PURIFICATION OF β-GLUCOSIDASE PRODUCED FROM Trichoderma viride USING COW DUNG AS CARBON SOURCE

β-glucosidases have characteristics of biotechnological interest and have thus become important industrial enzymes.In this study, β-glucosidase produced by Trichoderma viride from cow dung was subjected to a three step purification process involving ammonium sulphate precipitation, gel filtration by Sephadex G-100 and ion exchange chromatography by DEAE-Sephadex A-25. The elution profile on Sephadex G-100 resulted in a single broad peak (fractions 9-21) which had a yield of 3.7% and a purification fold of 4.29 with a specific activity of 25.70 µmol/min/mg proteins while the elution profile on DEAE-Sephadex A-25 resulted in a single broad peak (fraction 8-14) which had a yield of 2.76% and a purification fold of 22.14 with a specific activity of 132.41µmol/min/mg of protein. The purified enzyme was obtained as a single band and had a molecular mass of 51.8 kDa on SDS-PAGE. This results provide support for further studies of this enzyme towards revealing its potential biotechnological applications.

Expression of Immune Regulatory Genes in the Porcine Internal Genital Tract Is Differentially Triggered by Spermatozoa and Seminal Plasma

Mating or cervical deposition of spermatozoa or seminal plasma (SP) modifies the expression of genes affecting local immune defense processes at the oviductal sperm reservoir in animals with internal fertilization, frequently by down-regulation. Such responses may occur alongside sperm transport to or even beyond the reservoir. Here, immune-related gene expression was explored with cDNA microarrays on porcine cervix-to-infundibulum tissues, pre-/peri-ovulation. Samples were collected 24 h post-mating or cervical deposition of sperm-peak spermatozoa or SP (from the sperm-peak fraction or the whole ejaculate). All treatments of this interventional study affected gene expression. The concerted action of spermatozoa and SP down-regulated chemokine and cytokine (P00031), interferon-gamma signaling (P00035), and JAK/STAT (P00038) pathways in segments up to the sperm reservoir (utero-tubal junction (UTJ)/isthmus). Spermatozoa in the vanguard sperm-peak fraction (P1-AI), uniquely displayed an up-regulatory effect on these pathways in the ampulla and infundibulum. Sperm-free SP, on the other hand, did not lead to major effects on gene expression, despite the clinical notion that SP mitigates reactivity by the female immune system after mating or artificial insemination.

Abstract 547: Proteomic Correlation of Gel Filtration Lipoprotein Subfractions with Atheroprotective Functions of HDL

HDL has been shown to possess a variety of cardio-protective functions, including removal of excess cholesterol from the periphery, inhibition of oxidation, and stimulation of endothelial function. It has been proposed that various HDL sub-particles exist, each with distinct protein and lipid compositions. We hypothesized that fractionation of plasma would separate HDL particles with different protein compositions into distinct populations responsible for different HDL functions. Plasma from 10 healthy adults was fractionated by gel filtration and the protein composition of phospholipid containing fractions was analyzed by mass spectrometry. Each fraction was assessed for its ability to efflux cholesterol from macrophages, as well as its ability to inhibit LDL oxidation. Correlations were made between individual proteins in the HDL fractions and their ability to participate in both functional assays. One peak of activity was found in the cholesterol efflux assay when analyzing fractions in the HDL range. Cholesterol efflux activity did not correlate strongly with any protein in the HDL range of fractions, though there was a weak correlation with Protein S (r=0.337, p<0.01). However, the phospholipid (PL) and cholesterol (CH) concentration of the fractions correlated strongly with efflux (r=0.75. and r=0.61 respectively, both p<0.00001) across all fractions (both LDL and HDL). In contrast, the PL or CH content were not correlated with anti-oxidation activity. However, there were 2 strong peaks of anti-oxidation activity in the HDL range that did correlate with specific proteins (all values of r>0.3 and p<0.05). Activity in the first peak, fraction 25, in the small HDL range, correlated most strongly with ceruloplasmin and inter-α-trypsin inhibitor 4, while activity in the second peak, fraction 28, in the minimally lipidated/free protein range, correlated with gelsolin and albumin. In conclusion, we have shown that PL and CH correlate more strongly with cholesterol efflux than any protein in the HDL sub-fractions, while the protein composition of particular HDL sub-fractions is a better indicator of its anti-oxidative capacity than the PL or CH concentration.

Pre-Clinical Characterization and Analysis of Long Lasting Recombinant Factor IXFc

Abstract 4415 A recombinant Factor IXFc fusion molecule has been demonstrated to prolong half-life in several species (normal mice, rats, monkeys and FIX-deficient mice and dogs). The recombinant fusion molecule consists of a single rFIX moiety fused to the Fc region of Immunoglobulin G. A number of post-translational modifications exist on the rFIX portion of the fusion protein. These PTMs consist of γ-carboxylation, in the Gla domain, of the first 12 glutamic acids; the majority of which occupy glutamic acids 1–10 and partial occupation of glutamic acids 11–12. Other possible modifications include: hydroxylation of an aspartic acid residue, sulfation of a tyrosine residue and phosphorylation of a serine residue. In addition, there are several N- and O- linked glycosylation sites. One of the more important PTMs that may impact biological activity is γ-carboxy-glutamic acid (Gla) content thus several analytical methods were developed to monitor Gla content. Analytical methods include: amino acid analysis, AAA, (total Gla content), peptide mapping by HPLC-MS (Gla occupation), FIX coagulation activity by aPTT and analytical anion-exchange HPLC (iso-form separation). Material from the polishing ion-exchange step; purified peak and strip peak fractions, were analyzed. The average Gla content, measured by AAA and quantified on a molar basis relative to plasma derived FIX, of the purified peak fraction was 11.0 (3.9 % RSD) and, relative to plasma derived FIX which was fixed at 12.0 Gla. The average Gla content and biological activity of the strip peak fraction was reduced relative to the purified peak fraction. Gla occupancy was determined by digesting FIXFc with Lys-C and separating the peptides by HPLC with detection by a Mass Spectrometer (HPLC-MS). The primary peptides monitored were K1K2 and K3, each peptide has a maximum occupancy of 6 Gla's. The K1K2 peptide with 6 Gla was detected in the purified peak fraction, and 5 Gla was below detection whereas the strip peak fraction exhibited a reduced level of 6 Gla's and consequently K1K2 peptide with 5 Gla's was detected. The K3 peptide exhibits a distribution of Gla's ranging from 4 to 6, for both purified and strip peak fractions. An anion-exchange HPLC method was used to separate intact rFIXFc iso-forms. Purified peak fraction was injected onto an anion-exchange column and was separated into three species. Each species was collected and analyzed by aPTT, AAA and peptide mapping. Each species retained biological activity as well as Gla content ≥9.5. Peptide mapping indicated 6 Gla's in the K1K2 peptide (5 Gla's if present were below detection), while 4, 5 and 6 Gla's were detected in the K3 peptide. Strip peak fraction was also injected on to the anion-exchange column and two primary species were separated, each species was collected and analyzed as the purified peak fraction. No biological activity was measured for species 1, and species 2 had equivalent activity as the strip peak fraction (starting material) at 0.40 IU/nmol. Gla content by AAA indicated the strip peak fraction (starting material) to be reduced relative to the purified peak fraction; species 1 and 2 were determined to have a Gla content of 8.5 and 9.9, respectively. Peptide mapping of the strip peak fraction (starting material and species 1 and 2, derived from analytical anion-exchange HPLC), indicated peptides corresponding to K1K2 with 5 Gla's as well as 6 Gla's, and the K3 peptide exhibited 4, 5 and 6 Gla's. In summary, a number of analytical methods were developed and utilized to assess the Gla content of rFIXFc in purified peak fractions as well as the strip peak fraction. The data suggests the purified peak fraction is highly γ-carboxylated. Further fractionation of the rFIXFc iso-forms derived from the purified peak fraction was performed by analytical anion-exchange into several species. The separated species (species 1 and 2) retained biological activity with a reduction in Gla content, relative to the un-fractionated material and species 3. Analysis of the strip peak fraction exhibited a reduction of biological activity and Gla content, as well as an increased population of K1K2 5 Gla's. These analytical techniques were used to provide analytical as well characterization methodologies to support process development for designing a manufacturing process for production of clinical material. Disclosures: No relevant conflicts of interest to declare.

Effect of epinephrine or cAMP on cAMP-bound protein kinase holoenzymes in rat heart

Nonperfused or epinephrine-perfused rat hearts were used to examine the relative amounts of adenosine 3',5'-cyclic monophosphate (cAMP)-free and cAMP-bound holoenzymes of type II cAMP-dependent protein kinase (cAK). Crude tissue extracts of nonperfused hearts were chromatographed in the absence or presence of [3H]cAMP using DEAE-high-performance liquid chromatography. A partially resolved cAMP-free peak of cAK eluted at 0.17 M NaCl, and an asymmetric peak containing bound [3H]cAMP eluted at a slightly higher NaCl concentration. The first peak contained a tetrameric holoenzyme [2 regulatory (R) subunits and 2 catalytic (C) subunits]. From analysis of R-to-C ratios, the [3H]cAMP-bound peak contained a mixture of tetrameric and trimeric (R2C) forms. Both cAMP-free and cAMP-bound holoenzyme forms were virtually inactive without added cAMP under the conditions used. [3H]cAMP dissociation rate studies revealed that the bound cAMP in the peak fraction was equally distributed in the two different binding sites of the enzyme. Compared with the cAMP-free form, the cAMP-bound enzyme in the peak fraction exhibited enhanced binding in nonequilibrium [3H]cAMP binding assays. The cAMP-bound holoenzymes were estimated to represent at least 64% of the total type II cAK in control extracts, and the cAMP-free form was largely converted to the cAMP-bound forms by perfusing hearts with epinephrine.

A squid dynein isoform promotes axoplasmic vesicle translocation.

Axoplasmic vesicles that translocate on isolated microtubules in an ATP-dependent manner have an associated ATP-binding polypeptide with a previously estimated relative molecular mass of 292 kD (Gilbert, S. P., and R. D. Sloboda. 1986. J. Cell Biol. 103:947-956). Here, data are presented showing that this polypeptide (designated H1) and another high molecular mass polypeptide (H2) can be isolated in association with axoplasmic vesicles or optic lobe microtubules. The H1 and H2 polypeptides dissociate from microtubules in the presence of MgATP and can be further purified by gel filtration chromatography. The peak fraction thus obtained demonstrates MgATPase activity and promotes the translocation of salt-extracted vesicles (mean = 0.87 microns/s) and latex beads (mean = 0.92 microns/s) along isolated microtubules. The H1 polypeptide binds [alpha 32P]8-azidoATP and is thermosoluble, but the H2 polypeptide does not share these characteristics. In immunofluorescence experiments with dissociated squid axoplasm, affinity-purified H1 antibodies yield a punctate pattern that corresponds to vesicle-like particles, and these antibodies inhibit the bidirectional movement of axoplasmic vesicles. H2 is cleaved by UV irradiation in the presence of MgATP and vanadate to yield vanadate-induced peptides of 240 and 195 kD, yet H1 does not cleave under identical conditions. These experiments also demonstrate that the actual relative molecular mass of the H1 and H2 polypeptides is approximately 435 kD. On sucrose density gradients, H1 and H2 sediment at 19-20 S, and negatively stained samples reveal particles comprised of two globular heads with stems that contact each other and extend to a common base. The results demonstrate that the complex purified is a vesicle-associated ATPase whose characteristics indicate that it is a squid isoform of dynein. Furthermore, the data suggest that this vesicle-associated dynein promotes membranous organelle motility during fast axoplasmic transport.

The Use of HPLC for the Purification of the Qв-Protein

The Qв-protein is as a hydrophobic integral membrane protein firmly bound in the reaction center complex of photosystem II. A new method was developed to purify the SDS extracted protein using reversed-phase chromatography with two binary linear gradient systems. The identification of the Qв-protein was achieved by its rapidly labeling during photoassimilation of [35S]sulfate and by its reaction with the photoaffinity label azido-[14C]atrazine. Furtherm ore, antisera against the purified Qв-protein reacted with a single peak fraction, the second peak of the chrom atogram , which was identical with the labeled protein peak fraction.