Larger Molecular Weight Species Research Articles

Using Symmetrical Organic Cation Solutions to Study P2X7 Ion Permeation.

P2X7 receptors are ATP-gated ion channels permeable to metal cations, such as Na+, K+, and Ca2+. They also exhibit permeability to various large molecular weight species, reaching up to 900Da, in a process known as macropore formation, which is a unique functional hallmark across the P2X family. While well-documented in a range of different cell types, the molecular mechanism underlying this phenomenon is poorly understood, and has been clouded through the use of electrophysiological methodology prone to artifacts as a result of significant changes in ionic concentrations in asymmetric conditions. In this chapter, we discuss the permeation properties of P2X7, the related methodological challenges and the use of symmetrical organic cation solutions as a useful technique for probing P2X7 permeation.

RNA size and 3-dimensional structure determine ultrafiltration behavior of small RNA molecules

Successful commercialization of RNA therapeutics will require the development of appropriate manufacturing and purification processes suitable for the production of large quantities of high quality and high purity RNA with well-defined tertiary structures. Ultrafiltration has the potential to play a major role in RNA processing, both for removal of large molecular weight species (with the RNA collected in the permeate) and for RNA concentration or buffer exchange (with the RNA collected in the retentate). Previous studies have demonstrated the potential of using tangential flow filtration (TFF) for RNA separations, but most of these data were obtained with crude RNA mixtures containing RNA with a range of size and structure. The objective of this study was to directly evaluate the effects of RNA size and tertiary structure on the ultrafiltration behavior over a range of solution conditions (e.g., added salt and urea). Transmission of a hairpin RNA with 70 nucleotides was much greater than that of a linear RNA having the same number of nucleotides, consistent with the smaller size as determined by dynamic light scattering. RNA transmission increased at high NaCl concentrations and in the presence of urea, particularly for the linear RNA. These results provide new insights into RNA ultrafiltration as well as a framework for the application of ultrafiltration for the purification of RNA therapeutics.

Passively Sampled Environmental Films Show Geographic Variability and Host a Variety of Microorganisms

Environmental films are ubiquitous coatings that form on virtually all surfaces exposed to the troposphere. This work analyzes large molecular weight species in native environmental films acquired at three collection sites in the vicinity of Iowa City, Iowa, USA that include an urban residence, a wooded area, and a metropolitan building rooftop. The films are developed for up to twelve months on polished silicon wafer substrates housed in flat-plate passive samplers. Film composition was assessed by Fourier transform ion cyclotron resonance secondary ion mass spectrometry (FTICR-SIMS), and spatially resolved chemical morphology was analyzed via time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging. Mass analysis spanning up to m/z 2,000 included detection of a variety of organic and inorganic compounds. We characterize MS data with PCA to identify variability between the collection sites and the respective films generated therein, where we detected a variety of organic and biotically generated...

Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants.

BACKGROUNDA current challenge in bioprocessing is the ability to analyse critical quality attributes such as aggregation without prior purification. This study evaluated the use of fluorescent dyes (Bis‐ANS, SYPRO Orange, Thioflavin T and ProteoStat) to characterise mAb aggregates in Chinese hamster ovary clarified cultures.RESULTSThe null and mAb culture supernatants showed an increase in fluorescence intensity over the duration of the culture. The null cultures on day 14 saw a rapid increase in fluorescence intensity; day 10 to day 14, Bis‐ANS and Thioflavin T had average increases of 21% and 48%, respectively, whereas ProteoStat and SYPRO Orange showed an average increase of 60%. Higher fluorescence intensity on day 14 with the null cultures, also correlated with loss of viability.CONCLUSIONFluorescent dyes are not a specific indicator of mAb aggregation, but rather an indicator of overall protein aggregation or high molecular weight species. SYPRO Orange was more sensitive at detecting very large molecular weight species and ProteoStat seemed better suited to smaller aggregates. Although the assay cannot be used to measure mAb aggregates in cell culture, it could be used to aid cell line selection in maximising viabilities and minimising the amount of aggregates. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

PHARMACOLOGICAL CHARACTERIZATION OF BAN2401-MEDIATED Aβ PROTOFIBRIL CLEARANCE BY MICROGLIA

Amyloid β (Aβ) peptides aggregate into soluble, large molecular weight species (protofibrils) that eventually form the insoluble fibrils deposited in plaques. Aβ protofibrils are the dominant form of soluble Aβ in Alzheimer disease (AD) brains and are thought to play a role in the pathogenesis of AD through their deleterious effects on a number of physiological processes. BAN2401 is a humanized monoclonal IgG1 antibody with high selectivity for Aβ protofibrils that is currently in clinical development for early AD. mAb158, the mouse precursor, has previously been shown to remove insoluble amyloid plaque in mice, neutralize large soluble Aβ aggregates in vivo/in vitro and facilitate their clearance via a shift toward FcγR-dependent microglial phagocytosis. The present work aims to further characterize the mechanism of action of BAN2401-mediated Aβ protofibril clearance. Protofibril uptake was assessed in cultured human primary microglia prepared from AD brain by visualizing fluorescent tagged antibody under high power microscopy. Protofibril uptake was also assessed in an EOC-20 microglia cell antibody dependent cellular phagocytosis (ADCP) assay by measuring intracellular Aβ via ELISA in the absence or presence of FcγR blocker or F(ab’)2 fragments. Aβ protofibrils were readily taken up in human AD-derived microglia in an FcγR-independent manner, while BAN2401 facilitated further uptake of protofibrils in FcγR-mediated fashion. BAN2401 facilitated protofibril uptake in EOC-20 microglia cells was blocked with an FcγR blocker or F(ab’)2 fragments (EC50=257±61 ng/mL; corrected for background uptake). Aβ can be taken up by microglia via a non-specific pattern-recognizing receptor pathway or through an FcγR-mediated process. Previous work has demonstrated that BAN2401 facilitates protofibril clearance, and this clearance was inhibited by an FcγR blocker to levels lower than those seen in the absence of BAN2401, suggesting a shift from non-specific uptake toward microglial activation and FcγR-dependent microglial phagocytosis. The present studies isolate the FcγR-dependent component of BAN2401-mediated protofibril uptake and provide further support for antibody dependent cellular phagocytosis of Aβ protofibrils as the primary mechanism of action for BAN2401.

"Inside-Out" PEGylation of Bovine β-Cross-Linked Hemoglobin.

The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside-out PEGylation, has been used here to conjugate a multiarm maleimide-PEG (Mal-PEG) to β-cross-linked (βXL-Hb) hemoglobin (Hb) tetramers through the Cys β93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three βXL-Hb to one Mal-PEG. Thermal denaturation studies showed that the cross-linked and PEGylated species were more stable than native Hb. Cross-linking under oxy-conditions produced a high oxygen affinity Hb species (P50 =9.18Torr), but the oxygen affinity was not significantly altered by PEGylation (P50 =9.67Torr). Inside-out PEGylation can be used to produce a hemoglobin-based oxygen carrier and potentially for other multiprotein complexes.

The soluble, periplasmic domain of OmpA folds as an independent unit and displays chaperone activity by reducing the self-association propensity of the unfolded OmpA transmembrane β-barrel

OmpA is one of only a few transmembrane proteins whose folding and stability have been investigated in detail. However, only half of the OmpA mass encodes its transmembrane β-barrel; the remaining sequence is a soluble domain that is localized to the periplasmic side of the outer membrane. To understand how the OmpA periplasmic domain contributes to the stability and folding of the full-length OmpA protein, we cloned, expressed, purified and studied the OmpA periplasmic domain independently of the OmpA transmembrane β-barrel region. Our experiments showed that the OmpA periplasmic domain exists as an independent folding unit with a free energy of folding equal to − 6.2 (± 0.1) kcal mol -1 at 25 °C. Using circular dichroism, we determined that the OmpA periplasmic domain adopts a mixed alpha/beta secondary structure, a conformation that has previously been used to describe the partially folded non-native state of the full-length OmpA. We further discovered that the OmpA periplasmic domain reduces the self-association propensity of the unfolded barrel domain, but only when covalently attached (in cis). In vitro folding experiments showed that self-association competes with β-barrel folding when allowed to occur before the addition of membranes, and the periplasmic domain enhances the folding efficiency of the full-length protein by reducing its self-association. These results identify a novel chaperone function for the periplasmic domain of OmpA that may be relevant for folding in vivo. We have also extensively investigated the properties of the self-association reaction of unfolded OmpA and found that the transmembrane region must form a critical nucleus comprised of three molecules before undergoing further oligomerization to form large molecular weight species. Finally, we studied the conformation of the unfolded OmpA monomer and found that the folding-competent form of the transmembrane region adopts an expanded conformation, which is in contrast to previous studies that have suggested a collapsed unfolded state.

Photocatalytic oxidation, GAC and biotreatment combinations: an alternative to the coagulation of hydrophilic rich waters?

This study investigated the photocatalytic oxidation of a raw water rich in hydrophilic natural organic matter (NOM) and the impact on the removal of: dissolved organic carbon (DOC), UV absorbance at 254 nm (UV254) and trihalomethanes formation potential (THMFP). Dissolved organic carbon and UV254 removals were 40% and 55%, respectively, after 1 min irradiation time and 1 g L−1 dose of TiO2. The THMFP content was reduced from 305 µg L−1 in raw water to 144 µg L−1 after 10 min treatment, whereas chlorine reactivity was stable with treatment. The results showed that larger molecular weight species were preferentially degraded during the process. Dissolved organic carbon and THMFP removals reached 60% and 70%, respectively, after photocatalytic oxidation and granular activated carbon (GAC) columns.

Conditioning saliva for use in a microfluidic biosensor

This report details an approach to saliva conditioning for compatibility of raw patient samples with microfluidic immunoassay components, principally biosensor surfaces susceptible to fouling. Stimulated whole human saliva spiked with a small molecule analyte (phenytoin, 252 Da) was first depleted of cells, debris and high molecular weight glycoproteins (mucins) using membrane filtration. This process significantly reduced but did not eliminate fouling of biosensor surfaces exposed to the sample. An H-filter, which separates solutes from mixed samples based on their diffusion in laminar flow, was used to extract the analyte from the remaining large molecular weight species in the filtered saliva sample. Patient samples treated in this way retained 23% of the analyte with 97% and 92% reduction in glycoproteins and proteins, respectively, and resulted in 3.6 times less surface fouling than either untreated or filtered saliva alone. These sample conditioning steps will enable the use of fouling-sensitive detection techniques in future studies using clinical saliva samples.

Regulation of NGF and NT-3 protein expression in peripheral targets by sympathetic input

Nerve growth factor (NGF) and neurotrophin-3 (NT-3) are target-derived proteins that regulate innervating sympathetic neurons. Here, we used western blot analysis to investigate changes in NGF and NT-3 protein in several peripheral tissues following loss of sympathetic input. Following removal of the superior cervical ganglion (SCG), large molecular weight (MW) NGF species, including proNGF-A, were increased in distal intracranial SCG targets, such as pineal gland and extracerebral blood vessels (bv). Mature NGF was a minor species in these tissues and unchanged following sympathectomy. Large MW NGF species also were increased when sympathectomy was followed by in vivo NGF administration. Mature NT-3, which was abundant in controls, was significantly decreased in these targets following sympathetic denervation. The decrease in mature NT-3 was enhanced following NGF administration. The trigeminal ganglion, which provides sensory input to these targets, showed increased NGF, but decreased NT-3, in these treatments, demonstrating that decreased NT-3 at the targets did not result from enhanced NT-3 uptake. Unlike pineal gland and extracerebral bv, the external carotid artery, an extracranial proximal SCG target, showed no change in NGF following denervation, and mature NT-3 was significantly increased. Following NGF administration, NT-3 was significantly decreased. We provide evidence for sympathetic regulation of NGF and NT-3 in peripheral targets and that elevated NGF can depress NT-3. The differential response in distal and proximal adult targets is consistent with the idea that neurons innervating proximal and distal targets may serve different roles in regulating neurotrophin protein. In addition, we conclude that previous ELISA results showing increased NGF protein following sympathetic denervation may have resulted from increases in large MW species, rather than an increase in mature NGF.

Rheological and Light Scattering Properties of Flaxseed Polysaccharide Aqueous Solutions

Polysaccharides isolated from flaxseed meals using ethanol consisted of a soluble ( approximately 7.5% w/w) and an insoluble fraction (2% w/w). The soluble fraction was dialyzed in various salt concentrations and characterized using viscometry and light scattering techniques. Observations using a size-exclusion column coupled to a multiangle laser light scattering (SEC-MALLS) revealed three molecular weight fractions consisting of a small amount ( approximately 17%) of large molecular weight species (1.0 x 10(6)) and a large amount ( approximately 69%) of small molecular weight species (3.1 x 10(5) Da). Dynamic light scattering measurements indicated the presence of very small molecules (hydrodynamic radius approximately 10 nm) and a very large molecular species (hydrodynamic radius in excess of 100 nm); the latter were probably aggregates. The intrinsic viscosity, [eta], of the polysaccharide in Milli-Q water was 1030 +/- 20 mL/g. The viscosity was due largely to the large molecular weight species since viscosity is influenced by the hydrodynamic volume of molecules in solution. The Smidsrod parameter B obtained was approximately 0.018, indicating that the molecules adopted a semi-flexible conformation. This was also indicated by the slope ( approximately 0.56) from the plot of root-mean-square (RMS) radius versus molar mass (M(w)).

Iron-Mediated Aeration: Evaluation of Energy-Assisted Enhancement for In Situ Subsurface Remediation

Laboratory tests using ultraviolet radiation and sonocation energy were found to kinetically enhance an iron-mediated aeration (IMA) process under development to remove chelated metals and radionuclides and associated organics, from groundwater and soils. A model inorganic contaminant ( Cd2+ ) chelated with ethylenediamine tetraacetic acid (EDTA) was used. The IMA process breaks the complex, releasing the target metal for removal. Overall experimental results indicate that the EDTA degradation mechanism can be accelerated compared to nonenergized IMA, by a factor of 2 using sonocation energy and by a factor of 3–4 using photochemical energy at circumneutral pH. No differences in the by-products were indicated in chemical analyses. For both sonocation and photochemical tests, the major breakdown products detected were glyoxylic acid and formaldehyde. Only minor amounts of larger molecular weight species (iminodiacetic acid, nitrilotriacetic acid, and ethylenediamine triacetic acid) were detected.

Chemical characterization of humic‐like substances (HULIS) formed from a lignin‐type precursor in model cloud water

A representative lignin‐type component from biomass burning aerosol has been shown to react with OH radicals in model cloud water yielding colored organic species. In this paper we investigated the chemical properties of the complex reaction products formed from 3,5‐dihydroxybenzoic acid. The reaction was followed by UV‐VIS spectrophotometry, liquid chromatography, electrospray‐mass spectrometry, thermally assisted hydrolysis and methylation‐gas chromatography/mass spectrometry and a thermal method. This paper provides experimental proofs that actually larger molecular weight species are formed in the aqueous phase by free radical oligomerization. The features observed by all analytical techniques closely resemble those found for natural humic acids and HULIS found in rural and biomass burning aerosol. Therefore such processes are assumed to produce the ubiquitous humic‐like substances (HULIS) in atmospheric aerosol. Since these species show intense absorbance in the lower visible to UV range, they might also be important in atmospheric absorption of solar radiation.

Carbonization of anthracene in a batch reactor

The coking of liquid anthracene in a batch tubular reactor was followed by measuring the formation of pyridine insolubles and the disappearance of anthracene as a function of time over the temperature range 465–525°C. Conditions, that can affect coking behavior and rates, like partition of the feed between liquid and vapor, agitation of the reactor, and whether the gas pressure in the reactor is autogeneous or imposed are considered. Carbonization primarily involves hydrogen-transfer reactions and condensation (polymerization) of free radicals into larger molecular weight species. Cleavage (cracking) of hydroaromatic species, giving smaller liquid and gas phase molecules, is at a minimum under the conditions used in this study.

Antiport mediated rounding and endocytosis are enhanced by sulphate

Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na +/H + antiporter via HCO 3 −-dependent H + pumping, is significantly enhanced by the addition of Na 2SO 4 (FTR + SO 4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive. At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol·wt which is a 455-fold diversity. This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol·wt species that measures up to 5 μm in width. Two million mol·wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion. Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages. However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz. epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202). The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change. However the cell surface reversion is not associated with efflux of FDx which are sorted out into ‘granular patches’, the later stage endosomes without membrane outlines in AME. FDxloaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions. Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches. AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an alternate membrane recycling capability where granular patches are instrumental in sorting. It appears to be not a simple endocytosisexocytosis pathway.

Immunoreactive growth hormone-releasing hormone in rat leukocytes

In the present study, we evaluated whether mononuclear leukocytes could synthesize and secrete growth hormone-releasing hormone (GHRH) in vitro. By using RNA slot-blot analysis, we detected maximum basal levels of specific GHRH mRNA in the cytoplasm of rat leukocytes after an 8 h in vitro incubation. Northern gel analysis demonstrated that the specific GHRH RNA was polyadenylated and had a molecular mass of approximately 0.8 kDa. Further studies using antibody affinity chromatography followed by size separation on high-performance liquid chromatography (HPLC) columns showed two peaks of immunoreactive (ir) material, a large molecular weight species, and a smaller molecular weight species at approximately 5 kDa. The smaller molecular weight irGHRH appeared to be de novo synthesized since it could be radiolabeled with tritiated amino acids. Both molecular species were detectable in enzyme-linked immunosorbent assay (ELISA) with specific antibodies made to the first 23 amino acids as well as specific antibody obtained commercially made to the entire molecule (1–43). Although the larger molecular weight form appeared to be the more predominant, only the lower molecular weight form could block the binding of 125I-hGHRH to pituitary cells. Most importantly, the lower molecular weight leukocyte-derived GHRH stimulated an increase in the level of GH RNA in the pituitary. We conclude that lymphocytes produce an irGHRH that is similar to hypothalamic GHRH in terms of bioactivity, antigenicity, and molecular weight. The findings demonstrate a potential regulatory loop between the immune and neuroendocrine tissues.

Purification and properties of xylanase from the thermophilic fungus, Humicola lanuginosa (Griffon and Maublanc) Bunce

An extracellular xylanase was purified to homogeneity from the culture filtrate of the thermophilic fungus, Humicola lanuginosa (Griffon and Maublanc) Bunce and its properties were studied. A fourfold purification and a yield of 8% were achieved. The molecular-weight of the protein was found to be 22,500 based on electrophoretic mobility and 29,000 by gel filtration behavior. The protein is rich in acidic amino acids, glycine and tyrosine, and poor in sulfur-containing amino acids. The kinetic properties of the enzyme are similar to those of other fungal xylanases. The enzyme shows high affinity toward larchwood xylan ( K m = 0.91 mg/ml) and hydrolyzes only xylan. The enzyme becomes inactivated when stored for more than 2 months at −20 °C in the dry state. Such an inactivation has not been reported so far for any xylanase. Using chromatographic techniques, one species of protein differing from the native protein in charge but enzymatically active was isolated in low yields. However, a large molecular-weight species of the protein devoid of enzyme activity was isolated in substantial quantities and further characterized. Based on ultracentrifugation and gel electrophoretic studies, it was concluded that this species may be an aggregate of the native protein and that such an aggregation might be taking place on storage in the dry state at −20 °C, leading to loss in activity.

Heparin modulates the secretion of a major excreted protein-like molecule by vascular smooth muscle cells

Previous work from our laboratory has shown that heparin specifically induces the release of a pair of proteins of approximately 35 000 and 37 000 Da into the culture medium of vascular smooth muscle cells (SMC). In this report, we demonstrate that the previously identified 37 000-Da smooth muscle protein is composed of two protein species with very similar molecular weights based on migration pattern in SDS-polyacrylamide gels. The larger molecular weight species in this doublet has a similar molecular weight and shares antigenic determinants with major excreted protein (MEP), a lysosomal proteinase previously shown to be secreted by normal and transformed fibroblasts and epidermal cells. Antisera to MEP precipitated the higher molecular weight band from the doublet; preimmune serum was not reactive with the smooth muscle protein. Exposure of smooth muscle cells to heparin resulted in decreased amounts of immunoprecipitable protein released into the medium. Thus, it now appears that three proteins in the 35 000–38 000 molecular weight range are modulated by heparin, and that the largest of the heparin-modulated vascular SMC proteins has a similar molecular weight and is immunologically related to MEP. The release of MEP-like protein from SMC is decreased by heparin, while the remaining two heparin-modulated proteins are increased in the presence of heparin.

Characterization of the reactions of platinum antitumor agents with biologic and nonbiologic sulfur-containing nucleophiles

Substitution reactions with biologic nucleophiles appear to govern the antitumor and toxic properties of platinum complexes. In this paper we have characterized the reactions of several platinum antitumor agents with sulfur-containing amino acids, peptides, proteins, and nonbiologic nucleophiles. The rate constants for the reactions of trans-diamininedichloroplatinum(II) ( trans-DDP), cis-diamminedichloroplatinum(II) (DDP),diammine (1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) and cis-diisopropylamine-cis-dichloro-trans-dihydroxy platinum(IV) (CHIP) with cysteine (Cys), methionine (Met), and glutathione (GSH) were determined at 37°. A reactivity ratio of 1:1.5:22:6500 was determined for the reaction of GSH with CHIP, CBDCA, DDP, and trans-DDP respectively. The rate constant for the binding of DDP to DNA, 7.4 × 10 −5 sec −1, decreased to 5.9 × 10 −5 sec −1and 1.7 × 10 −5 sec −1 in the presence of 0.5 and 5mM GSH respectively. The products formed in the reaction of GSH with trans-DDP, DDP, and CBDCA were also examined. Under conditions of high platinum concentration (2–3 mM), CBDCA and DDP form large molecular weight species with GSH as indicated by 1H-NMR and ultrafiltration experiments. The complex [Pt(GSH) 2 · 3H 2O] n was isolated from the reaction of 3 mM DDP with 6 mM GSH. The product formed in the reaction of 3 mM trans-DDP with 6 mM GSH was not macromolecular in nature, and 1H-NMR spectra revealed that platinum was bound to the Cys sulfhydryl group. Rate constants were determined for the reactions of these platinum complexes with diethyldithiocarbamate (DDTC) and thiosulfate, two agents known to reduce platinummediated nephrotoxicity. DDTC, but not thiosulfate, was shown to rapidly chelate platinum from [Pt(GSH) 2·3H 2O] n. The effects of DDP, CBDCA, and CHIP on the sulfhydryl-dependent rat renal proximal tubule membrane enzymes alkaline phosphatase (AP), γ-glutamyltranspeptidase (GGTP), leudne aminopeptidase (LAP), and the Na +/K +- and Mg 2+-adenosine-5'-triphosphatases (ATPases) were also investigated in vitro. The ability of platinum complexes to inhibit these enzymes parallels their reactivity with other nucleophiles. DDTC and thiourea were shown to restore activity to platinum-inhibited enzymes. Chloride ion was found to reduce platinum-mediated enzyme inhibition in an unpredictable manner, the greatest effect being observed with LAP and GGTP and the least with the ATPases. None of these renal enzymes was directly inhibited by DDP in vivo.

Chicken intestinal receptor for 1,25-dihydroxyvitamin D3. Immunologic characterization and homogeneous isolation of a 60,000-dalton protein.

The chick 1,25-dihydroxyvitamin D3 receptor has been identified via immunoblot analysis and isolated to homogeneity via positive immunoselection and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cytosolic extracts of intestinal mucosa, as well as purified samples highly enriched for receptor by nonimmunologic methodology were electrophoresed on denaturing gels, transferred to nitrocellulose, and probed utilizing a purified monoclonal antibody against the chick receptor. Two protein signals were detected by this approach, a major species of 60,300 daltons and a minor form at 58,600 daltons. Both immunologically identified species were present in receptor-positive tissues but were absent in receptor-negative liver extracts. The two immunoreactive cytosolic proteins comigrated with two polypeptides detected via Coomassie Blue staining as well as by immunoblot analysis after enrichment utilizing DNA-cellulose, blue dextran-Sepharose, and other chromatographic separation techniques. Increasing concentrations of the minor form during purification suggest it arises from the larger molecular weight species via proteolysis. Finally, both forms of the receptor were isolated to near homogeneity employing positive immunoselection and each individually purified to homogeneity employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These experiments show that the chick receptor exists as a major species of 60,300 as well as a minor form of 58,600 and that both forms can be purified to homogeneity via immunoaffinity chromatography.