Sephadex Column Research Articles

Fatty acid-binding protein in bovine Longissimus dorsi muscle

Experiments were conducted to purify and obtain amino acid sequence data for fatty acid-binding protein from bovine longissimus dorsi muscle. 1. 1. Separation of sarcoplasmic proteins by fractionation on Sephadex and DEAE cellulose columns indicated that a low abundance fatty acid-binding protein exists in bovine l. dorsi muscle. 2. 2. Fatty acid-binding protein in bovine l. dorsi muscle cannot be separated from myoglobin by standard protein purification procedures. However, partial sequencing of a partially purified protein fraction indicated the presence of an NH 2-terminally blocked protein. 3. 3. Myoglobin bound palmitate under the in vitro conditions of these experiments.

Digitalis-like compounds in the toad Bufo viridis: interactions with plasma proteins.

Digitalis-like compounds (DLC), normal constituents of animal tissues, are possible regulators of the Na+,K(+)-ATPase implicated in water and salt homeostasis. DLC are present in toad (Bufo viridis) tissues. Although DLC highest levels were found in toad skin, it was also detected in plasma and many internal organs. The abundant distribution and the different levels of DLC in various tissues exclude the possibility that toxicity is the only function of these compounds in the toad. The concentration of DLC in toad plasma is 30 microM, out of which 25-30% is bound to plasma proteins. Fractionation of toad plasma proteins on a G-100 Sephadex column followed by the extraction of DLC from the plasma proteins revealed that DLC are bound primarily to proteins of 48,000-53,000 Da. These results establish the existence of bufodienolide-binding protein(s) in animal plasma.

Chitinase Purified from Alfalfa Infected by Clover Proliferation Mycoplasmalike Organism.

This study focused on the purification and characterization of chitinase from alfalfa (Medicago sativa L.) infected by CP-MLO. Chitinase (EC 3.2.1 14) was purified from leaves and shoots of MLO-infected alfalfa plants by ammonium sulfate precipitation, chitin affinity chromatography and Sephadex column chromatography. Three chitinase isoenzymes were identified and shown to have molecular masses of approximately 40, 35 and 30 kilodaltons as determined by SDS-polyacrylamide gel electrophoresis, while only one chitinase isoenzyme was detected in healthy plants. The endochitinase activity in CP-MLO infected plants was 24 times higher than that in healthy plants. The optimum temperature range of the enzyme activity was from 35°C to 50°C. Ag and Hg ions significantly inhibited the enzyme activity.

Regulation of the activity of glyceraldehyde 3-phosphate dehydrogenase by glutathione and H2O2.

The activity lost during storage of a solution of muscle glyceraldehyde 3-phosphate dehydrogenase was rapidly restored on adding a thiol compound, but not arsenite or azide. On treatment with H2O2, the enzyme was partially inactivated and complete loss of activity occurred in the presence of glutathione. Samples of the enzyme pretreated with glutathione followed by removal of the thiol compound by filtration on a Sephadex column showed both full activity and its complete loss on adding H2O2, in the absence of added glutathione. Most of the activity was restored when the H2O2-inactivated enzyme was incubated with glutathione (25 mM) or dithiothreitol (5 mM) whereas arsenite or azide were partly effective and ascorbate was ineffective. The need for incubation for a long time with a strong reducing agent for restoration of activity suggests that the oxidized group (disulfide or sulfenate) must be in a masked state in the H2O2-inactivated enzyme. Analysis by SDS-PAGE gave evidence for the formation of a small quantity of glutathione-reversible disulfide-form of the enzyme. Circular dichroic spectra indicated a decrease in alpha-helical content in the inactivated form of the enzyme. The evidence suggest that glutathione and H2O2 can regulate the active state of this enzyme.

Calcium-dependent pro-cholecystokinin V-9-M immunoreactive peptide release from rat brain slices and CCK-secreting rat medullary thyroid carcinoma cells in culture

The release of peptides immunoreactive for a synthetic peptide (V-9-M) contained in the amino-terminal of pro-CCK was examined. The potassium-evoked release of V-9-M immunoreactive peptides from rat cerebral cortical slices in vitro was calcium dependent. Cholecystokinin-secreting rat medullary thyroid carcinoma cells also secreted significant quantities of these peptides. Sephadex column chromatography of the release media from slices and cells showed two V-9-M immunoreactive peptides, one larger and one smaller than V-9-M itself. Previous behavioral studies have suggested that V-9-M has a distinct neuropharmacological profile. These results demonstrate that V-9-M-like peptides are released along with CCK-8 and are consistent with the hypothesis that V-9-M-like peptides may be neurotransmitters or neuromodulators or may be involved in the sorting or transport of CCK-8.

An improved and rapid method for the isolation of rat lymph node or spleen T lymphocytes for T cell proliferation assays

To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating Ia-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of Ia-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays.

Subcellular distribution of tissue radiocopper following intravenous administration of 67Cu-labeled Cu-PTSM

The subcellular distribution of radiocopper in the brain and liver of rats has been determined following i.v. administration of Cu-PTSM, pyruvaldehyde bis( N 4-methylthiosemicarbazonato)copper(II), labeled with copper-67. Homogenized tissue samples were separated by differential centrifugation into four subcellular fractions: (I) cell membrane + nuclei; (II) mitochondria; (III) microsomes; and (IV) cell cytosol. Upon sacrifice at 10 min post-Cu-PTSM injection, brain fractions, I, II, III and IV contain 35 ± 12, 11 ± 3, 2.8 ± 1.3 and 51 ± 7% of brain activity, respectively ( n = 4). In animals sacrificed 24 h post-injection the subcellular fractions of brain tissue show little change from the radiocopper distribution seen at 10 min post-injection, although the mitochondrial fraction may contain slightly more tracer and the cytosolic fraction slightly less (I, 40 ± 10%; II, 18 ± 5%; III, 3.4 ± 1.5%; and IV, 38 ± 5%; n = 5). Subcellular fractions I, II, III and IV of liver contain 25 ± 5, 12 ± 3, 17 ± 4 and 46 ± 6% of 67Cu tracer in animals sacrificed 10 min post-Cu-PTSM injection. An identical subcellular distribution of 67Cu, was found in the liver following i.v. administration of ionic radiocopper (as Cu-citrate). The liver and brain cytosolic fractions at 10 min post-injection were further separated by Sephadex column chromatography. In liver cytosol, three different radiocopper components with molecular weights of about 140,000, 41,000–46,000 and 10,000–16,000 Da were found. In the brain supernatant fraction, most of the radiocopper was bound to a single low molecular weight cytosolic component (14,000–16,000 Da). These results suggest that the intracellular decomposition of tracer Cu-PTSM may result in the radiocopper entering the normal cellular pools for copper ions.

Differential elution from a Sephadex G-15 column of sodium and phosphate ions of sodium phosphate with sodium or potassium phosphate buffer

When a sample solution containing sodium-22-labelled sodium chloride and carrier-free phosphorus-32-labelled phosphoric acid was eluted from a Sephadex G-15 column with either 0.025 M sodium or potassium phosphate buffer (pH 7.0), the labelled phosphate ion was eluted earlier than the sodium-22. The presence of cold 0.72 M sodium chloride with the sodium-22-labelled sodium chloride in the sample did not affect the elution sequence. When 1 M monosodium phosphate was eluted with distilled water from fresh and phosphate-treated Sephadex columns, the sodium and phosphate ions were eluted together in approximately the same fractions in both instances. From these observations, it is concluded that sodium ion repeatedly exchanges its partner phosphate ion with that in the eluent during its elution from Sephadex.

Effects of short-term 17 beta-estradiol treatment on the properties of T4-binding proteins in the plasma of immature rainbow trout, Oncorhynchus mykiss.

To determine the effects of 17 beta-estradiol (E2) on the properties of the plasma proteins that bind L-thyroxine (T4) immature rainbow trout, Oncorhynchus mykiss, were injected intraperitoneally on days 0 and 3 with 0.5 mg E2-3-benzoate/100 g body weight, and plasma was sampled on days 4, 7, or 12. Control trout received peanut oil alone. E2 caused a small but significant decrease in the free T4 index. Saturation analysis on miniature G-25 Sephadex columns revealed at least two major T4-binding sites. Filtration on agarose Bio-gel A 1.5 also indicated two major T4-binding protein fractions with molecular weights of 150 and 55 kDa with a small proportion of T4 binding to a 1,500-kDa site presumed to be lipoprotein. Addition of unlabeled T4 displaced [125I]T4 from the 55-kDa site and unmasked an adjacent site of higher molecular weight. E2 increased the proportion of T4 bound to the low-affinity (150 kDa) site relative to that bound to the high-affinity (55 kDa) site, increased the level of protein associated with the 1,500-kDa site and its T4 binding, and also initiated the production of presumed vitellogenin (VTG), which bound a small amount of T4. It is concluded that the E2-induced depression in FT4 is caused by a shift in T4 binding between high-affinity and low-affinity sites, and also by binding of small amounts of T4 to presumed lipoprotein and VTG.

Renin and angiotensin converting enzyme in elasmobranchs

Renin-like activity (RLA) and angiotensin I converting enzyme-like activity (ACELA), the two key enzymes of the renin-angiotensin system (RAS), were sought in the elasmobranch Scyliorhinus canicula. Renal extracts were desalted in a G-25 and eluted in a G-100 Sephadex column (calibration 15,000–70,000). The fractions were concentrated in a vacuum device. A 48,000-MW fraction incubated with synthetic and porcine angiotensinogen generated angiotensin I estimated by RIA. This same fraction was vasopressor in rats and dogfish. ACELA was sought in gill, heart, liver, spleen, pancreas, intestine, kidney, gonads, brain, skin, and muscle of dogfish using a spectrophotometric assay. The highest level of ACELA was found in the gills followed by spleen, kidney, and brain (33.79 ± 2.3, 29.56 ± 1.0, 14.62 ± 1.0, and 13.80 ± 2.3 nmol hippurate/min/mg protein, respectively). Intestine, gonads, skin and muscle contained no measurable amounts of ACELA. Captopril inhibited enzymatic activity from all ACELA containing tissues.

ATP and glutathione mediated inhibition of lipid peroxidation in rat liver systems.

The role of glutathione (GSH) in cellular antioxidant systems has long been recognised especially for protection against membrane lipid peroxidation (LPO). Several enzymes using glutathione have been implicated in such protection including GSHPx, PHGPx and GSH Stransferases. Such enzymes may or may not be susceptible to acute regulation [ 1-3 1 . We have further explored GSH mediated inhibition of LPO and describe here the previously unreported phenomenon of an ATP and GSH mediated lipid antioxidant system in rat liver homogenate and microsomes f gel sieved micromolecule-free cytosol. Male Sprague-Dawley rats were killed by cervical dislocation and 10% w/v liver homogenates were made in 0.25 M sucrose/83 mM NaF : twice washed conventional microsomes were made and suspended in 25 mM MOPS/0.15 M KC1115 mM NaF at pH 1.4. Liver post-microsomal supernatant was freed of small molecules, including ATP and GSH, by gel filtration through a G-25 Sephadex column. Samples of homogenate and microsomes f gel filtered PMSN were preincubated aerobically for 30 min at 37 C with a solution containing MOPS/KCl buffer, magnesium chloride, ATP and/or GSH. LPO activity was assessed as rates of formation of thiobarbituric acid reactive substances (TBARS) when preincubated samples were subsequently challenged with a nonenzymatic LPO initiation system ( 0.018 mM FeS04, 1.6 mM ADP, 0.2 mM ascorbate ) . The TBA reagent contained 0.04% BHT. To some assays bromosulphophthalein (BSP) was added and some preincubated samples were heat denatured. Protein was assayed by the Lowry method and GSH using DTNB [ 4 I . All concentrations stated below are those in the LPO assay. In homogenate 0.15 mM GSH inhibited LPO 33% relative to controls ( n= 15 ) although such inhibition was inconsistent ( ranging from 80%). However 1 mM ATP + 0.15 mM GSH inhibited LPO 92% and such inhibition was consistent ( pc 0.001 : n=12 ) . ATP alone only inhibited 23% ( n=3 1 . Similar results were seen using microsomes + gel-filtered PMSN, with 0.15 mM GSH inhibiting 55% ( p< 0.01 : n=7) but again inconsistently. However 1 mM ATP + 0.15 mM GSH inhibited 92% ( pc 0.001 : n=7 ) with ATP alone inhibiting by 3% (n=2). Microsomes alone showed consistently low inhibition by 0.15 mM GSH ( 12% : n=5 ) , but when added with 1 mM ATP this caused 91% inhibition ( pr 0.001 : n=5 ) . ATP alone inhibited 10% ( n=2 1 . Assays confirmed that gel filtration removed 97% of the cytosolic GSH while washed microsomes contained trivial levels ( 1% of unsieved cytosol 1 . Hence the

Patterns of thyroxine and triiodothyronine in serum and follicle-bound oocytes of the tilapia, Oreochromis mossambicus, during oogenesis

This study describes simultaneous measurements of thyroid hormones, thyroxine (T 4) and triiodothyronine (T 3), in the oocytes and serum of a female teleost fish over a complete reproductive cycle. We have identified patterns in circulating T 4 and T 3 levels as well as their accumulation into oocytes during the reproductive cycle of the tilapia ( Oreochromis mossambicus). This is the first description of the patterns with which thyroid hormones accumulate in teleost oocytes. The sampling strategy used in the study eliminated the possible influences of covarying environmental factors that may affect thyroid hormone levels independently of reproductive events. Hormones in serum and oocytes were measured by radioimmunoassay utilizing miniature Sephadex columns. The total content of both thyroid hormones in the oocytes increased throughout most of the ovarian cycle as the oocytes increased in size from <2 mg to approximately 6.5 mg by ovulation. By contrast, concentrations of thyroid hormones in the oocytes rose only during the first third of post-spawning oocyte growth (up to ∼2 mg) before attaining plateaus at ∼6 ng/g for T 4 and 13 ng/g for T 3. Serum concentrations of T 4 and T 3 varied in cyclical patterns during oogenesis, dropping to lows of 3.4 ng/ml (T 4) and 2.7 ng/ml (T 3) when the oocytes were 1.5 and 2 mg, respectively, and then increasing to 6.5 ng/ml (T 4) and 4.8 ng/ml (T 3) when the oocytes reach ∼6 mg. The concentrations of both hormones decreased shortly before spawning. Maximum concentrations of thyroid hormones in the oocytes were reached approximately 10 days prior to those in the serum. Although the serum levels of T 4 were greater than those of T 3, the reverse was found in the oocytes. Triiodothyronine appears to be accumulated selectively over T 4 and the patterns with which both thyroid hormones accumulate in the oocytes of the tilapia do not appear to be tied to serum levels.

Effect of swim-up, Percoll and Sephadex sperm separation methods on the hypo-osmotic swelling test.

The separation of spermatozoa from the seminal plasma is required to prepare the spermatozoa for intrauterine insemination, in-vitro fertilization and sex selection. This study evaluated the effects of sperm preparation techniques on the functional integrity of the sperm membrane, as measured by the hypo-osmotic swelling test (HOS). Thirty-four semen specimens obtained from the male partner of infertile couples were evaluated. A semen analysis and HOS test were performed on each specimen. The remainder of the specimen was divided into three equal aliquots, the first prepared using Percoll, the second using a swim-up method and the third using a Sephadex column. After the preparation, a semen analysis and HOS test was performed on each aliquot. The mean and standard deviation for the HOS test was 72.9 +/- 8.5% initially, 71.2 +/- 13.1 after Percoll, 75.2 +/- 15.1 after swim-up and 62.4 +/- 14.5 after Sephadex. Analysis of variance showed that the mean HOS score was the same after Percoll and swim-up as it was initially but significantly lower after preparation with Sephadex. There was also a higher proportion of abnormal semen specimens (HOS less than 50%) after preparation with Sephadex than after the other preparation methods. We recommend the use of the HOS test as part of a screening panel for sperm separation.

Purification and characterization of an inactivator of glucosamine-6-phosphate synthetase from rat submandibular gland.

Glucosamine-6-phosphate synthetase (G6P synthetase) inactivator was purified from rat submandibular gland by a combination of Sephadex G-100 gel filtration and column isoelectric focusing. The purified inactivator showed a single protein band by polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 26, 000 by gel chromatography. It caused an apparent inactivation of G6P synthetase partially purified from rat liver. The purified inactivator was found to be a trypsin-like enzyme because it specifically hydrolyzed the trypsin substrate, Nα-benzoyl-L-arginine-p-nitroanilide, and was inhibited by soybean trypsin inhibitor. Thus, the action of G6P synthetase inactivator may be due to the degradation of the synthetase through the proteolytic activity of the inactivator.

Monoamine-mediated enhancement of acetylcholine release in rat hippocampus by 6 R- l-erythro-5, 6, 7, 8-tetrahydrobiopterin

Recently, we reported that intracerebroventricular administration of 6 R- l-erythro-tetrahydrobiopterin (6R-BH 4), a common natural cofactor for phenylalanine, tyrosine and tryptophan hydroxylases, enhances in vivo exocytotic release of acetylcholine (ACh) from the rat hippocampus, as monitored by brain microdialysis. In the present study, we developed a method for separation of ACh from 6R-BH 4 on small columns of Sephadex G-10, which allowed us to determine by high pressure liquid chromatography (HPLC) the changes of ACh levels recovered in dialysates following addition of 6R-BH 4 to the dialyzing fluid. Infusions of 6R-BH 4 (final concentration, 0.25, 0.5 and 1 mM in the perfusing fluid) produced concentration-dependent increases in ACh levels in dialysates (1.5-, 3- and 8-fold of the basal levels, respectively), but was without effect on choline levels in dialysates. Infusion of 6 S- l-erythro-5, 6, 7, 8-tetrahydrobiopterin (6S-BH 4), an unnatural diastereoisomer of 6R-BH 4, or l-erythro-biopterin (biopterin), an oxidized form of 6R-BH 4, did not affect the ACh levels in dialysates. The 6R-BH 4-induced increase in ACh levels persisted after pretreatment with atropine, a muscarinic antagonist, but did not occur in rats pretreated with reserpine to deplete monoamines. These results show that 6R-BH 4 stimulates exocytotic ACh release in the hippocampus through action on a local monoaminergic system in the hippocampus.

New conformationally restricted technetium-99m N2S2 complexes as myocardial perfusion imaging agents

In developing 99mTc complexes as potential myocardial imaging agents, a new series of ligands based on a conformationally restricted N2S2 system were investigated. Using piperazine or homopiperazine as the starting material, two N2S2 ligands (4a and 4b) with additional conformation restriction between the two nitrogen donor atoms were synthesized. The 99mTc complexes were prepared by a direct labeling method with tin(II) tartrate as the reducing agent for [99mTc]pertechnetate. The resulting 99mTc complexes were purified through a sulfonpropyl Sephadex column and further purified by HPLC with a reverse-phase column eluting with a solvent system of acetonitrile/buffer. Biodistribution studies in rats showed initial uptake in the heart (0.21%, 0.42% dose/order for [99mTc]4a and 4b at 2 min postinjection). Carrier-added preparation of [99mTc]4b was successful. NMR, IR, UV, crystallographic, and elemental analysis of the [99Tc]4b complex suggest that it contains a TcVO3+ center core and is 1+ charged. The results suggest that this series of 1+ charged 99mTc complexes may have potential as myocardial imaging agents, and further study of the complexes is warranted.

Characterization of chitinase and chitobiase produced by the entomopathogenic fungus Metarhizium anisopliae

Extracellular fluids from Metarhizium anisopliae grown on chitin as the sole carbon source contained distinct chitinase ( pH optimum 5.3, MW 33 kDa) and chitobiase ( pH optimum 5, MW 110 kDa, pI 6.4) activities. Chitinase activity was stabilized against extremes of pH and temperature by the presence of its chitin substrate. Chitinase fractions eluted from a sephadex column had activity against chitosan as well as locust cuticle chitin, crystalline chitin, and colloidal chitin. Products of enzymolysis were analyzed by descending paper chromatography. Chitinase had no activity against chitobiose, only trace activity against chitotriose, but preferentially cleaved chitotetraose in the middle bond releasing chitobiose. N-Acetyglucosamine was the major (vs colloidal chitin) or only (vs crystalline chitin) product detected following chitin hydrolysis. The chitobiase (also called β- N-acetylglucosaminidase) possessed simple glycosidase activity against p-nitrophenyl-acetylglucosaminide and hydrolyzed di-, tri-, and tetrasaccharides to N-acetylglucosamine. Chitobiase activity was significantly inhibited by its reaction product, N-acetylglucosamine. The mechanism of action of chitinolytic enzymes is discussed.

Galanin immunoreactivity in the blowfly nervous system: Localization and chromatographic analysis

In this study chromatographic, immunochemical, and immunocytochemical methods provide evidence of a galanin-like peptide(s) in an invertebrate, the blowfly Phormia terraenovae. The major portion of the galanin-like immunoreactivity (GAL-LI) in fly heads was extractable in acetic acid but not in boiling water, which suggests that the peptide(s) may be highly basic in nature. GAL-LI was present both in the head and body portion of the blowfly in roughly the same amounts. Initial gel filtration data, using a G-50 Sephadex column and a weak phosphate-buffer (pH 6.5) as eluent, suggested that a fly GAL-LI peptide(s) from fly heads, eluting as an apparent single peak, was smaller than porcine GAL(1-29) and GAL(1-15). However, concomitant analysis using a G-25 Sephadex column and acetic acid (0.2 M) as eluent, spread the immunoreactive material over a great portion of the chromatogram, although the main portion of the material eluted in the same size range as porcine GAL(1-29). Taken together, the gel filtration data thus suggest that fly GAL-LI peptide(s) may be highly basic but presumably similar in size to vertebrate GAL(1-29). However, the hydrophobic properties of the fly GAL-LI peptide(s) differ from that of porcine GAL as demonstrated by the presence of several immunoreactive components eluting both early as well as late in the chromatogram when using reverse-phase high performance liquid chromatography (HPLC); early peaks may represent highly basic and/or possibly smaller GAL-immunoreactive peptide(s), whereas later peaks may represent less basic and possibly elongated forms. Immunocytochemistry indicated that GAL-LI was present in the nervous system of the blowfly. About 160 GAL-immunoreactive neurons were found in the brain and subesophageal ganglion, 26 in the fused thoracic ganglion and 30 in the fused abdominal ganglion. In the brain, GAL-immunoreactive fibers supply specific subdivisions of the central body, optic lobe, superior protocerebrum, and tritocerebrum as well as neuropil in the subesophageal ganglia. In the thoracico-abdominal ganglia, GAL-immunoreactive neuron processes are found inside synaptic neuropil as well as in the neural sheath of the ganglia and several of the dorsal nerve roots. Many of the GAL-immunoreactive neurons react also with an antiserum against porcine galanin message associated peptide, a peptide present in the preprogalanin protein. Immunocytochemical double-labeling indicated that some GAL-immunoreactive neurons also reacted with antisera against the molluscan peptides FMRFamide and SCPB, whereas no evidence could be found for colabeling with antisera against tyrosine hydroxylase, substance P and physalaemin.(ABSTRACT TRUNCATED AT 400 WORDS)

Vanadobin, a Vanadium-Binding Substance, Extracted from the Blood Cells of an Ascidian, Can Reduce Vanadate(V) to Vanadyl(IV)

Ascidians specifically accumulate high levels of vanadium from seawater in their blood cells. Almost all of the vanadium is present in a reduced form in the blood cells, although the metal exists in a +5 oxidation state in seawater. It has, therefore, been assumed that agents that cause the reduction of vanadate(V) to vanadyl(IV) must be present within ascidian blood cells. In this regard, we have extracted a vanadium-binding substance, which we have called vanadobin, from the vanadocytes of ascidians. We examined whether vanadobin is involved in the reduction of vanadate(V) accumulated from seawater. Data obtained by spectrophotometry and ESR spectrometry revealed that not only a crude homogenate of vanadium-rich blood cells but also a purer form of vanadobin eluted from a column of Sephadex G-15 could reduce vanadate(V). Our experiments demonstrate that vanadobin, a vanadium-binding substance extracted from ascidian blood cells, can reduce vanadate(V) to vanadyl(IV) and maintain it in the reduced form.

Tc-99m labeled tissue-type plasminogen activator: Preparation, stability and preliminary imaging of thrombus-bearing rats

Tissue-type plasminogen activator (t-PA) is a thrombolytic agent that directly binds to fibrin formed in clots. In terms of radiolabeling and nuclear imaging, t-PA has several advantages in Tc-99m labeling: it is stable in acidic solution at pH 3, which is suitable for labeling Tc-99m by a method of stannous reduction and blood disappearance after administration is rapid, which is desirable for imaging targets using short-lived radionuclides. Recombinant t-PA was labeled with Tc-99m by a method of stannous reduction without significant degradation of biochemical activity, over 95% of which was retained after the labeling procedure. Labeling efficiency in paper chromatography was over 98%. The moiety of hydrolyzed Tc-99m that was not eluted through the Sephadex column was estimated to be less than 10%. Tc-99m labeled t-PA, however, appeared to become unstable when diluted with normal saline. Nevertheless, in in vitro fibrin binding, Tc-99m labeled t-PA showed high affinity with fibrin: 80% of 100 ng/ml of Tc-99m t-PA bound to 10(-5) mol of the fibrinogen. Preliminary animal studies also showed a concentration of Tc-99m labeled t-PA at fresh thrombi formed in the inferior vena cava. Tc-99m labeled t-PA appears to have potential for thrombus imaging and the preparation of an instant kit.