity in leukemic myeloblasts

The Mg2+ATPase activity of the myosin of a myeloid leukemia cell line (Ml) was not activated by purified Ml actin or by muscle actin alone. Activation required the presence of a cellular fraction as a cofactor in addition to the actin, when Mg2+ATPase was stimulated as much as 20-fold. The cofactor was partially purified and characterized. 1) Its molecular weight was estimated as 45,000 to 55,000 daltons by gel filtration and as 45,000 daltons by SDS polyacrylamide gel electrophoresis. 2) The cofactor was a light chain kinase that phosphorylated both the L1 and L2 light chains of the Ml cell myosin, but not the L3 or heavy chain.     

Purification and characterization of natural and recombinant human plasminogen activator inhibitor-1 (PAI-1)

Human plasminogen activator inhibitor-1 (PAI-1) was purified from the conditioned medium of endotoxin-stimulated umbilical vein endothelial cell cultures by combinations of zinc-chelate-Sepharose chromatography, gel filtration on Sephacryl S-300 and immunoadsorption on an insolubilized murine monoclonal antibody (MA-7D4). The final product was obtained with a recovery of approximately 20% from conditioned medium containing about 3 micrograms/ml PAI-1. The yield of PAI-1 was 15-100 micrograms/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with Mr = 46,000 both under reducing and non-reducing conditions. On gel filtration on Sephacryl S-300, however, the material was separated in two fractions, one eluting at the void volume, which contains active PAI-1, and one with Mr = 46,000 containing inactive material that could be reactivated with 12 M urea. SDS gel electrophoresis of the isolated high-Mr fraction revealed several bands including a main 46,000-Mr component, which reacted with anti-(PAI-1) antibodies on immunoblotting and neutralized tissue-type plasminogen activator (t-PA). The active high-Mr fraction and the reactivated low-Mr fraction of PAI-1 inhibited t-PA very rapidly with an apparent second-order rate constant of (1.5-4) x 10(7) M-1 s-1. The cDNA of endothelial cell PAI-1 was cloned and expressed in Chinese hamster ovary cells. The translation product, purified from conditioned medium of transfected cells, also revealed a high-Mr and a low-Mr fraction on gel filtration, which were indistinguishable from the natural proteins by physicochemical, immunochemical and functional analysis. On reduced SDS gel electrophoresis, the high-Mr fraction was separated into the Mr-46,000 low-Mr PAI-1 and two other components with Mr 65,000 and one barely entering the gel. When reactivated low-Mr PAI-1 was added to plasma, PAI activity and PAI-1 antigen eluted with an apparent Mr greater than or equal to 300,000 on gel filtration, indicating that active PAI-1 complexes with one or more binding proteins in plasma.     

An enzyme mediating the conversion of zeatin to dihydrozeatin in phaseolus embryos

A reductase catalyzing the conversion of zeatin to dihydrozeatin was detected in soluble fractions of immature Phaseolus vulgaris embryos. The enzyme was partially purified by ammonium sulfate fractionation and affinity, gel filtration, and anion exchange chromatography. NADPH was the only cofactor required for enzyme activity, and the pH optimum was 7.5 to 8.0. The enzyme did not recognize compounds closely related to zeatin, such as ribosylzeatin, cls-zeatin, O-xylosylzeatin, N⁶-(Δ²-isopentenyl)adenine, or N⁶-(Δ²-isopentenyl)adenosine. No conversion of dihydrozeatin to zeatin by the enzyme was observed. Two forms of the reductase could be separated by either gel filtration or anion exchange high performance liquid chromatography. The high molecular weight isozyme (M_r 55,000 ± 5,000) eluted as the second peak from the anion exchange column, while the low molecular weight isozyme (M_r 25,000± 5000) was less negatively charged. The results suggest that side chain reduction occurs at the free base level. In addition, Phaseolus embryos are useful for the detection of zeatin-specific metabolic enzymes.     

Auxin-Stimulated NADH Oxidase Purified from Plasma Membrane of Soybean

NADH oxidation by plasma membrane vesicles purified from hypocotyls of etiolated soybean seedlings by two-phase partition was stimulated 2- to 3-fold by auxins, indole-3-acetic acid, 2,4-dichlorophenoxy acetic acid (2,4-D), and α-naphthaleneacetic acid. The stimulation was concentration dependent in the presence or absence of detergent with a maximum for 2,4-D at 1 micromolar. The NADH oxidation activity was solubilized with the zwitterionic detergent CHAPS and purified by ion exchange chromatography and gel filtration approximately 2000-fold over the total homogenate. Both the partially purified fraction and an active band from nondenaturing gel electrophoresis revealed the same three bands when analyzed by denaturing gel electrophoresis. When obtained from plasma membrane vesicles from the region of rapid cell elongation, the NADH oxidase complex retained auxin responsiveness throughout purification (3- to 5-fold stimulation by 1 micromolar 2,4-D).     

2'-Deoxy-3'-O-(4-benzoylbenzoyl)- and 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate, fluorescent photoaffinity analogues of adenosine 5'-diphosphate. Synthesis, characterization, and interaction with myosin subfragment 1

Two new fluorescent nucleotide photoaffinity labels, 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate (Bz2 epsilon ADP) and 2'-deoxy-3'-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate [3'(Bz2)2'd epsilon ADP], have been synthesized and used as probes of the ATP binding site of myosin subfragment 1 (SF1). These analogues are stably trapped by the bifunctional thiol cross-linker N,N'-p-phenylenedimaleimide (pPDM) at the active site in a manner similar to that of ATP [Wells, J.A., & Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970], and nonspecific photolabeling can be minimized by removing free probe by gel filtration prior to irradiation. Both probes covalently photoincorporate with high efficiency (40-50%) into the central 50-kDa heavy chain tryptic peptide, as found previously for the nonfluorescent parent compound 3'(2')-O-(4-benzoylbenzoyl)adenosine diphosphate [Mahmood, R., & Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959]. The solution conformations of Bz2 epsilon ADP and 3'(Bz2)-2'd epsilon ADP were analyzed by steady-state and time-resolved fluorescence spectroscopy. These data indicated that the benzoylbenzoyl rings in both analogues were stacked over the epsilon-adenine ring. The degree of stacking was greater with the 2' isomer than with the 3' isomer. Fluorescence quantum yields and lifetimes were measured for Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP reversibly bound, stably trapped, and covalently photoincorporated at the active site of SF1. These values were compared with those for 3'(2')-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6-ethenoadenos ine diphosphate (CBH epsilon ADP) and 2'-deoxy-3'-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6- ethenoadenosine diphosphate [3'(CBH)2'd epsilon ADP]. These derivatives were synthesized as fluorescent analogues of the expected product of the photochemical reactions of Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP, respectively, with the active site of SF1. The fluorescence properties of the carboxybenzhydrol derivatives trapped at the active site by pPDM were compared with those of the Bz2 nucleotide-SF1 complexes. These properties were consistent with a photoincorporation mechanism in which the carbonyl of benzophenone was converted to a tertiary alcohol attached covalently to the protein. The specific, highly efficient photoincorporation of Bz2 epsilon ADP at the active site will allow it to be used as a donor in distance measurements by fluorescence resonance energy transfer to acceptor sites on actin.     

Human pituitary tryptase: molecular forms, NH2-terminal sequence, immunocytochemical localization, and specificity with prohormone and fluorogenic substrates

A human pituitary-derived serine protease, immunologically identical to human lung tryptase (Smith, T. J., Hougland, M.W., and Johnson, D.A. (1984) J. Biol. Chem. 259, 11046-11051), was found immunohistochemically to be associated with mast cells present in pituitary connective tissue. Western blotting combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of multiple forms: a major Mr 36,300 form and three minor forms with Mr 32,400, 33,400, and 34,600. Two major forms with Mr 35,600 and 34,100 were detected by affinity labeling with 125I-D-Tyr-Glu-Phe-Lys-Arg-CH2Cl. Treatment of the pituitary tryptase preparation with N-glycosidase F indicated that some of the molecular weight heterogeneity results from N-linked glycosylation. The multiple molecular weight forms appear to have the same NH2-terminal sequence: Ile-Val-Gly-Gly-Gln-Glu-Ala-Pro. Pituitary tryptase has an apparent Mr = 110,000 by gel filtration on Sephadex G-200 in the presence of 0.3 M NaCl, indicating that the enzyme may be a tetramer of Mr = 32,400-36,300 subunits. However, this quaternary structure was not stable to gradient polyacrylamide gel electrophoresis. Human pituitary tryptase was so reactive toward synthetic tripeptide coumarin-containing substrates containing a pair of basic amino acids at the site of cleavage such as benzyloxylcarbonyl-L-Ala-L-Lys-L-Arg-4-methylcoumarin-7-amide (k cat/Km = 2.38 X 10(8) M-1 s-1) that Briggs-Haldane kinetics may apply. The reversible inhibitor NaCl at a concentration of 1 M decreased the k cat/Km for benzyloxylcarbonyl-L-Ala-L-Lys-L-Arg-4-methylcoumarin-7-amide to 6.53 X 10(6) M-1 s-1, which reflected a 100-fold increase in apparent Km. Based on active site titration with fluorescein mono-p-guanidinobenzoate hydrochloride, NaCl had no effect on the number of accessible active sites. Substrate specificity studies with prohormones indicated that pituitary tryptase has a preference for cleaving COOH-terminal to arginine or lysine residues which are preceded by a proline residue 4 or 6 residues NH2-terminal to the site of cleavage.     

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 identification of prostaglandin I 2 stimulating factor from human plasma

To isolate and identify the plasma factor which stimulates prostaglandin I 2 production by rat aortic ring, a human plasma fraction which showed a major stimulating activity on prostaglandin I 2 production was purified by ultrafiltrate, Sephadex G-10 gel filtration and QAE-Sephadex column chromatography. The purified plasma factor was identified as acid by its ultraviolet and infrared absorption spectroscopy, and ¹H nmr and ¹³C nmr spectroscopy. The stimulating activity of the purified plasma factor and that of authentic uric acid coincided with each other. The stimulating potency of uric acid at its physiological concentration in human plasma (about 50 μg/ml) was half of the deproteinized human plasma, and was about 30 fold stronger than that of L-tryptophan, a cofactor of prostaglandin hyperoxidase.     

Purification and subunit structure of rat-liver phosphoprotein phosphatase, whose molecular weight is 260000 by gel filtration (phosphatase IB)

1. Phosphoprotein phosphatase IB is a form of rat liver phosphoprotein phosphatase, distinguished from the previously studied phosphoprotein phosphatase II [Tamura et al. (1980) Eur. J. Biochem. 104, 347-355] by earlier elution from DEAE-cellulose, by higher molecular weight on gel filtration (260000) and by lower activity toward phosphorylase alpha. This enzyme was purified to apparent homogeneity by chromatography on DEAE-cellulose, aminohexyl--Sepharose-4B, histone--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200. 2. The molecular weight of purified phosphatase IB was 260000 by gel filtration and 185000 from S20,W and Stokes' radius. Using histone phosphatase activity as the reference for comparison, the phosphorylase phosphatase activity of purified phosphatase IB was only one-fifth that of phosphatase II. 3. Sodium dodecyl sulfate gel electrophoresis revealed that phosphatase IB contains three types of subunit, namely alpha, beta and gamma, whose molecular weights are 35000, 69000 and 58000, respectively. The alpha subunit is identical to the alpha subunit of phosphatase II. While the beta subunit is also identical or similar to the beta subunit of phoshatase II, the gamma subunit appears to be unique to phosphatase IB. 4. When purified phosphatase IB was treated with 2-mercaptoethanol at -20 degrees C, the enzyme was dissociated to release the catalytically active alpha subunit. Along with this dissociation, there was a 7.4-fold increase in phosphorylase phosphatase activity; but histone phosphatase activity increased only 1.6-fold. The possible functions of the gamma subunit are discussed in relation to this activation of enzyme.     

The purification of luteinizing-hormone-releasing factor with some observations on its properties

Bullock median-eminence tissue was used as a source of luteinizing-hormone-releasing factor for small-scale experiments to explore methods for its isolation. The presence of luteinizing-hormone-releasing factor was detected by the ovulation response in rabbits after intrapituitary infusion of the extract. Gel filtration was found to be suitable for the purification of these extracts. The releasing factor appeared to be a basic peptide of molecular weight in the range 1200-2500. On a larger scale, an extract of hypothalamic tissue from sheep was used to establish a multi-stage isolation procedure that resulted in a 200000-fold purification of luteinizing-hormone-releasing factor. After the initial extraction the isolation process consisted of: (1) two cycles of gel filtration; (2) anion-exchange chromatography; (3) gel filtration in a partially organic medium; (4) thin-layer chromatography on cellulose. Stage (3) separated two zones of activity each containing peptides. One of these was purified further by stage (4) to give a preparation that was active at a dose of 6mug. of peptide/animal, although activity diminished seriously during storage. This preparation contained only five or six components, but the small amounts of peptides obtained at this stage of purity were insufficient for full characterization.