Characterization of apolipoprotein E genetic variations in Taiwanese: assocation with coronary heart disease and plasma lipid levels

Apolipoprotein E (apoE, protein; APOE, gene) is important in lipoprotein metabolism. Three isoforms, apoE2 (Cys112 Cys158), apoE3 (Cys112 Arg158), and apoE4 (Arg112 Arg158), are present in the general population. This report investigates the frequency distribution of apoE isoforms and the association of APOE genotypes with plasma lipid profile and coronary heart disease (CHD) in a population of Taiwan. ApoE isoforms were determined genetically by polymerase chain reaction and HhaI restriction enzyme digestion in control and coronary heart disease (CHD) patients. Plasma lipid and lipoprotein concentrations were also determined. The control group exhibited frequencies of 84.6% APOE3, 7.9% APOE4, 7.5% APOE2, 70.6% APOE3E3, 14.4% APOE3E4, 13.6% APOE2E3, and 1.4% APOE2E4. Comparable frequencies were observed in the CHD group. In both APOE2 carrier and APOE3E3 groups, the CHD patients expressed abnormal lipid profiles while the control group expressed normal lipid profiles. The APOE4 carriers, however, expressed abnormal lipid profiles in both normal control and CHD groups. Extremely high apoE levels in the hypertriglyceridemic group (TG > 400 mg/dL) seemed to be undesirable and were often observed in CHD patients

Characterization of proADAMTS5 processing by proprotein convertases.

ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in arthritis, is synthesized as a zymogen, proADAMTS5. We report a detailed characterization of the propeptide excision mechanism and demonstrate that it is a major regulatory step with unusual characteristics. Using furin-deficient cells and a furin inhibitor, we found that proADAMTS5 was processed by proprotein convertases, specifically furin and PC7, but not PC6B. Mutagenesis of three sites containing basic residues within the ADAMTS5 propeptide (RRR⁴⁶, RRR⁶⁹ and RRRRR²⁶¹) suggested that proADAMTS5 processing occurs after ^Arg261. That furin processing was essential for ADAMTS5 activity was illustrated using the known ADAMTS5 substrate aggrecan, as well as a new substrate, versican, an important regulatory proteoglycan during mammalian development. When compared to other ADAMTS proteases, proADAMTS5 processing has several distinct features. In contrast to ADAMTS1, whose furin processing products were clearly present intracellularly, cleaved ADAMTS5 propeptide and mature ADAMTS5 were found exclusively in the conditioned medium. Despite attempts to enhance detection of intracellular proADAMTS5 processing, such as by immunoprecipitation of total ADAMTS5, overexpression of furin, and secretion blockade by monensin, neither processed ADAMTS5 propeptide nor the mature enzyme were found intracellularly, which was strongly suggestive of extracellular processing. Extracellular ADAMTS5 processing was further supported by activation of proADAMTS5 added exogenously to HEK293 cells stably expressing furin. Unlike proADAMTS9, which is processed by furin at the cell-surface, to which it is bound, ADAMTS5 does not bind the cell-surface. Thus, the propeptide processing mechanism of ADAMTS5 has several points of distinction from those of other ADAMTS proteases, which may have considerable significance in the context of osteoarthritis.

Purification and characterization of naringinase from a newly isolated strain of Aspergillus niger 1344 for the transformation of flavonoids

An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg2+, SDS, p-chloromercuribenzoate, Cu2+ and Mn2+ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca2+, Co2+ and Mg2+ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg2+ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.

Partial purification and characterization of limonoate dehydrogenase from Rhodococcus fascians for the degradation of limonin

An extracellular limonoate dehydrogenase was purified 10-fold from a cell-free extract of Rhodococcus fascians by ammonium sulfate precipitation, dialysis, and ultrafiltration. This purified dehydrogenase catalyzed the
conversion of limonoate to 17-dehydrolimonoate. The enzyme showed optimum activity at pH 8.0 and 40oC, with K_m value of 0.9 µM, and requires Zn ions and sulfhydryl groups for catalytic action. The enzyme activity was inhibited by Hg²⁺ and NaN₃ ions. The degradation of limonin (66%) in Kinnow mandarin juice was successfully demonstrated with partially
purified limonoate dehydrogenase. With scale-up preparation of limonoate dehydrogenase, a successful debittering operation of fruit juices appears feasible.

Partial purification and characterization of exoinulinase from kluyveromyces marxianus YS-1 for preparation of high-fructose syrup

An extracellular exoinulinase( ^{2, 1}- ß- D fructan fructanohydrolase, EC 3.2.1.7), which catalyzes the hydrolysis of inulin into fructose and glucose, was purified 23.5-fold by ethanol precipitation, followed by Sephadex G-100 gel permeation from a cell-free extract of Kluyveromyces marxianus YS-1. The partially purified enzyme exhibited considerable activity between pH 5 to 6, with an optimum pH of 5.5, while it remained stable(100%) for 3 h at the optimum temperature of 50º c. Mn²⁺ and Ca²⁺ produced a 2A-fold and 1.2-fold enhancement in enzyme activity, whereas Hg²⁺ and Ag²⁺  completely inhibited the inulinase. A preparation of the partially purified enzyme effectively hydrolyzed inulin, sucrose, and raffinose, yet no activity was found with starch, lactose, and maltose. The enzyme preparation was then successfully used to hydrolyze pure inulin and raw inulin from Asparagus racemosus for the preparation of a high-fructose syrup. In a batch system, the exoinulinase hydrolyzed 84.8% of the pure inulin and 86.7% of the raw Asparagus racemosus inulin, where fructose represented 43.6mg/ml and 41.3mg/ml, respectively.

Molecular characterization and enzymatic hydrolysis of flavanoid extracted from citrus waste

The citrus fruit processing industry generates substantial quantities of waste rich in phenolic substances, which is a valuable natural source of polyphenols (flavonoids) such as naringin and its disposal is becoming a major problem. In the US alone, the juice processing of oranges and grapefruit generates over 5 Mt of citrus waste every year. In the case of India, about 2.15 Mt of citrus peel out of 6.28 Mt of citrus fruits are produced yearly from citrus juice processing. In case of Australia, about 15-40% of citrus peel waste is generated by processing of citrus fruit (0.85 Mt). Thus Isolation of functional compounds (mostly flavanoids) and their further processing can be of interest to the food and pharmaceutical industry. This peel is rich in naringin and may be used for rhamnose production by utilizing α-L-rhamnosidase (EC 3.2.1.40), an enzyme that catalyzes the cleavage of terminal rhamnosyl groups from naringin to yield prunin and rhamnose. We recently purified recombinant α-L-rhamnosidase from E. coli cells using immobilized metal-chelate affinity chromatography (IMAC) and used it for naringin hydrolysis. The purified enzyme established hydrolysis of naringin extracted from citrus peel and thus endorses its industrial applicability for producing rhamnose. Infrared (IR) spectroscopy confirmed molecular characteristics of naringin extracted from citrus peel waste.

Structural and functional characterization of the oxidoreductase a-DsbA1 from wolbachia pipientis

The α-proteobacterium Wolbachia pipientis is a highly successful intracellular endosymbiont of invertebrates that manipulates its host's reproductive biology to facilitate its own maternal transmission. The fastidious nature of Wolbachia and the lack of genetic transformation have hampered analysis of the molecular basis of these manipulations. Structure determination of key Wolbachia proteins will enable the development of inhibitors for chemical genetics studies. Wolbachia encodes a homologue (α-DsbA1) of the Escherichia coli dithiol oxidase enzyme EcDsbA, essential for the oxidative folding of many exported proteins. We found that the active-site cysteine pair of Wolbachia α-DsbA1 has the most reducing redox potential of any characterized DsbA. In addition, Wolbachia α-DsbA1 possesses a second disulfide that is highly conserved in α-proteobacterial DsbAs but not in other DsbAs. The α-DsbA1 structure lacks the characteristic hydrophobic features of EcDsbA, and the protein neither complements EcDsbA deletion mutants in E. coli nor interacts with EcDsbB, the redox partner of EcDsbA. The surface characteristics and redox profile of α-DsbA1 indicate that it probably plays a specialized oxidative folding role with a narrow substrate specificity. This first report of a Wolbachia protein structure provides the basis for future chemical genetics studies.

Characterization of Type 1 ribosome inactivating proteins in edible plants

The ribosome inactivating proteins (RIPs) from plants possess RNA N-glycosidase activity that depurinates the major rRNA, thus damaging ribosome in an irreversible manner and arresting protein synthesis. RIPs occur in fungi, bacteria and plants and are abundant in angiosperms, where they appear to have defensive role. RIPs are presently classified as rRNA N-glycosidase in the enzyme nomenclature (EC 3.2.2.22) and do exhibit other enzymatic activities such as ribonuclease and deoxyribonuclease activities. RIPs are classified into two groups based on their difference in their primary structure. Type I RIPs consist of a single polypeptide chain of approximately 26–35 kDa that possess an RNA N-glycosidase activity. These proteins have attracted a great deal of attention because of their anti-viral, anti-tumor, and anti-microbial activities, which is useful in medical research and development. Here, we describe isolation of a novel protein from Momordica sp, a highclimbing vine from family Cucurbitaceae which is native to the tropical regions of Africa, Asia, Arabia and Caribbean. The purified protein has been verified by SDS-PAGE and mass spectrometry to contain only single chain Type-1 ribosome inactivating proteins (RIPs). With present experiments, we determined the presence of RIPs in edible plant materials, including some that are eaten raw by human beings. The novel protein is further characterized to validate its therapeutic potential.

Purification and characterization of a low molecular mass alkaliphilic lipase of Bacillus cereus MTCC 8372

A low molecular mass alkaliphilic extra-cellular lipase of Bacillus cereus MTCC 8372 was purified 35-fold by hydrophobic interaction (Octyl-Sepharose) chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 8 kDa. It is a homopentamer of 40 kDa as revealed by native-PAGE. The lipase was optimally active at 55 °C and retained approximately half of its original activity after 40 min incubation at 55 °C. The enzyme was maximally active at pH 8.5. Mg ²⁺ , Cu ²⁺ , Ca ²⁺ , Hg ²⁺ , Al ³⁺ and Fe ³⁺ at 1 mM enhanced hydrolytic activity of the lipase. Interestingly, Hg ²⁺ ions synergized and Zn ²⁺ and Co ²⁺ ions antagonized the lipase activity. Among surfactants, Tween 80 promoted the lipase activity. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was highly specific towards p -nitrophenyl palmitate and showed a V _max and K _m of 0.70 mmol.mg ⁻¹ .min ⁻¹ and 32 mM for hydrolysis of p NPP.