Biochemical and functional characterization of an L-amino acid oxidase isolated from Bothrops pirajai snake venom

In this work we describe the isolation of a new l-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity, and hydrophobic chromatography steps. BpirLAAO-I homodimeric acid glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively) displays high specificity toward hydrophobic/aromatic amino acids, while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a high similarity between a amino acid sequence with other LAAOs from: Bothrops spp., Crotalus spp., Calloselasma rhodostoma, Agkistrodon spp., Trimeresurus spp., Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp., and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.     

Proton translocating ATPase in lysosomal membrane ghosts. Evidence that alkaline Mg2+-ATPase acts as a proton pump

Membrane ghosts were prepared from purified lysosomes (tritosomes) of rat liver by hypo-osmotic treatment. Mg2+-ATP-driven acidification was observed in the membrane ghosts using acridine orange as a fluorescent probe of the transmembrane pH gradient (delta pH). Its properties were the same as those of intact lysosomes reported previously (Ohkuma, S., Moriyama, Y., & Takano, T. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762; Moriyama, Y., Takano, T., & Ohkuma, S. (1982) J. Biochem. 92, 1333-1336). The H+-pump was found to be electrogenic with use of bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol as a fluorescent membrane potential probe. Alkaline Mg2+-ATPase activity was also identified on the membranes. It showed a pH maximum of pH 8.0-8.5, a Km value for ATP of 0.36 mM and a Vmax of 0.41 units/mg protein at 30 degrees C. Its activity was inhibited by dicyclohexylcarbodiimide, tri-n-butyltin, azide and ADP, but not by ouabain or vanadate. It differed from mitochondrial F1F0-ATPase in sensitivities to N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin, and oligomycin. Since this alkaline Mg2+-ATPase activity is very similar to the H+-pump activity in its requirement for divalent cations, substrate specificity and sensitivities to various chemicals, it may act as a proton translocase (H+-pump). Possible mechanisms of action of some chemicals, such as 4-acetamide-4'-isothiocyanatostilbene-2,2'-disulfonic acid, that inhibited the H+-pump but not the alkaline Mg2+-ATPase, are discussed.     

Acid ribonuclease from HeLa cell lysosomes.

An acid ribonuclease has been purified from HeLa cell lysosomes. The specific activity of the RNase in lysosomes is 8-fold higher than that in nuclei and 15-fold higher than that in the postlysosomal fraction. The purified enzyme showed no detectable DNase, phosphodiesterase, phosphatase, or alkaline RNase activity. The acid RNase binds to Con A-agarose and is inferred to be a glycoprotein. It has a low isoelectric point at pH 3.0 to 3.5, and the optimal pH for activity is between 5.0 and 5.5. The enzyme requires no divalent cation for optimal activity and is totally inhibited by 1 mM Cu2+ or Hg2+. Monovalent cations including Na+, K+, and NH4+ stimulate the activity in low ionic strength buffer. The enzyme degrades rRNA faster than tRNA, and tRNA faster than poly(U); poly(A) and poly(C) are highly resistant. The products from rRNA are mostly oligonucleotides with 3'-phosphate ends. An acid RNase is also present in the lysosomes of L-cells grown in a medium free of serum; it is probably identical to the one described here.     

Origin and diversification of the L-amino oxidase family in innate immune defenses of animals

L-amino acid oxidases (LAOs), because they produce hydrogen peroxide as a by-product, function in innate immune defenses of both vertebrates and mollusks. Phylogenetic analysis revealed two major subfamilies of LAOs: (1) a subfamily including LAOs from vertebrates and mainly from Terrabacteria and (2) a subfamily including LAOs from mollusks and Hydrobacteria. These subfamilies thus originated early in the history of life, implying that their innate immune functions in vertebrates and mollusks have evolved separately. Mammalian LAOs were found to belong to three separate clades: (1) LAO1, (2) LAO2, and (3) IL4I1. Phylogenetic analysis supported the hypothesis that LAO1 and LAO2 arose by a gene duplication prior to the divergence of marsupials from placental mammals, while IL4I1 duplicated from the ancestor of the LAO1 and LAO2 prior to the divergence of tetrapods from bony fishes. Mammalian IL4I1 clustered with LAOs from bony fishes, and these molecules shared a number of unique sequence features, including both amino acid replacements and a unique two-codon deletion. It is certain such unique features may be functionally important, especially three unique amino acid replacements in close proximity to the putative active site.     

Spectral and kinetic studies of the oxidation of monosubstituted phenols and anilines by recombinant Synechocystis catalase-peroxidase compound I.

A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Synechocystis PCC 6803 is reported. Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the first examination of second-order rate constants for the oxidation of a series of aromatic donor molecules (monosubstituted phenols and anilines) by a bifunctional catalase-peroxidase compound I using the sequential-mixing stopped-flow technique. Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation. A >/=50-fold excess of peroxoacetic acid is required to obtain a spectrum of relatively pure and stable compound I which is characterized by about 40% hypochromicity, a Soret maximum at 406 nm, and isosbestic points between the native enzyme and compound I at 357 and 430 nm. The apparent second-order rate constant for formation of compound I from ferric enzyme and peroxoacetic acid is (8.74 +/- 0.26) x 10(3) M(-)(1) s(-)(1) at pH 7. 0. Reduction of compound I by aromatic donor molecules is dependent upon the substituent effect on the benzene ring. The apparent second-order rate constants varied from (3.6 +/- 0.1) x 10(6) M(-)(1) s(-)(1) for p-hydroxyaniline to (5.0 +/- 0.1) x 10(2) M(-)(1) s(-)(1) for p-hydroxybenzenesulfonic acid. They are shown to correlate with the substituent constants in the Hammett equation, which suggests that in bifunctional catalase-peroxidases the aromatic donor molecule donates an electron to compound I and loses a proton simultaneously. The value of rho, the susceptibility factor in the Hammett equation, is -3.4 +/- 0.4 for the phenols and -5.1 +/- 0.8 for the anilines. The pH dependence of compound I reduction by aniline exhibits a relatively sharp maximum at pH 5. The redox intermediate formed upon reduction of compound I has spectral features which indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme.     

Purification of mouse interleukin 2 to apparent homogeneity

A procedure has been developed for the rapid purification of mouse interleukin 2 (IL2) to apparent homogeneity, using gel filtration, anion exchange, hydrophobic chromatography, and reverse phase high pressure liquid chromatography (RP-HPLC). IL2 eluted at a high concentration of acetonitrile on HPLC (approximately 40%), well removed from other proteins. This protocol did not resolve isoelectric variant forms of IL2. Both the biological activity and protein migrated as a band of apparent molecular weight 22,000-23,000 on SDS-polyacrylamide gel electrophoresis. It had a high potency, producing 30% of the maximal response in T cell growth at a concentration of 2-4 X 10(-12) M. Mouse Il2 synthesized in a wheat germ cell-free translation system behaved similarly on RP-HPLC as the form secreted by EL4 cells. Thus, the hydrophobicity of mouse IL2, which facilitates its purification, is an intrinsic property of the protein, determined primarily by its amino acid sequence.     

Electrostatic role of aromatic ring stacking in the pH-sensitive modulation of a chymotrypsin-type serine protease, Achromobacter protease I.

Achromobacter protease I (API) has a unique region of aromatic ring stacking with Trp169-His210 in close proximity to the catalytic triad. This paper reveals the electrostatic role of aromatic stacking in the shift in optimum pH to the alkaline region, which is the highest pH range (8.5-10) among chymotrypsin-type serine proteases. The pH-activity profile of API showed a sigmoidal distribution that appears at pH 8-10, with a shoulder at pH 6-8. Variants with smaller amino acid residues substituted for Trp169 had lower pH optima on the acidic side by 0-0.9 units. On the other hand, replacement of His210 by Ala or Ser lowered the acidic rim by 1.9 pH units, which is essentially identical to that of chymotrypsin and trypsin. Energy minimization for the mutant structures suggested that the side-chain of Trp169 stacked with His210 was responsible for isolation of the electrostatic interaction between His210 and the catalytic Asp113 from solvent. The aromatic stacking regulates the low activity at neutral pH and the high activity at alkaline pH due to the interference of the hydrogen bonded network in the catalytic triad residues.     

Binding of fatty acids to the uncoupling protein from brown adipose tissue mitochondria

The uncoupling protein 1 (UCP1) is a H(+) carrier which plays a key role in heat generation in brown adipose tissue. The H(+) transport activity of UCP1 is activated by long-chain fatty acids and inhibited by purine nucleotides. While nucleotide binding has been well characterized, the interaction of fatty acid with UCP1 remains unknown. Here I demonstrate the binding of fatty acids by competition with a fluorescent nucleotide probe 2(')-O-dansyl guanosine 5(')-triphosphate (GTP), which has been shown previously to bind at the nucleotide binding site in UCP1. Fatty acids but not their esters competitively inhibit the binding of 2(')-O-dansyl GTP to UCP1. The fatty acid effect was enhanced at higher pH, suggesting the binding of fatty acid anion to UCP1. The inhibition constants K(i) were determined by fluorescence titrations for various fatty acids. Short-chain (C<8) fatty acids display no affinity, whereas medium-chain (C10-14) and unsaturated C18 fatty acids exhibit stronger affinity (K(i)=65 microM, for elaidic acid). This specificity profile agrees with previous functional data obtained in both proteoliposomes and mitochondria, suggesting a possible physiological role of this fatty acid binding site.     

Purification and characterization of an enzyme from Mycobacterium sp. Pyr-1, with nitroreductase activity and an N-terminal sequence similar to lipoamide dehydrogenase

Mycobacterium sp. Pyr-1 produces an enzyme with nitroreductase activity that reduces 1-nitropyrene and 4-nitrobenzoic acid to the corresponding aromatic amines. This enzyme was constitutive and required NADH; and its activity was enhanced by FAD. It was inhibited by antimycin A, dicumarol, and o-iodosobenzoic acid; and it was inactivated by ammonium sulfate precipitation. After purification to homogeneity, the protein produced a single band on native and SDS-polyacrylamide gels and had a single amino-terminal sequence. The N-terminal amino acid sequence was identical to the corresponding sequences of the lipoamide dehydrogenases of M. leprae, M. tuberculosis and Corynebacterium glutamicum. The amino-terminal sequence was also similar to lipoamide dehydrogenases from M. smegmatis and several other bacteria. The amino acid sequence of an internal peptide (12 of 13 amino acids) was nearly identical to the corresponding sequences of lipoamide dehydrogenases from M. leprae and M. tuberculosis and was similar to those of C. glutamicum, Streptomyces coelicolor and S. seoulensis. The data show that a unique lipoamide dehydrogenase in Mycobacterium sp. Pyr-1, which differs from classic (Type I) bacterial nitroreductases, reduces aromatic nitro compounds to aromatic amines.     

SO-LAAO, a novel L-amino acid oxidase that enables Streptococcus oligofermentans to outcompete Streptococcus mutans by generating H2O2 from peptone

We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H(2)O(2)) through lactate oxidase activity. In this study, we found that the lox mutant of S. oligofermentans regained the inhibition while growing on peptone-rich plates. Further studies demonstrated that the H(2)O(2) produced on peptone by S. oligofermentans was mainly derived from seven L-amino acids, i.e., L-aspartic acid, L-tryptophan, L-lysine, L-isoleucine, L-arginine, L-asparagine, and L-glutamine, indicating the possible existence of L-amino acid oxidase (LAAO) that can produce H(2)O(2) from L-amino acids. Through searching the S. oligofermentans genome for open reading frames with a conserved flavin adenine dinucleotide binding motif that exists in the known LAAOs, including those of snake venom, fungi, and bacteria, a putative LAAO gene, assigned as aao(So), was cloned and overexpressed in Escherichia coli. The purified protein, SO-LAAO, showed a molecular mass of 43 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and catalyzed H(2)O(2) formation from the seven L-amino acids determined above, thus confirming its LAAO activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known LAAOs in both substrate profile and sequence, suggesting that it could represent a novel LAAO. An aao(So) mutant of S. oligofermentans did lose H(2)O(2) formation from the seven L-amino acids, further verifying its function as an LAAO. Furthermore, the inhibition by S. oligofermentans of S. mutans in a peptone-rich mixed-species biofilm was greatly reduced for the aao(So) mutant, indicating the gene's importance in interspecies competition.     

Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells

Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP⁺RFP⁺ puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.     

斜带石斑鱼L-氨基酸氧化酶基因克隆及表达分析

鱼类的L-氨基酸氧化酶(L-amino acid oxidase, LAAO)具有广泛的抑菌杀虫效果, 为了解斜带石斑鱼(Epinephelus coioides)LAAO基因序列特征及其在刺激隐核虫(Cryptocaryon irritans)感染后的表达变化, 该试验克隆得到2个石斑鱼LAAO基因: EcLAAO-1和EcLAAO-2, 它们的ORF长度分别为1536和1569 bp, 编码511和522个氨基酸, 均含有氨基酸氧化酶(Amino_oxidase)结构域以及LAAO保守序列: DBM和GG motif。多重序列比对显示石斑鱼LAAO与其他鱼类LAAO具有较高的相似性。系统进化树分析表明, 斜带石斑鱼的LAAO与硬骨鱼类亲缘关系较近。实时荧光定量PCR检测结果显示EcLAAO-1和EcLAAO-2在石斑鱼各组织均有表达, 其中皮肤、鳃、胸腺、肝脏和肌肉中含量较丰富; 在感染刺激隐核虫后, 鳃和脾脏EcLAAO-1, EcLAAO-2表达量显著升高(P<0.05), 这些结果暗示了石斑鱼LAAO参与先天性免疫, 并在抗御刺激隐核虫感染中发挥重要作用。     

Functional and molecular adaptation of Cl/HCO3- exchanger to chronic alkaline media in renal cells.

The Cl(-)/HCO3- exchanger (AE) is one of the mechanisms that cells have developed to adjust pH Despite its importance, the role of AE isoforms in controlling steady-state pH during alkalosis has not been widely investigated. In the present study, we have evaluated whether conditions simulating acute and chronic metabolic alkalosis affected the transport activity and protein levels of Cl-/HCO3- exchangers in a rat cortical collecting duct cell line (RCCD1). pH(i) was monitored using the fluorescent dye BCECF in monolayers grown on permeable supports. Anion exchanger function was assessed by the response of pH(i) to acute chloride removal. RT-PCR and immunoblot assays were also performed. Our results showed that RCCD1 cells express two members of the anion exchanger gene family: AE2 and AE4. Functional studies demonstrated that while in acute alkalosis pH(i) became alkaline and was not regulated, after 48 h adaptation; steady-state pH(i) reached a value similar to the physiological one. Chronic treated cells also resulted in a 3-fold rise in Cl(-)/HCO3- exchange activity together with a 2.2-fold increase in AE2, but not AE4, protein abundance. We conclude that RCCD1 cells can adapt to chronic extracellular alkalosis reestablishing its steady-state pH(i) and that AE2 would play a key role in cell homeostasis.     

Hyperacidification of trans-Golgi network and endo/lysosomes in melanocytes by glucosylceramide-dependent V-ATPase activity

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H(+) -translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent K(i) for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.     

Cathepsin L. A new proteinase from rat-liver lysosomes.

1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondrial pellet, gel filtration of the lysosomal extract and chromatography on CM-Sephadex. 2. Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate its molecular weight was found to be 23000-24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8-6.1 ascertained by analytical isoelectric focusing. 3. Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6-7. In contrast to cathepsin B1, its capability of hydrolyzing N-substituted derivatives of arginine is low and it does not split esters. 4. Greatest activity is obtained close to pH 5.0 with 70-90% of maximal activity at pH 4.0 and pH 6.0 and 30-40% at pH 7.0. 5. The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl-lysine. Leupeptin acts as a pseudo-irreversible inhibitor. 6. The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep-frozen state.     

Effect of human carbonic anhydrase II on the activity of the human electrogenic Na/HCO3 cotransporter NBCe1-A in Xenopus oocytes

Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO3 cotransporter NBCe1-A, enhancing transport. After injecting oocytes with NBCe1-A cRNA (Day 0), we measured NBC current (I(NBC)) by two-electrode voltage clamp (Day 3), injected CA II protein + Tris or just Tris (Day 3), measured I(NBC) or the initial rate at which the intracellular pH fell (dpH(i)/dt) upon applying 5% CO2 (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured I(NBC) or dpH(i)/dt (Day 4). Because dpH(i)/dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. I(NBC) slope conductance was unaffected by injecting CA II. Moreover, EZA had identical effects in CA II versus Tris oocytes. Thus, injected CA II does not enhance NBC activity. In a second protocol, we made a fusion protein with enhanced green fluorescent protein (EGFP) at the 5' end of NBCe1-A and CA II at the 3' end (EGFP-e1-CAII). We measured I(NBC) or dpH(i)/dt (days 3-4), exposed oocytes to EZA, and measured I(NBC) or dpH(i)/dt (Day 3-4). dpH(i)/dt was greater in oocytes expressing EGFP-e1-CA II versus EGFP-e1, and EZA eliminated the difference. Thus, fused CA II was functional. Slope conductances of EGFP-e1-CAII versus EGFP-e1 oocytes were indistinguishable, and EZA had no effect. Thus, even when fused to NBCe1-A, CA II does not enhance NBCe1-A activity.     

The capacity of lysosomes of cultured mammalian cells to accumulate acridine orange is destroyed by hyperthermia

Summary Lysosomes of cultured mammalian cells, derived from a transplantable murine mammary adenocarcinoma, irreversibly lose their capacity to accumulate the fluorescent dye acridine orange after hyperthermia. As acridine orange may be regarded as a fluorescent probe of the internal pH of the lysosomes, we may conclude that the ability of lysosomes to maintain a low internal pH is destroyed by hyperthermia.The effects of hyperthermia on lysosome fluorescence and on cell survival show several similarities: in both cases hyperthermia is more effective at low pH, below pH 7.0, and CCP (carbonylcyanide-m-chlorophenylhydrazone) enhances effects at low pH, but has no clear effect at pH 8.0. This leads to the conclusion that effects on lysosomes are an important and early event in cellular injury caused by hyperthermia. The activation energy, however, obtained for the effects of hyperthermia on lysosome fluorescence is about a factor of two lower than the activation energy reported for cell survival after hyperthermia. This suggests that the effect on lysosomes is not directly caused by hyperthermia but is triggered by some other hyperthermia-induced cellular damage.Abbreviations CCP carbonylcyanide-m-chlorophenylhydrazone - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 2-(N-morpholino)propanesulfonic acid - Tricine N-tris(hydroxymethyl)methylglycine Supported in part by grants from the KWF (Koningin Wilhelmina Fonds) and the IRS (Interuniversitair Instituut voor Radiopathologie en Stralenbescherming)     

Phorbol ester induces elevated oxidative activity and alkalization in a subset of lysosomes

Background  

Lysosomes are acidic organelles that play multiple roles in various cellular oxidative activities such as the oxidative burst during cytotoxic killing. It remains to be determined how lysosomal lumen oxidative activity and pH interact and are regulated. Here, I report the use of fluorescent probes to measure oxidative activity and pH of lysosomes in live macrophages upon treatment with the tumor promotor phorbol 12-myristate 13-acetate (PMA), and provide novel insight regarding the regulation of lysosomal oxidative activity and pH.     

Existence of catalase-less peroxisomes in Sf21 insect cells

Catalase activity, a peroxisomal marker enzyme, was not detectable in any of the subcellular fractions of Spodoptera frugiperda (Sf) 21 insect cells, although marker enzymes in other organelles were distributed in the fractions in a manner similar to that seen in mammalian cells. When a green fluorescent protein fused with peroxisome targeting signal 1 at the C-terminal (GFP-SKL) was expressed in Sf21 cells, punctate fluorescent dots were observed in the cytoplasm. The fraction where GFP-SKL was concentrated exhibited long-chain and very-long-chain fatty acid beta-oxidation activities in the presence of KCN and the density of this fraction was slightly higher than that of mitochondria. Immunoelectron microscopy studies with anti-SKL antibody demonstrated that Sf21 cells have immunoreactive peroxisome-like organelles which are structurally distinct from mitochondria, endoplasmic reticulum, and lysosomes. In contrast to peroxisomal matrix proteins, adrenoleukodystrophy protein, a peroxisomal membrane protein, was not located to peroxisomes. This suggests that the targeting signal for PMP in insect cells is distinct from that in mammalian cells. These results demonstrate that Sf21 insect cells have unique catalase-less peroxisomes capable of beta-oxidation of fatty acids.