Purification and some properties of a novel alpha-L-fucosidase capable of acting on alpha-(1----6)-L-fucosidic linkages from Bacillus circulans M28

Two types of alpha-L-fucosidase (F-I and F-II), that differ in substrate specificity, were produced in the culture fluid by Bacillus circulans isolated from soil when the bacterium was cultivated on medium containing porcine gastric mucin. F-I was able to cleave the alpha-(1----2), alpha-(1----3), and alpha-(1----4)-L-fucosidic linkages in various oligosaccharides and glycoproteins, but not p-nitrophenyl alpha-L-fucoside, as previously reported [Y. Tsuji et al. (1990) J. Biochem. 107, 324-330]. F-II was purified from the culture fluid obtained with glucose medium by ammonium sulfate fractionation and various subsequent column chromatographies. The purified enzyme was found to be homogeneous on PAGE and its molecular weight was estimated to be approximately 250,000. The maximal activity was observed between pH 6.0 to 7.0, the stable pH range being 6.0 to 8.5. The enzyme specifically cleaved alpha-L-fucosidic bonds in low molecular weight substrates. The enzyme cleaved not only p-nitrophenyl alpha-L-fucoside, but also 2-fucosyllactose and 3-fucosyllactose. The enzyme was also able to act on the alpha-(1----6)-L-fucosidic linkages to N-acetylglucosamine in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine, and bi- and tetra-antennary oligosaccharides derived from porcine pancreatic lipase, which were not hydrolyzed by F-I.     

Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL

Two forms (F-I and F-II) of 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N⁶-(Δ²-isopentenyl)adenosine 5′-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5′-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The K_m values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5′-phosphates, AMP being the preferred substrate. N⁶-(Δ²-isopentenyl)Adenosine 5′-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3′-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5′-nucleotidases may play an important role in regulating levels of “active cytokinin” in plant cells.     

Lipoxygenase activities of human platelets and their subcellular fractions: Comparison between lipoxygenase-deficient platelets and normal platelets

Lipoxygenase activities were estimated in washed platelets (intact platelets) and their subcellular fractions obtained from 7 patients with deficient platelet lipoxygenase activities and 9 normal subjects. From sonicated platelet preparations, 12,000 g supernatant (F-I), cytosol (F-II) and microsomal fractions (F-III) were prepared by differential centrifugation. When 12-hydroxyeicosatetraenoic acid (12-HETE) produced by the incubation of arachidonic acid with intact platelets or each of their subcellular fractions from normal subjects was measured by reversed-phase high-performance liquid chromatography analysis, the lipoxygenase activities of F-I, F-II and F-III were 87%, 31% and 17%, respectively, of the enzyme activity of intact platelets. One of the patients showed no detectable lipoxygenase activity in any preparation, while the other patients showed reduced enzyme activities in all preparations. The addition of CaCl2 significantly increased 12-HETE synthesis solely by F-I from these patients. In most of these patients, contrary to normal subjects, it appeared that the lipoxygenase activity was not fully expressed in intact platelets, since the F-I produced more 12-HETE than the intact platelets.     

Partial purification and properties of a phosphatidylinositol 4,5-bisphosphate hydrolyzing phospholipase C from the soluble fraction of soybean sprouts

Three soluble enzyme fractions (F-I, F-II, and F-III) that hydrolyze phophoinositides were separated from soybean sprouts by using Matrex green gel column chromatography. Among the three phosphatidylinositol (PI)-specific phopholipsase C (PLC) enzymes, only the third fraction (F-III) was able to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) as well as phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP) as substrates. The F-I and F-II fractions only showed enzymatic activities for PI and PIP. The PIP2-hydrolyzing PLC protein, F-III, was partially purified using the chromatographic steps of the Matrex green gel, phenyl Toyopearl, Matrex orange gel, Mono S cation exchange, and superose 6 gel filtration columns. The molecular weight of the F-III protein was estimated to be about 64 kDa on SDS-PAGE. The protein showed immunocross-reactivity with a polyclonal antibody that was prepared against the X and Y motifs of animal PLC enzymes, the conserved catalytic domains. Ca2+ ion critically affected the PIP2-hydrolyzing PLC activity of the F-III protein, representing maximal activity at 10 microM Ca2+ concentration. The PIP2-hydrolyzing PLC activity of the protein was also significantly increased by sodium deoxycholate (SDC) from 0.05 to 0.08%. However, the activity was greatly reduced above the concentration, and no activity was detected at 0.3% SDC. In addition, the protein exhibited maximal PIP2-hydrolyzing PLC activity at pH, in the range of 6.5-7.5.     

Isolation and chemical characterization of algal polysaccharides from the green seaweed Ulva clathrata (Roth) C. Agardh

In order to obtain information on the content and composition of the water-soluble polysaccharides from Ulva clathrata, an extraction at 60°C, in different media, was performed: water, EDTA and HCl (F-I), each followed by a sequential extraction in NaOH 0.1 M (F-II). The extracts were recovered and analyzed for total carbohydrates, proteins, rhamnose, uronic acids and sulfate content. Differences were obtained in the yield and composition in both fractions of the different media (F-I and F-II). Higher yields resulted in the first fraction on all media. HCl extraction was the best in both fractions (14.83 ± 1.5% and 5.96 ± 1.1%, F-I and F-II, respectively). In all cases, F-I was more sulfated ranging from 27.87% to 35.8% and higher in rhamnose content, whereas F-II had higher protein and slightly higher uronic acid content. FTIR spectra showed that soluble polysaccharides from the green seaweed U. clathrata are sulfated polysaccharides, similar to ulvan obtained from other Ulva species and confirmed by the ¹ H-NMR spectrum, where the characteristic signal for the deoxy sugar (rhamnose) is present.     

Stimulation of phosphatase release from cultured tobacco cells by divalent cations

Upon addition of divalent cations to the incubation medium ofcultured tobacco cells, the release of phosphatase into themedium increased and the time course of the release became biphasic.A rapid release (phase I release) occurred instantaneously afterthe addition and then a release at a constant rate (phase IIrelease) followed. Sodium and potassium ions did not affectthe enzyme release. Lanthanum ions caused the biphasic enzymerelease but inhibited the phase II release. The effects of temperature and metabolic inhibitors indicatedthat phase I release was limited by a diffusion process butphase II release was limited by an enzymatic reaction requiringmetabolic energy. From the results it was concluded that divalent cations enhancedthe enzyme release not only by stimulating the transport ofenzyme to the outside of the cell membrane, but also by liberatingthe enzyme retained on the exterior of the cells, e.g., thecell walls. The released phosphatase could be separated into two fractions,F-I and F-II. Only F-I was released by phase I release, whileboth F-I and F-II resulted from phase II release. This indicatedthat F-I was preferentially trapped on the exterior of the cells. ¹ These experiments were carried out at the Department of Botanyin the Faculty of Science of the University of Tokyo. (Received December 15, 1978; )     

Isolation and characterization of mannan-binding proteins from chicken liver

Two binding proteins which recognize and bind mannose and N-acetylglucosamine (mannan-binding proteins, MBP) have been isolated from chicken liver to near homogeneity mainly by affinity chromatography on a column of Sepharose 4B-mannan. The neutral binding protein (pI 7.0), which has a high glycine content, is an analog of mammalian liver MBP (F-I). F-I consists of a series of proteins composed of two subunits of 28,000 (A) and 32,000 (B) Da. The proteins have molecular weights ranging from 280,000 to 740,000 and subunit compositions ranging from 6A + 4B to 5A + 19B. With increasing molecular weight the specific activity of mannan binding increases gradually, accompanied by a slight change in specificity to a preference for mannose rather than N-acetylglucosamine. The acidic binding protein (pI 5.1) is a glycoprotein with a high glutamic acid content (F-II). The molecular weight of F-II was estimated to be 640,000, and it is composed of single subunits of 41,000 Da. The two MBPs isolated in this study are distinct from the liver lectin specific for N-acetylglucosamine-terminated glycoproteins isolated from the same source [T. Kawasaki and G. Ashwell (1977) J. Biol. Chem. 252, 6536-6543] in chemical properties and binding specificities.     

蚯蚓纤溶酶新基因EfP-0的电子克隆及其编码区序列RT-PCR验证

利用生物信息学手段,以期获得蚯蚓纤溶酶F-Ⅰ-0组分的基因。根据从粉正蚓(Lumbricusrubellus)中分离的F-Ⅰ-0组分的N端氨基酸序列VVGGSDTTIGQYPHQL,利用DNAMAN软件通过电子克隆方法,从Lumbricidae的dbEST中获得该组分的核酸序列信息,设计特异引物,经过RT-PCR,成功地从赤子爱胜蚓(Eiseniafoetida)中克隆到一条蚯蚓纤溶酶新基因,命名为EfP-0。EfP-0基因全长678bp,编码225个氨基酸的成熟肽,属丝氨酸蛋白酶,胰蛋白酶家族,与F-Ⅰ-0组分的氨基酸组成非常接近。BLAST证明,EfP-0与已报道的蚯蚓纤溶酶基因之间的相似性均低于40%,因此为蚯蚓纤溶酶中的一个新基因,GenBank登录号为DQ836917。构建的pMAL-c2x-EfP-0重组质粒,在大肠杆菌TB1中获得融合蛋白MBP-EfP-0的可溶性表达,表达产物有酪蛋白平板溶解活性。     

Endo-β-N-acetylglucosaminidase from fig latex

Commercially available fig latex contains several endo-β-N-acetylglucosaminidases which catalyze the reaction: (Man)_nG1cNAcβ₁→4G1cNAcAsn → (Man)_nG1cNAc + G1cNAcAsn. Using (NH₄)₂SO₄ fractionation followed by chromatography on Sephadex G-100 and DEAE-Sephadex A-50, two distinct types of endo-β-N-acetylglucosaminidases have been partially purified and characterized. One, called F-I, hydrolyzes the di-N-acetylchitobiosyl linkage in the glycopeptide, (Man)₃(G1cNAc)₂Asn prepared from human IgG, much faster than that linkage in the glycopeptides, (Man)₅(G1cNAc)₂Asn and (Man)₆(G1cNAc)₂Asn both from ovalbumin. The other, called F-II, hydrolyzes the same linkage in (Man)₅(G1cNAc)₂-Asn and (Man)₆(G1cNAc)₂Asn, but not that in (Man)₃(G1cNAc)₂Asn.     

Control of glycogen synthase by insulin and isoproterenol in rat adipocytes. Changes in the distribution of phosphate in the synthase subunit in response to insulin and beta-adrenergic receptor activation

Rat adipocytes were incubated with [32P]phosphate to label glycogen synthase, which was rapidly immunoprecipitated from cellular extracts and cleaved using either CNBr or trypsin. All of the [32P]phosphate in synthase was recovered in two CNBr fragments, denoted CB-1 and CB-2. Isoproterenol (1 microM) rapidly decreased the synthase activity ratio (-glucose-6-P/+glucose-6-P) and stimulated the phosphorylation of both CB-1 and CB-2 by approximately 30%. Insulin opposed the decrease in activity ratio and blocked the stimulation of phosphorylation by isoproterenol. Incubating cells with insulin alone changed the 32P content of neither CB-1 nor CB-2. Trypsin fragments were separated by reverse phase liquid chromatography and divided into peak fractions, denoted F-I-F-VII in order of increasing hydrophobicity. F-V contained almost half of the [32P]phosphate and was phosphorylated when synthase was immunoprecipitated from unlabeled fat cells and incubated with [gamma-32P]ATP and the cAMP-independent protein kinase, FA/GSK-3. That F-V also had the same retention time as the skeletal muscle synthase fragment containing sites 3(a + b + c) suggests that it contains sites 3. Muscle sites 1a, 5, 1b, and 2 eluted with F-I, F-II, F-VI, and F-VII, respectively. F-V was increased approximately 25% by isoproterenol, but the largest relative increases were observed in F-I (4-fold), F-III (4-fold), and F-VI (2-fold). These results indicate that beta-adrenergic receptor activation results in increased phosphorylation of multiple sites on glycogen synthase. Insulin plus glucose decreased the overall 32P content of synthase by approximately 30%, with the largest decrease (40%) occurring in F-V. Without glucose, insulin decreased the [32P]phosphate in F-V by 17%, an effect which was balanced by increases in F-I, F-II, and F-III so that no net change in the total 32P contents of the fractions was observed. Thus, activation of glycogen synthase by the glucose transport-independent pathway seems to involve a redistribution of phosphate in the synthase subunit.     

毁灭柱孢菌中一种人参皂苷Rb1水解酶的纯化及酶学性质研究

通过DEAE-纤维素阴离子交换层析、30％～80％（NH3）2SO3盐析、Sepharose CL-6B凝胶过滤层析和Mono Q HR5／5阴离子交换层析,从毁灭枉孢菌培养液中部分纯化出一种能够水解人参皂苷Rb,的β-葡萄糖苷酶F-I。F—I具有较好的pH稳定性和热稳定性,在pH4．0～11．0范围内和55℃以下表现出良好的β-葡萄糖苷酶活性,其最适pH为5．0,最适温度为55℃。EDTA、Cu^2＋和Zn^2＋对该酶活性有较强的抑制作用。底物专一性分析表明,F—I能高特异性水解人工合成的底物pNPG,还能水解β-葡萄糖苷键连接的二糖如纤维二糖和龙胆二糖,说明此酶为一种β-葡萄糖苷酶。F—I对人参皂苷Rb1表现了较强的水解活性,而对人参皂苷Rb2和Rc的水解活性较低。该酶水解人参皂苷Rb1的路径为Rb1→Rd→F2→C—K。F—I对人参皂苷Rb1的这种高效水解为稀有人参皂苷的工业制备奠定了基础。     

Physiological correlation between nucleoside-diphosphate kinases and the 21-kDa guanine-nucleotide binding proteins copurified with the enzymes from the cell membrane fractions of Ehrlich ascites tumor cells

The physiological correlation between nucleoside-diphosphate kinases (NDP-kinases) and the 21-kDa guanine nucleotide-binding proteins (G1 and G2) which are copurified with the enzymes from the cell membrane fractions of Ehrlich ascites tumor cells has been biochemically investigated in vitro. We found that: incubation of the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinases (F-I and F-II) with one of the nucleoside 5'-diphosphates in the presence of 1 mM Mg2+ or 0.25 mM Ca2+ results in the rapid formation of nucleoside 5'-triphosphates without strict base specificity; GDP on the guanine nucleotide-binding proteins (G1, G2 and recombinant v-rasH p21) acts as a phosphate acceptor for the high-energy phosphates of the phosphoenzyme in the presence of 0.25 mM Ca2+; and [32P]GTP is preferentially formed from the 32P-labelled phosphoenzyme F-I and GDP-bound G1 or GDP-bound recombinant v-rasH p21 protein, even if any other nucleoside 5'-diphosphates are present in the reaction mixture. Although [32P]GTP formed was bound with the guanine nucleotide-binding proteins, it was immediately hydrolyzed by the proteins themselves in the presence of 5 mM Mg2+, but not in the presence of 0.25 mM Ca2+. Available evidence suggests that NDP-kinase may be responsible for the activation of the guanine nucleotide-binding proteins (G1, G2 and p21 proteins) through phosphate transfer by the enzyme.     

Characterization of nucleoside-diphosphate kinase-associated guanine nucleotide-binding proteins from HeLa S3 cells

Nucleoside-diphosphate (NDP) kinase-associated [alpha-32P]GTP-incorporating proteins from HeLa S3 cells have been biochemically characterized. Two distinct NDP-kinases (F-I and F-II) had been partially purified from HeLa S3 cells by Sephacryl S-300 gel filtration and DEAE-cellulose column chromatography. The [alpha-32P]GTP-incorporating proteins (approx. Mr 20,000) could be separated from NDP-kinases (approx. Mr 80,000) by 5-25% glycerol density-gradient centrifugation analysis after treatment with 7 M urea in the presence of 1 mM EDTA. [alpha-32P]GTP incorporation into these two proteins (G1 and G2) from NDP-kinases required 5 mM Mg2+ and was highly inhibited by either GDP or GTP analogues, such as guanylyl imidodiphosphate and guanylyl methylenediphosphate. [3H]GDP, but no other nucleoside 5'-diphosphates, was also bound to these two proteins in the presence of Mg2+ (5 mM). Moreover, incubation of [alpha-32P]GTP with either G1 or G2 in the presence of Mg2+ (5 mM) resulted in the formation of [32P]GDP and Pi. The data presented here indicated that the guanine nucleotide-binding activity, the GTPase activity, and the molecular weight (approx. Mr 20,000) of NDP-kinase-associated proteins from HeLa S3 cells are similar to those reported for ras oncogene products (p21 proteins).     

Xylanase activity of an endo-cellulase of carboxymethyl-cellulase type from Irpex lacteus (Polyporus tulipiferae).

An endo-cellulase [EC 3.2.1.4.] of carboxymethyl-cellulase type (F-1) which was fractionated from culture filtrate of Irpex lacetus and purified to electrophoretic and ultracentrifugal homogeneity, was found to show xylanase [EC 3.2.1.8.] activity. The activity was not removed from any of the intermediate fractions during the purification of the initial F-I peak, and the radio of xylanase to cellulase activity remained almost unchanged through the purification processes. The xylanase activity of F-I showed not only the same optiomal pH, heat stability, and pH stability as its cellulase activity, but also the same mobility as the cellulase activity upon cellulose acetate film and starch zone electrophoreses. The overall rates of hydrolysis of mixtures of variouis concentrations of CM-cellulose and xylan by F-1 coincided well with those calculated from the Michaelis-Menten treatment of two substances competing for the same active site of the enzyme. These results indicate that the xylanase activity of F-1 is intrinsic to the cellulase itself.     

The mechanisms of H2 activation and CO binding by hydrogenase I and hydrogenase II of Clostridium pasteurianum

Hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridium pasteurianum have been investigated by electron paramagnetic resonance (EPR) spectroscopy, in the presence and absence of the inhibitor, CO. These hydrogenases contain both a novel type of iron-sulfur cluster (H), which is the proposed site of H2 catalysis, and ferredoxin-type [4Fe-4S] clusters (F). The results show that the H clusters of these two hydrogenases have very different properties. The H cluster of oxidized hydrogenase II (Hox-II) exhibits three distinct EPR signals, two of which are pH-dependent. Hox-II binds CO reversibly to give a single, pH-independent species with a novel, rhombic EPR spectrum. The H cluster of reduced hydrogenase II (Hred-II) does not react with CO. In contrast, the EPR spectrum of Hox-I appears homogeneous and independent of pH. Hox-I has a much lower affinity for CO than Hox-II, and binds CO irreversibly to give an axial EPR signal. Hred-I also binds CO irreversibly. The EPR spectra of Fred-I and Fred-II show little or no change after CO treatment. Prior exposure to CO does not affect the catalytic activity of the reduced or oxidized hydrogenases when assayed in the absence of CO, but both enzymes are irreversibly inactivated if CO is present during catalysis. Mechanisms for H2 activation by hydrogenase I and hydrogenase II are proposed from the determined midpoint potentials (Em, pH 8.0) of H-I and H-II (Em approximately -400 mV, -CO; approximately -360 mV, +CO), F-I (Em = -420 mV, +/- CO), and F-II (Em = -180 mV, +/- CO). These allow one to rationalize the different modes of CO binding to the two hydrogenases and suggest why hydrogenase II preferentially catalyzes H2 oxidation. The results are discussed in light of recent spectroscopic data on the structures of the two H clusters.     

A halophilic extracellular protease from a halophilic archaebacterium strain 172 P1

An unidentified halophilic archaebacterium strain 172 P1 produced three extracellular proteases in media containing 15-27% salts. One component, F-II, was purified to homogeneity. It is a serine protease that can be inhibited by phenylmethylsulfonyl fluoride and chymostatin. A high concentration of NaCl was required for its stability; in the presence of 25% NaCl, only 4% of the activity was lost by incubating at 60 degrees C for 30 min, while complete inactivation occurred in the presence of 5% NaCl. F-II is a thermophilic and halophilic protease. High activity was obtained at 75-80 degrees C when F-II was assayed in the presence of 25% NaCl. The optimal concentration of NaCl required was 10-14% when assayed at 70 degrees C with azocasein as substrate, though a halophilic characteristic was not distinct at lower temperatures. Hydrolyses of the synthetic substrates succinyl-alanyl-alanyl-prolyl-phenylalanyl-4-methylcoumaryl-7-amide or succinyl-alanyl-alanyl-alanyl-p-nitroanilide at 26 degrees C were maximal at 25 and 30% NaCl, respectively. F-II was most stable at pH 6-7, and its optimal pH was 10.7. Its molecular weight was estimated as 44,000-46,000 by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and by gel filtration--high-pressure liquid chromatography. The sequence of the 35 N-terminal amino acid residues was determined and compared with that of other serine proteases.     

Structure of the complex oligosaccharides of fetuin.

The complete structure of the complex oligosaccharides of fetuin has been established. The three fractions of complex oligosaccharide which were isolated by ion exchange chromatography following pronase digestion (F-I, F-II, and F-III) had identical molar ratios of sialic acid (Sia), galactose, mannose, and N-acetylglucosamine of 3:3:3:5. A combination of methylation analyses, Smith periodate degradations, and endoglycosidase and exoglycosidase digestions were utilized to establish the structure which is proposed to be: (see article of journal). Features of this structure not previously established include the presence of 2 residues of alpha2,3- and 1 residue of alpha2,6-linked sialic acid and their location relative to the mannose branch points. Also unusual is the presence of an alpha-linked branch mannose with substituents at positions 2 and 4 which is in turn linked to position 6 of the beta-linked, branch mannose. These features result in unexpected resistance to specific exoglycosidases.     

Molecular Properties of Multiple Forms of Plant Myrosinase

Four proteins (F-IA, B, F-IIA &; F-IIB), having myrosinase activity, were separated and purified from mustard powder. Each enzyme was shown to be homogeneous chromatographically, ultracentrifugally and Disc electrophoretically. Molecular weights obtained by gel-filtration and sedimentation equilibrium were 153,000 (F-IA, F-I B &; F-IIA) and 125,000 (F-IIB). Sedimentation coefficients were 6.8 S (F-IA, B &; F-IIA) and 5.8 S (F-IIB). Stokes radius (Å), diffusion coefficient (cm²/sec) and frictional ratio (f/fo) were 47, 4.28 ×10^?7 and 1.33 (F-IA, B &; F-II1), and 43, 4.67 × 10^?7 and 1.29 (F-IIB), respectively. Isoelectric points were pH 4.6 (F-IA, B &; F-IIA) and pH 4.8 (F-IIA). The enzymes were glycoprotein with 9 ~ 22% carbohydrate. Amino acid composition of F-IA, B and F-IIA were very similar, but in case of F-IIB, glutamic acid, arginine and methionine contents were higher and aspartic acid and histidine contents were lower than others. The molecular weights estimated from SDS-polyacrylamide gel electrophoresis were 40,000 (F-IA, B &; F-IIA) and 30,000 (F-IIB), respectively, and hence the enzymes are considered to have at least 4 subunits. From these results, it may be confirmed that F-IA, B &; F-IIA have striking resemblances and only F-IIB is rather different.     

Evidence for the phosphorylation of the type II insulin-like growth factor receptor in cultured cells

The ATP pools of monolayer cultures of rat embryo fibroblasts and rat liver cells (BRL-3A2) were labeled with [32P]H3PO4. The type II insulin-like growth factor (IGF) receptor was purified by affinity chromatography on wheat germ lectin-Sepharose and IGF-II-Sepharose columns. A phosphorylated species having the expected size of the type II receptor (Mr = 220,000 without reduction, Mr = 260,000 with reduction) was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. IGF-II stimulated phosphorylation of the type II receptor in BRL-3A2 rat liver cells. Lability of the receptor phosphate bonds to alkaline pH suggests that the bulk of phosphorylation was occurring on serine residues.     

Purification and properties of a new cathepsin from rat liver.

1) A lysosomal protease, a new cathepsin that inactivates glucose-6-phosphate dehydrogenase [EC 1.1.1.49] and some other enzymes and differs from cathepsin B [EC 3.4.22.1] was purified about 2,200-fold from crude extracts of rat liver by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, and DEAE Sephadex and CM-Sephadex column chromatographies. 2) The new cathepsin was markedly activated by the thiol-reagent, 2-mercaptoethanol and inhibited by monoiodoacetate. 3) The molecular weight of the new cathepsin was found by Sephadex G-75 column chromatography to be 22,000, which is smaller than that of cathepsin B. 4) The optimum pH of the enzyme for inactivation of glucose-6-phosphate dehydrogenase was pH 5.0--5.5. The enzyme was unstable in alkali and on heat treatment. 5) The rates of inactivation of glucose-6-phosphate dehydrogenase, apo-ornithine aminotransferase [EC 2.6.1.13], apo-tyrosine aminotransferase [EC 2.6.1.5], apo-cystathionase [EC 4.4.1.1], glucokinase [EC 2.7.1.2], glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12], and malate dehydrogenase [EC 1.1.1.37] by the new cathepsin were higher than those by cathepsin B. However aldolase [EC 4.1.2.13] was inactivated more rapidly by cathepsin B than by the new cathepsin. Lactate dehydrogenase [EC 1.1.1.27], glutamate dehydrogenase [EC 1.4.1.2] and alcohol dehydrogenase [EC 1.1.1.1] were not inactivated by either cathepsin. Unlike cathepsin B, the new cathepsin scarcely hydrolyzes N-substituted derivatives of arginine.