Centrifugal Countercurrent Distribution Research Articles

Revealing surface changes associated with maturation of ram spermatozoa by centrifugal counter-current distribution in an aqueous two-phase system

Centrifugal counter-current distribution (CCCD) in an aqueous two-phase system was used to detect changes associated with maturation of ejaculated ram spermatozao. Spermatozoa obtained form three successive ejaculates of rams maintained in abstinence for one, two and three days were fractionated by CCCD. The results show that these ejaculates are relatively enriched in a cell population which presents a very high enhanced affinity to the lower dextran-rich phase. This cell population is not associated with loss of acrosomal integrity. In addition, it tends to disappear with longer abstinence periods, or after successive ejaculations at the same abstinence period, strongly suggesting that it is composed of immature cells. Therefore, phase partitioning can detect surface changes accompanying sperm maturation and offers a new possibility for sperm quality analysis.

Acquisition of viable-like surface properties of sperm cells by adsorption of seminal plasma proteins revealed by centrifugal countercurrent distribution

By using centrifugal countercurrent distribution in dextran/polyethylene glycol two-phase system, we show that the acquisition of adsorbed seminal plasma proteins by the sperm cell surface modifies the partition behaviour of bull spermatozoa reproducing that of alive cells. Thus, addition of increasing concentrations of proteins from the seminal plasma promoted a saturable enhanced affinity of the cells to the polyethylene glycol-rich phase in which alive cells preferentially partition. On the contrary, treatment with Tween provoked the opposite effect.

Sperm cell heterogeneity revealed by centrifugal counter-current distribution in an aqueous two-phase system

In recent years, partitioning in aqueous two-phase systems has been developed as a mild separation procedure of high selectivity for studying cell surface properties. This work was undertaken to study the surface heterogeneity of bull and ram sperm cells by using centrifugal counter-current distribution in dextran—poly(ethylene glycol) two-phase systems. The revealed heterogeneity appears to be associated with different viability states of the fractionated cells. Various strands of evidence support this conclusion: (a) assessment of viability by fluorescence probes showed a different enrichment of live cells in the different fractions tested, dead spermatozoa showing enhanced affinity to the dextran-rich phase; (b) freezing or thermic shock of sperm cells, known methods of promoting loss of viability, provoked an increase in the number of cells with enhanced affinity to this phase; (c) the same effect was observed when sperm cells were obtained from ejaculates after longer periods of abstinence. The results presented here strongly suggest that loss of semen viability results in a decreased hydrophobicity of the cell surface. Thus this may be suitable for detecting sperm surface changes and to separate spermatozoa populations enriched in some functional and/or structural surface features.

Management of sedimentation in centrifugal counter-current distribution of sperm cells in an aqueous 2-phase system

It is generally assumed that centrifugal counter-current distribution (CCCD) in aqueous two-phase systems cannot be employed for analyzing or fractioning cell populations, due to large particles of sediment in the system caused by enhanced gravity. The present work was undertaken to find out whether addition of Percoll to a two-phase system would be a useful method to avoid this cell sedimentation. The results obtained show that bull spermatozoa partition as a unique peak in a CCCD using a Dextran T500-poly(ethylene glycol) 6000 system, and that sedimentation takes place significantly in the upper phase during the process. Addition of increasing concentrations of Percoll made this unique peak wider and two different populations of bull spermatozoa were finally obtained when Percoll concentration rose to 13.6%. This management of cell sedimentation in CCCD could be of great interest for analyzing cell heterogeneity, since the shortening of the time required for counter-current distribution should prevent the loss of cell viability during the separation process. Finally, the results obtained suggest that an increase of viscosity rather than of density is the phase feature which has greater influence on managing cell sedimentation in CCCD.

Extraction of xanthine oxidase from milk by counter-current distribution in an aqueous two-phase system

By comparing the partitioning of total milk protein with that of pure xanthine oxidase, it was established that a two-phase system containing 7% (w/w) Dextran T 500 and 5% (w/w) polyethylene glycol 6000 in the range of 5–25 m M sodium phosphate is suitable for the extraction of the enzyme from milk. In this system, the bulk of milk protein partitioned in the upper phrase, whereas the pure enzyme showed a preferential affinity for the lower phase. The enzyme appears also to be released from the membrane of the milk fat globules as a consequence of the phase partitioning itself, as the enzyme activity (undetectable in the boud form) increased when successive partitionings of milk were carried out. After 57 transfers, centrifugal counter-current distribution (CCCD) of milk in this two-phase system allowed the separation of the xanthine oxidase from the bulk of milk protein. The enzyme thus isolated was mostly resolved as an unique peak, identified densitometrically after sodium dodecyl sulphate polyacrylamide gel electrophoresis. The results obtained show that CCCD of milk in a two-phase system is an useful procedure to achieve the release and isolation of xanthine oxidase from milk.

Heterogeneity of a crude synaptosomal preparation, studied by affinity partitioning using hexaethonium-poly(ethylene glycol).

The heterogeneity of a synaptosomal preparation was studied by the use of affinity partitioning in combination with centrifugal counter-current distribution. Hexaethonium-poly(ethyleneglycol) was used as the extracting agent. The fractions were analyzed for: light scattering, protein, choline acetyltransferase, L-glutamate decarboxylase, glutamine synthetase, 2',3'-cyclicnucleotide-3'-phosphohydrolase, acetylcholinesterase and succinate dehydrogenase. The material was fractionated into three main fractions which differed in their content of marker-enzymes.

An affinity-ligand gradient technique for purification of enzymes by counter-current distribution

Enzymes related to the glycolysis, present in an extract of baker's yeast, have been fractionated by counter-current distribution using an aqueous dextran—polyethyleneglycol two-phase system containing affinity ligands. To minimize the peak width a gradient of a general affinity ligand (triazine dye) was included in the stationary (liquid) phase. A centrifugal counter-current distribution machine was used to speed up the settling of the aqueous phases after equilibration. With 55 transfer cycles several of the measured enzymes increased 4–15 times in purity. Furthermore, a number of the enzymes showed clear heterogeneity indicating two or more isoenzymic forms. The use of this multistep liquid—liquid extraction in large scale for specific isolation of intracellular enzymes is discussed.

Liquid—liquid extraction of membranes from calf brain using conventional and centrifugal counter-current distribution techniques

Neural membranes isolated from calf brain have been partitioned in aqueous two-phase systems containing dextran and polyethyleneglycol. When the partition was repeated several times, using counter-current distribution technique, the distribution of the membranes between the upper phase and the interface changed in a non-ideal manner and in favour of the interface. By using a centrifugal counter-current distribution device the time for the experiment could be reduced by a factor of 7–8 and the distribution was similar to what could be expected for ideally behaving membranes. The time-dependent change of the membranes is discussed in terms of aggregation and lateral membrane perturbations. Despite this effect a certain fractionation has been achieved as deduced from analysis of cholesterol content, opiate receptor activity and acetylcholinesterase activity along the counter-current distribution row of fractions. Compared to the starting material these activities were enriched some two-fold in certain fractions.