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At least 451 records · Page 25

Electrophoresis and isoelectric focusing of whole cell and membrane proteins from the extremely halophilic archaebacteria

The subunits from two purified halobacterial membrane enzymes (ATPase and nitrate reductase) behaved differently with respect to isoelectric focusing, silver staining and interaction with ampholytes. Differential behavior was also observed in whole cell proteins from Halobacterium saccharovorum regarding resolution in two-dimensional gels and silver staining. It is proposed that these differences reflect the existence of two classes of halobacterial proteins.

Stan-Lotter, Helga↗

The origin of polynucleotide-directed protein synthesis

If protein synthesis evolved in an RNA world it was probably preceded by simpler processes by means of which interaction with amino acids conferred selective advantage on replicating RNA molecules. It is suggested that at first the simple attachment of amino acids to the 2'(3') termini of RNA templates favored initiation of replication at the end of the template rather than at internal positions. The second stage in the evolution of protein synthesis would probably have been the association of pairs of charged RNA adaptors in such a way as to favor noncoded formation of peptides. Only after this process had become efficient could coded synthesis have begun.

Orgel, Leslie E.↗

Electrostatic Stabilization Of Growing Protein Crystals

Proposed technique produces large crystals in compact, economical apparatus. Report presents concept for supporting protein crystals during growth in microgravity. Yields crystals larger and more-nearly perfect than those grown on Earth. Combines best features of sandwich-drop and electrostatic-levitation methods of support. Drop of protein solution inserted between pair of glass or plastic plates, as in sandwich-drop-support method. Electrostatically charged ring confines drop laterally and shapes it, as in electrostatic technique. Apparatus also made to accommodate several drops simultaneously between same pair of supporting plates. Drops can be inserted and crystals removed through ducts in plates.

Shlichta, Paul J.↗

Integrated Protein-Crystal-Growing Apparatus

Proposed apparatus for research on growth of protein crystals dispenses drops of protein and precipitating solutions, provides controlled environment for crystalization, and stores crystals. Intended for use in microgravity of outer space, concept of apparatus also useful in design of self-contained terrestrial experiments for remote and/or automatic execution.

Rhodes, Percy H.↗

Definition study for temperature control in advanced protein crystal growth

Some of the technical requirements for an expedient application of temperature control to advanced protein crystal growth activities are defined. Lysozome was used to study the effects of temperature ramping and temperature gradients for nucleation/dissolution and consecutive growth of sizable crystals and, to determine a prototype temperature program. The solubility study was conducted using equine serum albumin (ESA) which is an extremely stable, clinically important protein due to its capability to bind and transport many different small ions and molecules.

Nyce, Thomas A.↗

Stretch-induced prostaglandins and protein turnover in cultured skeletal muscle

The purpose of the study is to determine whether mechanical stimulation of cultured muscle cells influences prostaglandin efflux rates and whether they are related to stretch-induced alterations in protein turnover rates. The materials and methods of the experiment, including cell cultures, mechanical stimulation, protein synthesis, and degradation assays are outlined, and emphasis is placed on the effect of short-term mechanical stimulation in basal medium prostaglandin efflux from cultured skeletal muscle and stretch-induced alterations in prostaglandins efflux in complete medium. The major finding of the study is that mechanical stimulation of tissue-cultured skeletal-muscle cells under conditions inducing skeletal-muscle hypertropy increases the efflux of PGE(2) and PGE(2-alpha) but not 6-keto-PGF(1-alpha), the prostacyclin product.

Vandenburgh, Herman H.↗

Protein solubilities determined by a rapid technique and modification of that technique to a micro-method

A simple, rapid method for determination of protein solubilities has been developed which is based upon maximization of the free solution volume to be brought into equilibrium. The tetragonal lysozome solubility diagram has been determined from pH 4.0 to 5.2 (0.1 M sodium acetate), 2-7 percent NaCl, 3-25 C, and portions of the orthorhombic solubility diagram using this technique. Both tetragonal and orthorhombic solubilities were found to increase smoothly with decreasing salt concentration and increasing temperature; no retrograde solubilities were observed. Using column volumes of 75, 300, and 900 microliters, identical tetragonal lysozyme solubility diagrams were obtained. Chymotrypsinogen solubilities have also been determined using this apparatus, being retrograde over the temperature range tested. It is noted that the primary limiting factor in reducing the crystalline volume is the minimum solution sample size needed to accurately quantitate the protein.

Cacioppo, Elizabeth↗

The effect of microgravity on protein crystal growth

Based on the results of microgravity crystallization experiments using the protein canavalin aboard four separate U.S. Space Shuttle missions, visual observations and diffraction data are presented that support the contention that protein crystals of improved quality can be obtained in a microgravity environment. With canavalin, no significant increase in resolution was noted, but an overall improvement in diffraction quality, as judged by statistical analyses of the data, was clear. This improvement in quality may be due primarily to the elimination of defects and dislocations rather than an overall enhancement of order. The mechanism for this improvement may be microgravity-stabilized depletion zones that develop around growing crystals that establish and maintain optimal growth conditions more rapidly following nucleation. Such zones would be destroyed by convective flow effects in earth's gravity.

Mcpherson, Alexander↗

Protein crystal growth in low gravity

The objective of this research is to study the effect of low gravity on the growth of protein crystals and those parameters which will affect growth and crystal quality. The application of graphoepitaxy (artificial epitaxy) to proteins is detailed. The development of a method for the control of nucleation is discussed. The factor affecting the morphology of isocitrate lyase crystals is presented.

Feigelson, Robert S.↗

FNAS/advanced protein crystal growth

A scintillation method is presented for determination of the temperature dependence of the solubility, S(T), of proteins in 50-100 micro-l volumes of solution. S(T) data for lysozyme and horse serum albumin were obtained for various combinations of pH and precipitant concentrations. The resulting kinetics and equilibrium information was used for dynamic control, that is the separation of nucleation and growth stages in protein crystallization. Individual lysozyme and horse serum albumin crystals were grown in 15-20 micro-l solution volumes contained in x-ray capillaries.

Rosenberger, Franz↗

Protein crystal growth tray assembly

A protein crystal growth tray assembly includes a tray that has a plurality of individual crystal growth chambers. Each chamber has a movable pedestal which carries a protein crystal growth compartment at an upper end. The several pedestals for each tray assembly are ganged together for concurrent movement so that the solutions in the various pedestal growth compartments can be separated from the solutions in the tray's growth chambers until the experiment is to be activated.

Carter, Daniel C.↗

Thermophysical properties of lysozyme (protein) solutions

Thermophysical properties of protein solutions composed of the lysozyme crystals with a 0.1 M sodium acetate and 5 percent NaCl solution as the buffer (pH = 4.0) are determined. The properties being measured include specific heat, thermal conductivity, dynamic viscosity, and surface tension. The protein concentrations are varied. Thermal diffusivity is calculated using the measured results. The purpose of the research is to measure thermophysical properties of lysozyme solutions which would serve as the data bank for controlling and modeling the crystal growth process on earth as well as in space.

Liu, Jiaching↗

Micro-apparatus for rapid determinations of protein solubilities

We have developed a column-based micro-technique for rapid determinations of protein solubilities. While retaining a large crystal surface area, the column dead volume has been reduced to equal to or less than 5 micro liters. The technique was tested with tetragonal lysozyme (pH 4.5, 0.1 M acetate, 3.0 percent NaCl, 5-25 C) and column volumes of about 60, 300, and 900 micro liters. Identical solubility data were obtained, indicating that equilibration was obtained even in the smallest columns. In addition, solubility data for Br- and I- salts of lysozyme (pH 4.5, 0.1 M acetate buffer, 0.5 M salt concentrations) were obtained. It appears that the technique can be further miniaturized. The limit in further reducing the crystalline column volume is determined by the minimum solution sample size needed to determine the protein concentration.

Pusey, Marc L.↗

Selection of dietary protein and carbohydrate by rats: Changes with maturation

Weaning (21-day-old; 40-50 g) male rats given simultaneous access to foods, containing 18 percent casein and 15 or 70 percent carbohydrate (dextrin), tended to consume only 29-35 percent as much protein as carbohydrate (i.e., protein/carbohydrate ratios were 0.29-0.35). With maturation, when animals weighed 100 g or more, about half continued this pattern of nutrient choice, but the others abruptly began to consume considerably larger proportions of protein, exhibiting protein/carbohydrate ratios as high as 0.80-1.00. Each adult animal's protein/carbohydrate ratio tended to vary only slightly (s.e. = 3 percent of means). Adult protein/carbohydrate ratios were not correlated with fasting brain 5-HT or 5-HIAA levels. These marked differences among rats in eating behavior would not be observed when--as is usually the case--animals are given access to only one diet.

Yokogoshi, Hidehiko↗

Breadboard activities for advanced protein crystal growth

The proposed work entails the design, assembly, testing, and delivery of a turn-key system for the semi-automated determination of protein solubilities as a function of temperature. The system will utilize optical scintillation as a means of detecting and monitoring nucleation and crystallite growth during temperature lowering (or raising, with retrograde solubility systems). The deliverables of this contract are: (1) turn-key scintillation system for the semi-automatic determination of protein solubilities as a function of temperature, (2) instructions and software package for the operation of the scintillation system, and (3) one semi-annual and one final report including the test results obtained for ovostatin with the above scintillation system.

Rosenberger, Franz↗

Convective flow effects on protein crystal growth

The experimental setup for the in-situ high resolution optical monitoring of protein crystal growth/dissolution morphologies was substantially improved. By augmenting the observation system with a temperature-controlled enclosure, laser illumination for the interferometric microscope, and software for pixel by pixel light intensity recording, a height resolution of about two unit cells for lysozyme can now be obtained. The repartitioning of Na(+) and Cl(-) ions between lysozyme solutions and crystals was studied. Quite unexpectedly, it was found that the longer crystals were in contact with their solution, the lower was their ion content. The development of a model for diffusive-convective transport and resulting distribution of the growth rate on facets was completed. Results obtained for a realistic growth cell geometry show interesting differences between 'growth runs' at 1g and 0g. The kinematic viscosity of lysozyme solutions of various supersaturations and salt concentrations was monitored over time. In contrast to the preliminary finding of other authors, no changes in viscosity were found over four days. The experimental setup for light scattering investigations of aggregation and nucleation in protein solutions was completed, and a computer program for the evaluation of multi-angle light scattering data was acquired.

Rosenberger, Franz↗

Automated protein crystal growth facility

A customer for the protein crystal growth facility fills the specially designed chamber with the correct solutions, fills the syringes with their quenching solutions, and submits the data needed for the proper growth of their crystal. To make sure that the chambers and syringes are filled correctly, a NASA representative may assist the customer. The data needed is the approximate growth time, the growth temperature, and the desired crystal size, but this data can be changed anytime from the ground, if needed. The chambers are gathered and placed into numbered slots in special drawers. Then, data is entered into a computer for each of the chambers. Technicians map out when each chamber's growth should be activated so that all of the chambers have enough time to grow. All of this data is up-linked to the space station when the previous growth session is over. Anti-vibrational containers need to be constructed for the high forces encountered during the lift off and the landing of the space shuttle, and though our team has not designed these containers, we do not feel that there is any reason why a suitable one could not be made. When the shuttle reaches the space station, an astronaut removes a drawer of quenched chambers from the growth facility and inserts a drawer of new chambers. All twelve of the drawers can be replaced in this fashion. The optical disks can also be removed this way. The old drawers are stored for the trip back to earth. Once inside the growth facility, a chamber is removed by the robot and placed in one of 144 active sites at a time previously picked by a technician. Growth begins when the chamber is inserted into an active site. Then, the sensing system starts to determine the size of the protein crystal. All during the crystal's growth, the customer can view the crystal and read all of the crystal's data, such as growth rate and crystal size. When the sensing system determines that the crystal has reached the predetermined size, the robot is told to pick up a syringe filled with the correct quenchant solution and inject it into the chamber to stop the crystal growth. The chamber is then removed from the active site and placed into its original storage slot. Another chamber is then placed into the active site and the process is repeated in all of the active sites until all of the chambers have complted their growth. After ninety days (the scheduled time between shuttle visits), the crystal growth is completed, and the old drawers are replaced with new ones. Once the customer extracts the crystals, the chambers are retrieved for future customers.

Donald, Stacey↗

Protein requirements for long term missions

A key component of the diet for a space mission is protein. This first part of this paper reviews the reasons for emphasizing protein nurtition and then discusses what the requirements are likely to be. The second part discusses potential advantages of modifying these requirements and describes potential potential approaches to effecting these modificatons based on well established ground based models.

Stein, T. P.↗