Biomedical subjects

V Katta

Publications and source records attributed to V Katta.

29 records · Page 2Linked to original sources

PubMed1995

Microheterogeneity of erythropoietin carbohydrate structure.

The microheterogeneity of the carbohydrate structures on recombinant human erythropoietin (rHuEPO) expressed in Chinese hamster ovary cells has been evaluated by electrospray ionization (ESI) mass spectrometry (MS) of glycopeptide fragments. The microheterogeneity is largely associated with the presence or absence of terminal N-acetylneuraminic acid (Neu5Ac) residues, varying amounts of O-acetylation of the Neu5Ac residues, and the presence or absence of N-acetyllactosamine extensions. The N-linked carbohydrate structures were structurally diverse; 52 different N-linked oligosaccharide structures were identified. Consistent structural assignments could be made from data obtained using different proteolytic digests, ESI solvent systems (aqueous/methanol systems with acetic or formic acid), and on-line or off-line LC/MS analysis. All glycosylation sites exhibited some level of O-acetylation of Neu5Ac residues. Interestingly, glycosylation site asparagine-83 exhibits mono-O-acetyl and di-O-acetyl Neu5Ac residues, while the other sites, asparagine-24, asparagine-38, and serine-126, exhibit mainly mono-O-acetyl Neu5Ac derivatization. This difference in O-acetylation may be site specific or due to sample handling of labile structures. However, mild base treatment of rHuEPO with NaOH on ice removed the O-acetyl groups associated with a given carbohydrate structure, without adversely affecting the underlying oligosaccharide structure, resulting in a simplified mass spectra. Nuclear magnetic resonance spectroscopy of Neu5Ac residues released by neuraminidase treatment of total rHuEPO indicated that Neu5,9Ac2 residues were present. Additional resonances were also observed that were consistent with other Neu5Ac O-acetyl linkages; these O-acetyl resonances could be removed by mild base hydrolysis of rHuEPO.

Amino Acid Sequence↗

PubMed1995

Site-specific conjugation of diethylenetriaminepentaacetic acid to recombinant human granulocyte-colony-stimulating factor: preservation of protein structure and function.

The chelating agent diethylenetriaminepentaacetic acid (DTPA) was conjugated site-specifically to the N-terminus of recombinant human granulocyte-colony-stimulating factor (rhG-CSF) by reaction of the protein with DTPA dianhydride at an initial pH of 6.0. The reaction was efficient in that 84% of the starting rhG-CSF was N-terminally modified and could be purified to homogeneity by cation-exchange chromatography. Chelation of 111In by the DTPA-rhG-CSF conjugate was demonstrated by cation-exchange HPLC and thin-layer chromatography. Metal contamination of conjugate preparations, as well as metal-loading onto the conjugate, could be monitored by either cation-exchange HPLC or isoelectric focusing. The 1:1 stoichiometric molar ratio of DTPA to protein for the DTPA-rhG-CSF conjugate was determined by thin-layer chromatography and mass spectrometry, and the localization of the conjugated DTPA moiety was resolved using a peptide mapping procedure. The secondary structure (i.e., alpha-helicity) of the protein was unmodified following conjugation as revealed by circular dichroism. Furthermore, the conjugate induced a similar induction of peripheral WBC counts as unmodified rhG-CSF when injected subcutaneously into hamsters, demonstrating preservation of protein bioactivity. These results reveal a simple and efficient method for conjugating DTPA to protein, via reaction with the dianhydride, to yield a homogeneous and well-defined product. The procedure may prove to be a useful method of labeling growth factors and related proteins while preserving structural and functional integrity.

Animals↗

PubMed1995

Post-translational processing of membrane-associated neu differentiation factor proisoforms expressed in mammalian cells.

Expression vectors constructed from human and rat pro-neu differentiation factor (NDF) cDNAs were transfected in Chinese hamster ovary cells for expression of recombinant NDF molecules. Soluble NDF forms were released into culture medium after post-translational processing of the membrane-bound pro-NDF forms. Different human and rat NDF isoforms, after being purified from the culture medium, were subjected to structural and biochemical characterizations. The isolated human and rat NDF isoforms have been proteolytically processed at a specific site at the N terminus, which is different from that observed for the processing of rat or human NDF molecule prepared from natural origins. The processing of each recombinant NDF isoform at its C terminus was heterogeneous but consistently occurred at nearby peptide bonds. Specific N- and C-terminal processing by Chinese hamster ovary cells has resulted in the production of two types (alpha and beta) of recombinant NDFs containing 222-225 amino acid residues. Both human and rat NDF molecules are heavily glycosylated at two of the three potential Asn-linked glycosylation sites and contain O-linked sugars at 11 of the Thr/Ser sites. Glycosylation occurs at a short, Ser/Thr-rich spacer region that connects the N-terminal immunoglobulin homology unit to the epidermal growth factor domain. Cellular phosphorylation assay indicated that these secreted forms contain similar biological activity in receptor tyrosine autophosphorylation of mammary tumor cells.

Amino Acid Sequence↗

PubMed1994

Prompt fragmentation of disulfide-linked peptides during matrix-assisted laser desorption ionization mass spectrometry.

During the analysis of an Asp-N digest of a recombinant hematopoietic growth factor by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we observed pseudomolecular ions corresponding to reduced forms of peptides known to be present only in single disulfide linkages. Chromatographic fractionation of the peptide digest, followed by MALDI-MS and electrospray ionization (ESI) MS, confirmed that the reduced peptides were not present in the map. Fragmentation of the disulfide-linked peptides into their reduced forms occurred upon ionization from different matrices (alpha-cyano-4-hydroxycinnamic acid,2,5-dihydroxybenzoic acid, and in some instances sinapinic acid) but only after increasing the laser fluence to above threshold. Analysis of the disulfide-linked peptide fractions by ESI-MS, before and after mixing and drying with matrix, indicated that the matrix did not cause reduction. In a low-energy tandem mass spectrometric experiment with one of the cystinyl peptides, fragmentation did not occur preferentially at the disulfide bond. The pseudomolecular ions exhibited the same m/z values by MALDI-MS as their chemically reduced counterparts, indicating that they arose due to prompt fragmentation or "in-source decay" rather than "post-source decay". This finding is important for MALDI-MS analysis of peptide maps of proteins and peptide fractions with intact disulfides.

Amino Acid Sequence↗

PubMed1991

Use of a single-quadrupole mass spectrometer for collision-induced dissociation studies of multiply charged peptide ions produced by electrospray ionization.

The feasibility of obtaining the collision-induced dissociation (CID) spectra of multiply charged peptide ions produced by electrospray ionization in a simple and inexpensive single-quadrupole mass spectrometer is demonstrated. Collisional activation was carried out in the high-pressure region between the capillary exit and the skimmer entrance to the mass analyzer. The CID of multiply charged peptide ions is very efficient, and the observed fragment ion intensities are typically 1-5% of the parent ion intensity prior to CID. About 70 pmol of the peptide is consumed in obtaining each CID spectrum. Spectra obtained by CID of multiply charged ions from bradykinin, angiotensin II, two peptides with features similar to tryptic peptides, and a synthetic analogue of a component of TGF-alpha containing two disulfide bonds are shown. The influence of the primary structure of the peptide on the observed fragmentation pathways is discussed. Although the present single-quadrupole configuration is simple and effective, the inability to choose a particular parent ion for collisional activation makes it less powerful than the triple-quadrupole configuration for mixtures of peptides and peptide samples that yield more than one charge state in the normal mass spectrum. However, it has the potential for inexpensively obtaining sequence information of proteins at high sensitivity by analyzing the pure tryptic peptides obtained by on-line or off-line chromatographic separation of tryptic digests.

Amino Acid Sequence↗

PubMed1991

Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.

Hydrogen-exchange electrospray-ionization mass spectrometry is demonstrated to be an effective new method for probing conformational changes of proteins in solutions. The method is based on the mass spectrometric measurement of the extent of hydrogen/deuterium exchange that occurs in different protein conformers over defined periods of time. Results are presented in which hydrogen-exchange electrospray-ionization mass spectrometry is used to probe conformational changes in bovine ubiquitin induced by the addition of methanol to aqueous acidic solutions of the protein.

Animals↗

PubMed1990

Retinol is essential for growth of activated human B cells.

When EBV-transformed human B cells are removed from conventional cell cultures, washed, and seeded at a low cell density in serum-free medium, their growth potential is greatly diminished. Fresh serum restores the growth of low density B cell cultures. We have traced this restorative effect to an essential factor present in the lipid fraction of serum and have identified it as all-trans retinol. The identification is based on the close similarities of the factor isolated from serum with authentic all-trans retinol as revealed by mass spectrometry, HPLC chromatography, and the ability to stimulate the growth of lymphoblastoid cells in the bioassay. Retinol is active at concentrations equal to its concentration in serum. Retinol is also a requirement for growth in suspension cultures at cell densities of 3 x 10(5)/ml. Cells removed at any time from such exponentially growing cultures and transferred to retinol-free medium cease to grow and consequently die, whereas in the continued presence of retinol, cell growth is unabated. All-trans retinal can substitute for retinol, but retinoic acid fails to stimulate the growth of lymphoblastoid cells at physiological concentrations. Normal human B lymphocytes also require retinol as a costimulator of proliferation after activation by anti-mu antibody or Staphylococcus aureus (Cowan strain) bacteria. In serum, retinol is bound to retinol-binding protein, which in turn forms a complex with prealbumin. Accordingly, we find that B cells respond to retinol bound to its physiological serum carrier, retinol-binding protein. In conclusion, human B cells are critically dependent for optimal growth in cell culture on an external supply of retinol.

B-Lymphocytes↗

PubMed1990

Electrospray ionization mass spectrometric peptide mapping: a rapid, sensitive technique for protein structure analysis.

Electrospray ionization mass spectrometric peptide mapping is demonstrated to be a useful new technique for protein structure analysis. The procedure involves the digestion of the protein with trypsin and subsequent analysis of the total unfractionated digest by electrospray ionization mass spectrometry. The utility of the technique for investigating protein structure is illustrated by a peptide mapping analysis of human apolipoprotein AI (Mr = 28 kDa). The technique is rapid, sensitive, and requires no prior separation of the peptides. The discrimination effects observed in other mass spectrometric methods are less important in the present procedure.

Apolipoprotein A-I↗

PubMed1990

An electrospray-ionization mass spectrometer with new features.

The construction and performance of an electrospray-ionization mass spectrometer with new features are described. The mass spectrometer consists of a newly designed electrospray ion-source that is plugged directly into a modified commercial quadrupole mass spectrometer with the ions entering the mass analyzer through a long metal capillary tube and three stages of differential pumping. The present ion source differs from previous designs in the combination of techniques employed in the transportation and desolvation of solvated biomolecule ions, prior to mass analysis. Transport of ionized entities between atmospheric pressure and vacuum is carried out through a 203 mm long stainless steel capillary tube with a 0.5 mm bore. Desolvation is effected by the use of controlled heat transfer through the long capillary tube and collisional activation in a region of reduced pressure between the capillary tube exit and the skimmer. Desolvation with this system is convenient and effective and does not involve the strong countercurrent flows of gases that have been used by all previous workers. The effects on the spectra of peptides of capillary tube temperature and desolvation collision energy are investigated. Electrospray-ionization mass spectrometric results are described for thirteen proteins with molecular masses ranging from 5000 to 77,000 Da. The performance of the present instrument, with respect to mass accuracy and sensitivity, is comparable with previously reported systems. The effect of protein concentration in solution on the electrospray mass spectrometric response and charge-state distribution is discussed.

Mass Spectrometry↗

PubMed1986

Inhibition of gonadotropin-releasing hormone receptors in rat anterior pituitary monolayer cell cultures by danazol.

To study possible cellular antigonadotropic effects of danazol, monolayer cultures of anterior pituitary cells from immature female rats were treated with danazol. Measurements of luteinizing hormone release in response to 10(-8) mol/L gonadotropin-releasing hormone challenge and iodine 125-labeled gonadotropin-releasing hormone binding activity were done after exposure to increasing concentrations of danazol and for increasing lengths of time. It was found that luteinizing hormone secreted by pituitary cells in response to gonadotropin-releasing hormone was inhibited after danazol treatment in a dose- and time-dependent manner when compared to controls. Also, a 45% decrease in gonadotropin-releasing hormone receptor binding capacity was observed in pituitary cells cultured in the presence of increasing concentrations of danazol in the range of 10(-8) to 10(-4) mol/L when compared to controls. Furthermore, exposure to danazol for 25 to 96 hours caused a marked decrease in gonadotropin-releasing hormone binding activity (p less than 0.005). Under these experimental conditions danazol treatment decreased the pituitary receptors for gonadotropin-releasing hormone in a dose- and time-dependent manner. Scatchard analysis of saturation curves for the binding of gonadotropin-releasing hormone to cellular gonadotropin-releasing hormone receptors indicated that the observed decrease in gonadotropin-releasing hormone binding in the danazol-treated group was due to a change in the number of gonadotropin-releasing hormone binding sites rather than a change in the affinity. It is therefore concluded that the antigonadotropic activity of danazol appears to be related to a decrease in gonadotropin-releasing hormone receptors in the pituitary.

Animals↗

PubMed1985

Estradiol potentiation of gonadotropin-releasing hormone responsiveness in the anterior pituitary is mediated by an increase in gonadotropin-releasing hormone receptors.

In order to investigate the mechanism by which 17 beta-estradiol potentiates the action of gonadotropin-releasing hormone on the anterior pituitary in vitro, cultured pituitary cells from immature female rats were used as the model system. Cultures exposed to estradiol at concentrations ranging from 10(-10) to 10(-6) mol/L exhibited a significant augmentation of luteinizing hormone release in response to a 4-hour gonadotropin-releasing hormone (10 mumol/L) challenge at a dose of 10(-9) mol/L compared to that of control cultures. The estradiol augmentation of luteinizing hormone release was also dependent on the duration of estradiol exposure. When these cultures were incubated with tritium-labeled L-leucine, an increase in incorporation of radiolabeled amino acid into total proteins greater than that in controls was observed. A parallel stimulatory effect of estradiol on iodine 125-labeled D-Ala6 gonadotropin-releasing hormone binding was observed. Cultures incubated with estradiol at different concentrations and various lengths of time showed a significant increase in gonadotropin-releasing hormone binding capacity and this increase was abrogated by cycloheximide. Analysis of the binding data showed that the increase in gonadotropin-releasing hormone binding activity was due to a change in the number of gonadotropin-releasing hormone binding sites rather than a change in the affinity. These results suggest that (1) estradiol treatment increases the number of pituitary receptors for gonadotropin-releasing hormone, (2) the augmentary effect of estradiol on luteinizing hormone release at the pituitary level might be mediated, at least in part, by the increase in the number of binding sites of gonadotropin-releasing hormone, and (3) new protein synthesis may be involved in estradiol-mediated gonadotropin-releasing hormone receptor induction.

Animals↗