PubMed Health⌕ Search

Biomedical subjects

K Kalghatgi

Publications and source records attributed to K Kalghatgi.

16 recordsLinked to original sources
PubMed1997

A biophysical study of integral membrane protein folding.

In order to characterize the thermodynamic constraints on the process of integral membrane protein folding and assembly, we have conducted a biophysical dissection of the structure of bacteriorhodopsin (BR), a prototypical alpha-helical integral membrane protein. Seven polypeptides were synthesized, corresponding to each of the seven transmembrane alpha-helices in BR, and the structure of each individual polypeptide was characterized in reconstituted phospholipid vesicles. Five of the seven polypeptides form stable transmembrane alpha-helices in isolation from the remainder of the tertiary structure of BR. However, using our reconstitution protocols, the polypeptide corresponding to the F helix in BR does not form any stable secondary structure in reconstituted vesicles, and the polypeptide corresponding to the G helix forms a hyperstable beta-sheet structure with its strands oriented perpendicular to the plane of the membrane. [The polypeptide corresponding to the C helix spontaneously equilibrates in a pH-dependent manner between a transmembrane alpha-helical conformation, a peripherally bound nonhelical conformation, and a fully water soluble conformation; the conformational properties of this polypeptide are the subject of the accompanying paper: Hunt et al. (1997) Biochemistry 36, 15177-15192.] Our observations suggest that the folding of alpha-helical integral membrane proteins may proceed spontaneously. However, the preference for a non-native conformation exhibited by two of the polypeptides suggests that the formation of some transmembrane substructures could require external constraints such as the links between the helices, interactions with the rest of the protein, or the involvement of cellular chaperones or translocases. Our results also suggest a strategy for improving the thermodynamic stability of alpha-helical integral membrane proteins, a goal that could facilitate attempts to overexpress and/or refold them.

Amides↗
PubMed1996

Effect of mobile phase additives on peptide retention in reversed-phase chromatography with pellicular and totally porous sorbents.

The effect of two mobile phase additives, trifluoroacetic acid and phosphoric acid, on the energetics of peptide retention in reversed-phase chromatography was investigated using Hy-Tach C18 micropellicular and Vydac C4 and C18 totally porous stationary phases. The effect of the relatively low phase ratio of columns packed with micropellicular sorbents was also examined. The logarithmic retention factors, of two model peptides, Ac-RGGGGLGLGK-amide and Ac-RGAGGLGLGK-amide, were evaluated with different columns and additives in a practical range of eluent strength. The dependence of the logarithmic retention factor on the concentration of acetonitrile in the mobile phase was linear in all cases. The higher sensitivity of the retention to the organic modifier concentration in the case of the Hy-Tach C18 column is attributed to the relatively low phase ratio of this column. Pairwise plots of the logarithmic retention factors were linear. The plots of data obtained with the two additives has unit slopes and thus reveal homoenergetic retention behavior. On the other hand data obtained on two different columns manifest homeoenergetic retention, the slopes of plots are different from unity. The analysis has yielded consistent results and validated the assumption that the retention free energy can be divided into two components arising from mobile phase and stationary phase contributions. The approach also allowed an estimation of the relative phase ratios of the columns and the Vydac C18 column was found to have an 3 and 8 times higher phase ratio than the Vydac C4 and the Hy-Tech C18 column, respectively.

Acetonitriles↗
PubMed1992

Rapid displacement chromatography of melittin on micropellicular octadecyl-silica.

Rapid high-performance liquid chromatographic analysis and displacement purification of melittin and its variants were carried out by reversed-phase chromatography. High speed of separation was achieved by the use of columns packed with a micropellicular stationary phase consisting of a thin C18 hydrocarbonaceous layer on the surface of 2-microns fluid-impervious silica microspheres at elevated temperature. In the case of melittin from bee venom or its synthetic variants the plots of the logarithmic retention factor against acetonitrile concentration in the eluent were straight lines whereas the van't Hoff plots in the temperature range from 20 to 80 degrees C were non-linear. Purification of melittins by displacement was carried out with benzyldimethylhexadecyl ammonium chloride as the displacer. In a 20-min displacement run at 40 degrees C about 5 mg of highly pure melittin were isolated from 10 mg of synthetic mixture by using a 105 x 4.6 mm column. The results demonstrate that columns packed with micropellicular sorbents not only facilitate rapid high-performance liquid chromatographic analysis but are also suitable for fast peptide purification with high recovery.

Amino Acid Sequence↗
PubMed1990

Micropellicular stationary phases for rapid protein analysis by high-performance liquid chromatography.

The high separating speed, efficiency and operational stability of various micropellicular stationary phases are demonstrated in the high-performance liquid chromatography (HPLC) of biopolymers. The micropellicular sorbents were prepared from 2-microns fluid-impervious silica microspheres as the support, with a thin layer of different retentive materials at the surface. These include a molecular fur of octyl or stearyl chains for reversed-phase chromatography as well as a hydrophilic layer with amino groups and polyethyleneglycol chains for anion-exchange and hydrophobic interaction chromatography, respectively. The use of appropriate micropellicular stationary phases for protein separation by metal-interaction and affinity chromatography is also illustrated. In most cases, operation at elevated column temperature was found to be preferable for rapid separations. Preliminary results show that the stability of micropellicular columns compares very favorably with that of columns conventionally used in HPLC and that they are easy to maintain.

Animals↗
PubMed1988

Rapid high-performance affinity chromatography on micropellicular sorbents.

Short columns (30 x 4.6 mm I.D.), packed with 2-micron fluid-impervious silica microspheres with surface-bound Protein A or a lectin were used for fast separation and quantitation of immunoglobulins and glycoproteins by biospecific interaction chromatography. With stepwise elution, the total analysis time including column reequilibration did not exceed 3 min. In the assay of IgG with a stepwise change in pH best results were obtained with citrate buffer, which facilitated not only fast but also very sensitive analysis. The calibration curve was linear in the range 0.5-40 micrograms of human IgG. By using morpholinoethanesulfonic acid-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-acetic acid buffer with a linear decrease in pH from 6.0 to 4.0 and an increase in magnesium chloride concentration to 200 mM for elution, the subclasses of human IgG were separated at 40 degrees C above pH 4.0 in 3 min. Micropellicular concanavalin A and wheat germ agglutinin were used for rapid affinity chromatography of horseradish peroxidase and fetuin, respectively. The results suggest that micropellicular affinity sorbents afford fast and sensitive high-performance liquid chromatographic analysis by biospecific interaction chromatography. Although developed primarily for rapid analysis, the micropellicular Protein A exhibited unexpectedly high adsorption capacity (e.g., 4.5 mg human IgG per ml of wet bed volume). This suggests that such columns could be employed in preparative protein chromatography as well.

Chromatography, Affinity↗
PubMed1988

Rapid peptide mapping by high-performance liquid chromatography.

Short columns, packed with pellicular sorbents made of 2-micron fluid-impervious silica microspheres, were used at elevated column temperatures for rapid peptide mapping by high-performance liquid chromatography (HPLC). Enzymic digests of various proteins were chromatographed by gradient elution. In many cases the time of analysis was 10 min or less. In order to increase the retention particularly, that of short, polar peptides under such conditions, 1 mM octyl sodium sulfate or 5 mM hexyl sodium sulfate were added to the starting eluent. The length of the 4.6 mm I.D. columns was 30 or 75 mm, the sample load was in the range of 10-1000 pmoles. Highest analytical sensitivity was obtained at a flow-rate of 0.5 ml/min and room temperature, whereas for rapid analysis flow-rates of up to 2 ml/min were used at 80 degrees C. This temperature allowed the use of relatively high flow velocities of the mobile phase without significant loss in efficiency. The method was highly reproducible, as shown by the results obtained by automated analysis of cyanogen bromide fragments of lysozyme at high speed. The quality of the rapid peptide maps compares favorably with that of maps obtained by standard reversed-phase HPLC methods, which require much longer analysis times.

Autoanalysis↗
PubMed1987

Extracorporeal enzyme reactors for depletion of phenylalanine in phenylketonuria.

Multitubular enzyme reactors with immobilized enzymes were developed to achieve depletion of circulating substrate by extracorporeal means. To act as prototypes, reactors were prepared with immobilized L-phenylalanine ammonia-lyase, an enzyme that metabolizes phenylalanine to trans-cinnamic acid and ammonia without the need for a coenzyme. We report the first application of phenylalanine ammonia-lyase reactors in an extracorporeal circulation system in a patient with phenylketonuria. A phenylalanine level of 1.82 mmol/L (for the last 6 years) decreased to 1.24 mmol/L after 5.5. hours of treatment, without the enzyme entering the circulation. Total phenylalanine depletion from blood and tissue stores was estimated at 1800 mg. The hemodialysis-like procedure proved to be without side effects, specific for phenylalanine, and suitable in the management of pregnant women with phenylketonuria and late-onset hyperphenylalaninemia. The extracorporeal use of enzyme reactors for temporary enzyme replacement represents a new, safe, and effective therapeutic modality.

Adult↗
PubMed1984

Determination of aromatic amino acids by ion-pair reversed-phase liquid chromatography in human sera from healthy and phenylketonuric individuals.

A simple chromatographic procedure is described for simultaneous analysis of aromatic amino acids in serum samples obtained from normal individuals and from phenylketonuric patients. Quantitative measurement of phenylalanine, tyrosine, histidine and tryptophan is made in samples as small as 10 microliter after deproteinization with trichloroacetic acid. With phenoxyacetic acid as the internal standard, samples are analyzed by reversed-phase chromatography isocratically by an ion-pairing agent in the eluent, and the amino acids are detected with the UV detector at 210nm. Total analysis time was about 30 minutes. Using this method in serum samples from phenylketonuric patients phenylalanine was increased (as expected), and histidine was decreased, at a statistically significant level.

Amino Acids↗
PubMed1983

Anisotropic membranes with carboxypeptidase G1.

Anisotropic polysulfone membranes were prepared with carboxypeptidase G1 embedded in the polymer structure. The enzymatically active flat and hollow-fiber membranes were obtained by precipitating the polymer from solution in an organic mixture in which an aqueous solution of the enzyme had been dispersed. The process has been found to be particularly suitable for the immobilization of enzymes in anisotropic hollow fibers that exhibited no detectable enzyme leakage upon perfusion. The pH profiles measured with the enzyme in free solution and in the embedded form were similar. Kinetic parameters of multitubular enzyme reactors were investigated by measuring the rate of hydrolysis of glutamate from folic acid or methotrexate at different flow rates and substrate concentrations. The relatively slow mass transfer in such reactors was found to affect strongly the observed kinetics. The results of in vitro experiments with 5000 fiber reactors suggest that hollow fiber cartridges prepared with such membranes have clinical potential for the extracorporeal removal of methotrexate from blood.

Carboxypeptidases↗
PubMed1983

In vivo safety of hollow fiber enzyme-reactors with immobilized phenylalanine ammonia-lyase in a large animal model for phenylketonuria.

Hollow fiber enzyme-reactors with immobilized phenylalanine ammonia-lyase (PAL) were developed for the in vivo depletion of phenylalanine (Phe) in circulating blood. A series of experiments was conducted with a large animal model in order to explore its safety for clinical use. The level of red blood cells, white blood cells and platelets did not change during a 2-hr application of the reactors in anesthetized, heparinized dogs and monkeys with experimental hyperphenylalaninemia. No increase in blood urea nitrogen was observed due to generation of ammonia from PAL-catalyzed Phe breakdown. The other metabolic product, trans-cinnamic acid, was reported to be nontoxic. Repeated application of the PAL-reactors to the same animals did not produce untoward physiological or immunological reactions. These data suggest that PAL-reactors may be safe for in vivo use to control excess Phe brought about by fever, infection or pregnancy in phenylketonuric individuals otherwise balanced by a Phe-poor diet. Application of PAL-reactors may serve as a model for extracorporeal enzyme replacement in enzyme-deficiency diseases.

Ammonia-Lyases↗
PubMed1982

Depletion of phenylalanine in the blood of phenylketonuric patients using a PAL-enzyme reactor. An in vitro study.

Blood samples obtained from eight adult phenylketonuric individuals had a mean phenylalanine level of 25 mg/dl. When these samples were circulated through multitubular enzyme-reactors prepared with immobilized phenylalanine ammonia lyase an average of 77% of phenylalanine was metabolized within 30 minutes. We conclude that phenylalanine in human phenylketonuric blood is just as susceptible to metabolism by PAL-enzyme reactors as phenylalanine that is added to normal blood, or that is circulating in dogs and monkeys made hyperphenylalaninemic by experimental means.

Adult↗
PubMed1980

Multitubular reactors with immobilized L-phenylalanine ammonia-lyase for use in extracorporeal shunts.

Hollow fiber cartridges of improved design have been loaded with L-phenylalanine ammonia-lyase [EC 4.3.1.5.] from Rhodotorula glutinis in order to prepare multitubular enzyme reactors for removal of L-phenylalanine from blood. The kinetics of the enzyme in free solution was investigated and the results showed a behavior similar to that reported for L-phenylalanine ammonia-lyase from other sources. On the other hand kinetic data with the reactors were obtained in a recirculating system by measuring the increase in trans-cinnamate concentration in the perfusate. The effect of changing the enzyme load in the hollow fiber cartridge, the length of the cartridge, the substrate concentration, pH as well as the flow rate and the temperature have been investigated. The results showed that the overall kinetics of the enzymic reaction in the multitubular reactor is not the same as that in free solution and the difference can be attributed to diffusion resistances in the hollow fiber system.

Ammonia-Lyases↗
PubMed1978

Immobilized carboxypeptidase G1 in methotrexate removal.

Carboxypeptidase G1, and enzyme capable of cleaving the glutamate moiety from a variety of folate analogs, has been immobilized on nylon tubes and hollow fibers for use in extracorporeal enzyme reactors for methotrexate (MTX) removal from blood. The stability and reactor parameters of the system have been investigated with the use of single tubes and a multitubular arrangement. The results are used to predict their MTX-removing capacity at flow rates and MTX concentrations of clinical interest. In vivo experiments with dogs demonstrate the potential applications of such extracorporeal shunts in "rescue" after administration of large doses of MTX. For dogs a carboxypeptidase G1 reactor with a clearance value of about 150 ml/min would be required in such application.

Animals↗