Molecular characterization of prophenoloxidase-1 (PPO1) and the inhibitory effect of kojic acid on phenoloxidase (PO) activity and on the development of Zeugodacus tau (Walker) (Diptera: Tephritidae).

Phenoloxidase (PO) plays a key role in melanin biosynthesis during insect development. Here, we isolated the 2310-bp full-length cDNA of PPO1 from Zeugodacus tau, a destructive horticultural pest. qRT-polymerase chain reaction showed that the ZtPPO1 transcripts were highly expressed during larval-prepupal transition and in the haemolymph. When the larvae were fed a 1.66% kojic acid (KA)-containing diet, the levels of the ZtPPO1 transcripts significantly increased by 2.79- and 3.39-fold in the whole larvae and cuticles, respectively, while the corresponding PO activity was significantly reduced; in addition, the larval and pupal durations were significantly prolonged; pupal weights were lowered; and abnormal phenotypes were observed. An in vitro inhibition experiment indicated that KA was an effective competitive inhibitor of PO in Z. tau. Additionally, the functional analysis showed that 20E could significantly up-regulate the expression of ZtPPO1, induce lower pupal weight, and advance pupation. Knockdown of the ZtPPO1 gene by RNAi significantly decreased mRNA levels after 24 h and led to low pupation rates and incomplete pupae with abnormal phenotypes during the larval-pupal interim period. These results proved that PO is important for the normal growth of Z. tau and that KA can disrupt the development of this pest insect.

Comparison of the efficacy of febuxostat vs. benzbromarone in the treatment of gout: a meta-analysis in Chinese gout patients.

OBJECTIVE: Febuxostat and benzbromarone are two common drugs for the treatment of gout, but the clinical efficacy of these two drugs is controversial. This meta-analysis aimed to compare the efficacy of febuxostat and benzbromarone in the treatment of gout. MATERIALS AND METHODS: PubMed, Embase, and the Cochrane Library were searched for articles related to febuxostat and benzbromarone in the treatment of gout from inception to January 7, 2023. Titles and abstracts were reviewed in accordance with predesigned inclusion and exclusion criteria, and data were extracted independently. The Newcastle-Ottawa Scale (NOS) was used to evaluate the quality of the studies, and the continuous variables were expressed as the standard mean square error (SMD) by STATA 16 (Stata Corp., College Station, TX, USA). The sensitivity analysis was conducted by randomly removing a study, and the heterogeneity was analyzed by funnel plots and Egger's test. RESULTS: According to the search strategy, a total of 1,043 publications were retrieved from the three aforementioned databases, of which 45 publications were excluded due to duplication. Fourteen studies remained after screening titles and abstracts, and a total of 7 studies met the inclusion criteria after a comprehensive evaluation of the 14 studies. Meta-analysis showed that the uric acid (UA)-reducing effect of febuxostat is better than that of benzbromarone, while febuxostat showed a better ability to improve the estimated glomerular filtration rate (eGFR) and reduce Cr and blood urea nitrogen (BUN). In terms of hepatotoxicity, benzbromarone was not as potent as febuxostat in increasing alanine transaminase (ALT) and aspartate transaminase (AST), suggesting that benzbromarone has less hepatotoxicity. Moreover, there was no significant difference in the effect on blood lipid levels between the two drugs. CONCLUSIONS: The beneficial effect of febuxostat on renal function-related indexes such as the eGFR, Cr and BUN is significant, while benzbromarone is more effective in reducing UA and has relatively less hepatotoxicity. The specific efficacy of the two drugs needs to be confirmed by further research.

A Ran-independent pathway for export of spliced mRNA.

All major nuclear export pathways so far examined follow a general paradigm. Specifically, a complex is formed in the nucleus, containing the export cargo, a member of the importin-beta family of transporters and RanGTP. This complex is translocated across the nuclear pore to the cytoplasm, where hydrolysis of the GTP on Ran is stimulated by the GTPase-activating protein RanGAP. The activity of RanGAP is increased by RanBP1, which also promotes disassembly of RanGTP-cargo-transporter complexes. Here we investigate the role of RanGTP in the export of mRNAs generated by splicing. We show that nuclear injection of a Ran mutant (RanT24N) or the normally cytoplasmic RanGAP potently inhibits the export of both tRNA and U1 snRNA, but not of spliced mRNAs. Moreover, nuclear injection of RanGAP together with RanBP1 blocks tRNA export but does not affect mRNA export. These and other data indicate that export of spliced mRNA is the first major cellular transport pathway that is independent of the export co-factor Ran.

Protocol optimization for long-term liquid storage of goat semen in a chemically defined extender.

A specific problem in the preservation of goat semen has been the detrimental effect of seminal plasma on the viability of spermatozoa in extenders containing egg yolk or milk. The use of chemically defined extenders will have obvious advantages in liquid storage of buck semen. Our previous study showed that the self-made mZAP extender performed better than commercial extenders, and maintained a sperm motility of 34% for 9 days and a fertilizing potential for successful pregnancies for 7 days. The aim of this study was to extend the viability and fertilizing potential of liquid-stored goat spermatozoa by optimizing procedures for semen processing and storage in the mZAP extender. Semen samples collected from five goat bucks of the Lubei White and Boer breeds were diluted with the extender, cooled and stored at 5 degrees C. Stored semen was evaluated for sperm viability parameters, every 48 h of storage. Data from three ejaculates of different bucks were analysed for each treatment. The percentage data were arcsine-transformed before being analysed with anova and Duncan's multiple comparison test. While cooling at the rate of 0.1-0.25 degrees C/min did not affect sperm viability parameters, doing so at the rate of 0.6 degrees C/min from 30 to 15 degrees C reduced goat sperm motility and membrane integrity. Sperm motility and membrane integrity were significantly higher in semen coated with the extender containing 20% egg yolk than in non-coated semen. Sperm motility, membrane integrity and acrosomal intactness were significantly higher when coated semen was 21-fold diluted than when it was 11- or 51-fold diluted and when extender was renewed at 48-h intervals than when it was not renewed during storage. When goat semen coated with the egg yolk-containing extender was 21-fold diluted, cooled at the rate of 0.07-0.25 degrees C/min, stored at 5 degrees C and the extender renewed every 48 h, a sperm motility of 48% was maintained for 13 days, and an in vitro-fertilizing potential similar to that of fresh semen was maintained for 11 days.

Comparative study between slow freezing and vitrification of mouse embryos using different cryoprotectants.

The objective of this study was to evaluate the effects of different cryoprotectants and different cryopreservation protocols on the development of mouse eight-cell embryos. Mouse eight-cell embryos were cryopreserved by using propylene glycerol (PROH), ethylene glycerol (EG), dimethyl sulfoxide (DMSO) or glycerol (G) as cryoprotectant with slow-freezing or Vit-Master vitrification protocol. After thawing, the survival rate, blastocyst formation rate and blastocyst hatching rate of the embryos were compared. When the mouse eight-cell embryos were cryopreserved by the slow-freezing, the survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with PROH were significantly higher than those of DMSO and G (p < 0.05, respectively), but not significantly different among those of DMSO, G and EG (p > 0.05, respectively), and not significantly different between those of PROH and EG (p > 0.05, respectively). When the mouse eight-cell embryos were cryopreserved by Vit-Master vitrification, the survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with EG were significantly higher than those of PROH, DMSO and G (p < 0.05, respectively). Yet, there were no significant differences among those of PROH, DMSO and G (p > 0.05, respectively). In conclusion, PROH was the optimal cryoprotectant for the cryopreservation of mouse eight-cell embryos by slow-freezing protocol. EG was the optimal cryoprotectant for the cryopresevation of mouse eight-cell embryos by Vit-Master vitrification protocol, which may be commonly used in clinical and laboratory practice.

Liquid storage of goat semen in chemically defined extenders.

The suitability of certain commercial and self-made chemically defined extenders for liquid storage of goat semen was tested and the effects of storage temperatures, dilution rates and sperm washing and pH of extenders on the goat sperm during liquid storage were observed. Semen was collected from nine goat bucks of the Lubei White and Boer breeds using an artificial vagina. Each ejaculate after initial evaluation was diluted with a specific extender, cooled and stored at a desired temperature. Stored semen was evaluated for sperm motility and other parameters every 24 or 48 h of storage. The ranking order of the existing milk- and yolk-free extenders in sustaining goat sperm motility was Androhep > Zorlesco > Beltsville thawing solution > the Tris-glucose medium. The new extender (mZA) which was formulated based on Zorlesco and Androhep was more suitable for goat sperm than Androhep. The mZAP extender with Bovine Serum Albumin (BSA) replaced with polyvinyl alcohol (PVA) worked as efficiently as the mZA in maintaining sperm motility, membrane integrity, acrosome intactness and capacitation status. Goat sperm motility was best maintained at 5 degrees C during liquid preservation, but decreased significantly as the temperature increased. When semen was sixfold diluted, sperm motility was maintained longer (p < 0.05) after centrifugation, but sperm motility did not differ between the centrifuged and non-centrifuged groups when semen was 11-fold diluted. When the extender pH was adjusted from 6.6 to 6.04, the efficiency increased significantly in both Androhep and mZAP. A forward sperm motility of 34% was maintained for 9 days when buck semen was 11-fold diluted and stored at 5 degrees C in mZAP, with pH adjusted to 6.04. It is concluded that for liquid storage of buck semen, the mZA extender was more suitable than other extenders; BSA can be replaced with PVA in mZA; centrifugation to remove seminal plasma can be omitted by adequate dilution; and the storage temperature and pH of extenders affected sperm motility significantly.

Clarification of an uncertain intron within the cDNA sequences of arrowhead proteinase inhibitors A and B.

An uncertain intron of 87 bp within the cDNA sequences of arrowhead proteinase inhibitors A and B was clarified. By site-directed mutation with either a stop codon inside the uncertain intron or mutated codons at both its 5' and 3' ends, it was proved that there was neither a translation intron nor a protein intron present in the cDNA sequences of proteinase inhibitors A and B. The primary structure of inhibitor B was then reexamined by mass spectrometry molecular weight determination and partial amino acid sequencing. A 38 residue peptide was derived by degradation of inhibitor B with lysylendopeptidase, and purified, which was not found in the previous work, and its N-terminal part was none other than the missed 29 residue peptide encoded by the uncertain intron. The 38 residue peptide was very hydrophobic, while the 29 residue peptide it included was even more hydrophobic. The N-terminal part of the missed peptide was also aligned within a BrCN-degraded fragment of the inhibitor. In this paper the cause of the overlooking of this 29 residue peptide in the previous work and some unexpected problems which arose during the former sequence analysis are explained.

Purification and sequence determination of a new neutral mammalian neurotoxin from the scorpion Buthus martensii Karsch.

A new neutral mammalian neurotoxin, designated BmK M4, with an isoelectric point (pI) of 7.6 and a relatively low toxicity (LD50 = 4.0 +/- 0.25 microgram/g mice, i.v.) was purified from the venom of scorpion Buthus martensii Karsch (BmK). The complete amino acid sequence of the toxin composed of 64 amino acid residues was determined by automated Edman degradation of the N-terminal part of the reduced and S-carboxamidomethylated protein (up to 30 amino acid residues) and its peptide fragments degraded by lysylendopeptidase or Staphylococcus aureus Va protease. The calculated mol. wt based on the amino acid composition was 7001. By comparison with the sequences of other basic BmK mammalian neurotoxins, it was concluded that the weaker toxicity and lower pI value of BmK M4 might be the result of mutations H10E, R18G and K28D. The sequence comparison of BmK M4 with an acidic toxin, BmK M8, showed that the weakest toxicity and acidic property of BmK M8 may be the consequence of mutations K8D, D53A, V55E and V59E. The substitution of 21 Gly in BmK M4 for Tyrin other BmK toxins may also be of importance. In their tertiary structures, these mutated charged residues are mainly distributed in the surface (face B) that is roughly opposite to the "conserved hydrophobic surface" (face A) proposed by Fontecilla-Camps et al. in 1982. Therefore the toxin-receptor interaction may take a multiposition mode.

The relationship between mitochondrial activation and toxicity of some substituted carboxylic acids.

The activation of 4-bromocrotonic acid, 4-bromo-2-octenoic acid, valproic acid, and 3-methylglycidic acid by conversion to their CoA thioesters and the effects of these carboxylic acids on palmitoylcarnitine-supported respiration were studied with rat liver and rat heart mitochondria. 4-Bromocrotonic acid was activated by both liver and heart mitochondria, whereas 4-bromo-2-octenoic acid and valproic acid were only activated by liver mitochondria. 3-Methylglycidic acid was not a substrate of mitochondrial activation. All of the carboxylic acids that were activated also inhibited palmitoylcarnitine-supported respiration. 3-Methylglycidoyl-CoA was found to irreversibly inhibit 3-ketoacyl-CoA thiolase in a concentration-dependent and time-dependent manner. Together, these results lead to the conclusion that substituted medium-chain carboxylic acids, which enter mitochondria directly, may inhibit beta-oxidation as long as they are activated and perhaps further metabolized in the mitochondrial matrix to compounds that sequester CoA and/or inhibit beta-oxidation enzymes. Liver is more susceptible to inhibition by such xenobiotic carboxylic acids due to the broader substrate specificity of its mitochondrial medium-chain acyl-CoA synthetase (EC 6.2.1.2).

Identification of differentially expressed genes in normal mucosa, adenoma and adenocarcinoma of colon by SSH.

AIM: To construct subtracted cDNA libraries and further identify differentially expressed genes that are related to the development of colorectal carcinoma(CRC). METHODS: Suppression subtractive hybridization(SSH) was done on cDNAs of normal mucosa, adenoma and adenocarcinoma tissues from the same patient. Three subtracted cDNA libraries were constructed and then hybridized with forward and backward subtracted probes for differential screening. Positive clones from each subtracted cDNA library were selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. RESULTS: By this way, there were about 3-4 X 10(2) clones identified in each subtracted cDNA library, in which about 85% positive clones were differentially screened. Sequencing and BLAST homology search revealed some clones containing sequences of known gene fragments and several possibly novel genes showing few or no sequence homologies with any known sequences in the database. CONCLUSION: All results confirmed the effectiveness and sensitivity of SSH. The differentially expressed genes during the development of CRC can be used to shed light on the pathogenesis of CRC and be useful genetic markers for early diagnosis and therapy.

The Inhibitory Activities of Recombinant Eglin C Mutants on Kexin and Furin, Using Site-directed Mutagenesis and Molecular Modeling.

Mammalian furin and yeast kexin are members of the proprotein convertase family involved in the proteolytic processing of many important precursor proteins. Here the gene coding for the subtilisin inhibitor eglin C was totally synthesized and expressed in E.coli. Substitution of residues at each position P(1), P(2) and P(4) of eglin C with a basic residue using protein engineering could make eglin C a very strong inhibitor for furin (K(i) around 10(-9) mol/L),and even more strong for kexin (K( i ) around 10(-11) mol/ L). Results indicated that (1) A basic residue Lys or Arg at P(1) site is prerequisite for the inhibitor. (2) The second mutation with basic residue at P(4) site drastically increase the inhibitory activity by two orders of magnitude. (3) A basic residue at P(2) site is favorable for the binding to the enzyme, but unfavorable for the stability of the inhibitor, resulting in a temporary inhibition. (4) A hydrophobic residue is preferential at P(3) site. Based on the known crystal structures of subtilisin and eglin C, the interaction between the enzyme and inhibitor was modeled, and their involved residues were predicted which gave a good explanation to the experimental results.

Splicing is required for rapid and efficient mRNA export in metazoans.

Pre-mRNA splicing is among the last known nuclear events before export of mature mRNA to the cytoplasm. At present, it is not known whether splicing and mRNA export are biochemically coupled processes. In this study, we have injected pre-mRNAs containing a single intron or the same mRNAs lacking an intron (Deltai-mRNAs) into Xenopus oocyte nuclei. We find that the spliced mRNAs are exported much more rapidly and efficiently than the identical Deltai-mRNAs. Moreover, competition studies using excess Deltai-mRNA indicate that different factor(s) are involved in the inefficient export of Deltai-mRNA vs. the efficient export of spliced mRNA. Consistent with this conclusion, spliced mRNA and Deltai-mRNA, though identical in sequence, are assembled into different messenger ribonucleoprotein particles (mRNP) in vitro. Strikingly, the mRNA in the spliced mRNP, but not in the Deltai-mRNP, is exported rapidly and efficiently. We conclude that splicing generates a specific nucleoprotein complex that targets mRNA for export. Our results, revealing a link between splicing and efficient mRNA export, may explain the reports that an intron is required for efficient expression of many protein-coding genes in metazoans.

Short-chain 3-hydroxy-2-methylacyl-CoA dehydrogenase from rat liver: purification and characterization of a novel enzyme of isoleucine metabolism.

Short-chain L-3-hydroxy-2-methylacyl-CoA dehydrogenase (SC-HMAD), a soluble mitochondrial enzyme, was purified 6000-fold from rat liver in 6% yield by a six-step purification procedure. The purified enzyme was homogenous as judged by gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular mass of this protein was estimated to be 28 kDa under denaturing conditions. Under nondenaturing conditions, the enzyme behaved on Sephacryl S-200 like serum albumin with a molecular mass of 66 kDa. Thus, SC-HMAD seems to be a dimer composed of two, most likely identical 28-kDa subunits. Immunoblotting with antibodies to pig heart L-3-hydroxyacyl-CoA dehydrogenase (HAD) (EC 1.1.1.35) revealed that SC-HMAD and HAD are immunologically unrelated proteins. SC-HMAD, but not HAD, catalyzes the NAD(+)-dependent dehydrogenation of L-3-hydroxy-2-methybutyryl-CoA, a metabolite of isoleucine, to 2-methylacetoacetyl-CoA. Relative activities with 3-hydroxy-2-methylacyl-CoA thioesters having acyl chains with 4, 5, 10, and 16 carbon atoms are 88, 100, 16, and 0%, respectively. Unbranched 3-hydroxyacyl-CoA thioesters are also substrates of SC-HMAD, although poorer ones as evidenced by apparent Km values of 5 and 19 microM for L-3-hydroxy-2-methylbutyryl-CoA and L-3-hydroxybutyryl-CoA, respectively. Maximal velocities observed with these two substrates were similar. It is concluded that SC-HMAD catalyzes the second dehydrogenation step during the beta-oxidation of the isoleucine metabolite 2-methylbutyryl-CoA. This enzyme may also be involved in the beta-oxidation of natural and xenobiotic branched chain carboxylic acids.

The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans.

In metazoans, most pre-messenger RNAs contain introns that are removed by splicing. The spliced mRNAs are then exported to the cytoplasm. Recent studies showed that splicing promotes efficient mRNA export, but the mechanism for coupling these two processes is not known. Here we show that Aly, the metazoan homologue of the yeast mRNA export factor Yralp (ref. 2), is recruited to messenger ribonucleoprotein (mRNP) complexes generated by splicing. In contrast, Aly does not associate with mRNPs assembled on identical mRNAs that already have no introns or with heterogenous nuclear RNP (hnRNP) complexes. Aly is recruited during spliceosome assembly, and then becomes tightly associated with the spliced mRNP. Aly shuttles between the nucleus and cytoplasm, and excess recombinant Aly increases both the rate and efficiency of mRNA export in vivo. Consistent with its splicing-dependent recruitment, Aly co-localizes with splicing factors in the nucleus. We conclude that splicing is required for efficient mRNA export as a result of coupling between the splicing and the mRNA export machineries.

Delta 3,5, delta 2,4-dienoyl-CoA isomerase from rat liver mitochondria. Purification and characterization of a new enzyme involved in the beta-oxidation of unsaturated fatty acids.

Mitochondrial delta 3,5, delta 2,4-dienoyl-CoA isomerase, which catalyzes the conversion of 3,5-octadienoyl-CoA to 2,4-octadienoyl-CoA, was purified from rat liver 370-fold at almost 30% yield by a six-step purification procedure. The final preparation appeared to be homogeneous as judged by gel electrophoresis. The molecular weights of the native enzyme and its subunit(s) were estimated to be 126,000 and 32,000, respectively. The purification of delta 3,5, delta 2,4-dienoyl-CoA isomerase completes the characterization of the enzymes functioning in the NADPH-dependent pathway for the beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms. This novel pathway may not be operative in peroxisomes because delta 3,5, delta 2,4-dienoyl-CoA isomerase was only detected in mitochondria. Substrates of this pathway are 2,5-dienoyl-CoAs formed from 5-enoyl-CoAs by acyl-CoA dehydrogenase. Two sequential isomerization reactions catalyzed by delta 3, delta 2-enoyl-CoAs isomerase and delta 3,5, delta 2,4-dienoyl-CoA isomerase, respectively, convert 2,5-dienoyl-CoAs to 2,4-dienoyl-CoAs, which are reduced by NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34) before reentering the beta-oxidation spiral.

Two reactive site locations and structure-function study of the arrowhead proteinase inhibitors, A and B, using mutagenesis.

The arrowhead (Sagittaria sagittifolia, Linn.) proteinase inhibitor A and B are double-headed and multifunctional, consisting of 179 amino acid residues with three disulfide bridges. Both their primary structures and cDNA sequences have been elucidated [Yang, H. L., Luo, R. S., Wang, L. X., Zhu, D. X., & Chi, C. W. (1992) J. Biochem. 111, 537; Xu, W. F., Tao, W. K., Gong, Z. Z., & Chi, C. W. (1993) J. Biochem. 113, 153; Luo, M. J., Lu, W. Y., & Chi, C. W. (1997) J. Biochem. (in press)]. Though they share 91% homology, they are different in inhibitory activities. Sequence analysis of their full-length cDNAs showed that there are seven extra residues in the C-terminal part which might be cleaved off by proteinase post-processing. To locate the reactive sites and study the structure-function relationship of the two forms A and B, the genes coding for the mature inhibitor B and its extended form were respectively cloned into the secretion expression vector, pVT102U/alpha, and expressed in Saccharomyces cerevisiae strain S-78. Both of the gene products were purified and characterized to have the same inhibitory activities as the natural one. The gene product of the extended form was a mixture with the extended C-terminal part of the inhibitor either completely or partially removed. The two previously predicted reactive site residues, Lys-44 and Arg-76 of inhibitor B, were then respectively substituted with Ala by site-directed mutagenesis and expressed. As compared with the natural inhibitor, each of the mutants could only inhibit one molecule, instead of two molecules of trypsin, and displayed an inhibitory activity against elastase, thus confirming the location of the two reactive sites in the inhibitors. The gene coding for inhibitor A, which for some reason could not be expressed in S. cereviciae, was successfully expressed in the reconstructed plasmid pET-1522bx in Escherichia coli strain BL21 with the expressed product existing in the inclusion body. After denaturation and renaturation, the active inhibitor A was obtained and purified by anhydrotrypsin affinity chromatography. Using site-directed mutagenesis, two residues of inhibitor A, namely, Ser-82 and Leu-87, prominently different from Leu-82 and Arg-87 in inhibitor B, were replaced by these two corresponding residues, respectively. As compared with the natural inhibitor A, its S82L mutant showed a lower inhibitory activity toward trypsin, whereas a higher activity was found in the L87R mutant. Meanwhile, both of their chymotrypsin inhibitory activities became weaker than the natural one. The important accessary role of the residue of position 87 in causing the difference in inhibitory properties between inhibitor A and B was discussed.

Purification and characterization of the trifunctional beta-oxidation complex from pig heart mitochondria.

The presence of a trifunctional beta-oxidation complex in pig heart and its relationship to the known long-chain enoyl-CoA hydratase (EC 4.2.1.74) from pig heart mitochondria were investigated. For this study, the complex was partially purified by chromatography on DEAE-cellulose in the absence of detergents and was purified to near homogeneity in the presence of Triton X-100. Both enzyme preparations contained long-chain specific activities of enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase but were virtually inactive toward short-chain (C4) substrates. Both preparations exhibited very low or no activities of delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) and 2,4-dienoyl-CoA reductase (EC 1.3.1.34). The molecular weights of the two subunits of the pig heart complex were estimated to be 81,000 and 45,000, respectively. The partially purified preparation, obtained in the absence of detergent, was identified as a membranous fraction enriched with respect to the inner mitochondrial membrane. It is concluded that long-chain enoyl-CoA hydratase is a component enzyme of the trifunctional beta-oxidation complex which is associated with the inner membrane of pig heart mitochondria.

Nonhistone protein purified from porcine kidney acts as a suicide substrate inhibitor on furin-like enzyme.

AIM: To search and purify a naturally occurring protein inhibitor of the furin-like enzyme from the porcine kidney. METHODS: Recombinant kexin, a furin-like enzyme, from the yeast secretion expression was used as a target enzyme. The inhibitor component was extracted and purified from the acetone powder of porcine kidney. The inhibitory activity was monitored using a fluorogenic peptide substrate Boc-Arg-Val-Arg-MCA at spectrofluorimeter. RESULTS: The purified inhibitor component is a basic protein with an isoelectric point over 9.5. Its partial N-terminal sequence of 22 residues was determined, showing a high homology with nonhistone chromosomal protein HMG-17 in which there are four sites composed of dibasic residues, susceptible to be cleaved by the furin-like enzyme. This nonhistone protein could strongly compete with the fluorogenic substrate. However, this nonhistone protein would be degraded as a substrate by kexin if it was incubated with the enzyme for long time before adding the fluorogenic substrate, and subsequently lost its temporary inhibitory activity. CONCLUSION: The nonhistone protein isolated from the porcine kidney functioned as a suicide substrate inhibitor for the furin-like enzyme.

Mitochondrial beta-oxidation of 2-methyl fatty acids in rat liver.

The mitochondrial beta-oxidation of 2-methyl fatty acids was studied with coupled rat liver mitochondria and purified enzymes. Measurements of mitochondrial respiration supported by 2-methyl fatty acids, straight chain fatty acids, or their coenzyme A (CoA) thioesters revealed that free short-chain and medium-chain 2-methyl fatty acids are oxidized nearly or as efficiently as are their straight chain analogs. Long-chain 2-methyl hexadecanoyl-CoA is also oxidized, although more slowly than its unbranched counterpart. However, medium-chain 2-methyldecanoyl-CoA, in contrast to its unbranched analog, is not oxidized at all. Of all acyl-CoA dehydrogenases only long-chain acyl-CoA dehydrogenase acts on medium-chain and long-chain 2-methylacyl-CoA thioesters. The resultant 2-methyl-2-enoyl-CoA thioesters are substrates of the mitochondrial trifunctional beta-oxidation complex which catalyzes the sequential hydration, dehydrogenation, and thiolytic cleavage of 2-methyl-substituted substrates to yield chain-shortened acyl-CoA thioesters and propionyl-CoA. The matrix enzymes L-3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase, in contrast to enoyl-CoA hydratase, are inactive with medium-chain and long-chain 2-methyl-substituted chain substrates. The specificity of the beta-oxidation enzymes toward 2-methyl-branched substrates forms the basis for assays of long-chain acyl-CoA dehydrogenase and the trifunctional beta-oxidation complex in the presence of their mitochondrial isozymes. It is concluded that rat liver mitochondria can oxidize 2-methyl fatty acids, but does so most effectively with medium-chain and short-chain ones that can enter mitochondria directly in a carnitine-independent manner.