James W. Drysdale

Isoelectric focusing in gels

Abstract This review deals primarily with analytical and preparative isoelectric focusing (IEF) in gel media. We have tried to cover new developments over the years 1972, 1973 and early 1974 since the previous years have already been reviewed. An introductory chapter on the properties and detection of Ampholines is followedby a section on analytical and preparative apparatus for gel IEF, with particular emphasis on thin-layer techniques and on continous-flow IEF. A chapter on sample detection deals with techniques for staining and destaining and for histochemical enzyme detection. New developments in pH measurements, such as the flat-membrane electrode and the antimony microelectrode, are reported. Among the various applications of IEF to the study of biological systems we report the analysis of glycoproteins, immunoglobulins, lipoprotein, membrane proteins, peptides and metalloproteins. Particular emphasis has been given to the use of IEF as a probe of interacting protein systems. Examples are given of studies on sub-unit exchange and ligand binding in haemoglobin. IEF can be effectively used in combination with other techniques for two-dimensional procedures. An IEF run in the first dimension can be followed by an immunodiffusion or immunoelectrophoresis, or by gel electrophoresis, or by electrophoresis in a gel gradient, or by SDS gel electrophoresis. Combined use of these methods will often define in a few simple experiments, several physico-chemical parameters of the proteins under study. The review ends with a chapter on transient state isoelectric focusing and with remarks with on future trends and developments.

Isoelectric points of proteins: A table

We have prepared a table of isoelectric points (pZ) of proteins selected from the literature. Initially, we retrieved a computerized bibliography from the MEDLARS data base covering the time period of January 1970-March 1976. This search, supplemented by an additional search of Biological Abstracts, produced about 3000 citations. From this bibliography we initially screened about 1000 papers and selected 400 for the table. These papers were chosen to be representative rather than comprehensive. Data obtained were collected according to the following scheme: 1. Only named proteins are recorded. Classes of proteins (ribosomal, membrane, etc.) are not included. 2. Only pZ’s obtained by isoelectric focusing in the presence of carrier ampholytes are included in the table. These values are taken directly from the text and have not been normalized for effects of temperature or other factors that might affect the apparent pZ. All values are rounded off to one decimal place. 3. pZ values were determined under nondenaturing conditions except where indicated (** denotes values obtained by isoelectric focusing in the presence of urea). pi’s for protein subunits have not been included. 4. In cases where there are several reports for the pZ of a given protein, we have selected only one of several references. This was usually the most accessible reference. 5. When only one or two major forms were present, those values are listed. With multiple forms, the pZ range is given. In a pZ range with one predominant component, that pZ is listed first and underlined, e.g., 5.7, 5.5-6.0 represents multiple forms with pi’s between 5.5 and 6.0, with the major component at pZ 5.7.

Human Isoferritins: Organ Specific Iron and Apoferritin Distribution

Summary. Ferritins from human liver, spleen, heart, pancreas and kidney were compared by electrophoresis and isoelectric focusing in polyacrylamide gels, by immunodiffusion against antisera to homologous and heterologous ferritin, and in some cases by their cyanogen bromide peptides. All ferritins appeared to consist of a single species on gel electrophoresis with the exception of heart ferritin which separated into two major components. Small differences in electrophoretic mobility were found in all tissue ferritins. By contrast, all tissue ferritins were found to consist of multiple forms when analysed by gel electrofocusing. At least five isoferritins were found in most tissues, several of which were common to most tissues. At least two were common to all tissues. Those ferritins which were most easily distinguishable electrophoretically, e.g. spleen and heart ferritin, showed the greatest differences on gel electrofocusing. The ferritin profile was characteristic of each organ and was reproducible both within individuals and between individual tissues. There were striking differences in the iron content of the various isoferritins within a tissue. Further, the iron content of isoferritins common to more than one tissue varied with the tissue of origin. Some isoferritins in several organs and all of the isoferritins in pancreas appeared to contain little, if any, iron. All five tissue ferritins contained antigenic determinants in common with liver ferritin. However, an additional antigenic determinant was found in liver ferritin which was not detectable in the ferritins from the other organs.

Human ferritin gene expression

Publisher Summary This chapter discusses human ferritin gene expression. Ferritin molecules consist of an approximately spherical shell of 24 subunits surrounding a variable core of an iron-oxide-phosphate complex. The variable iron content does not appreciably alter the surface charge of the protein. However, the molecules of different surface charge, called “isoferritins,” are readily displayed by a variety of electrophoretic or chromatographic procedures. These are hybrid molecules containing different proportions of two subunit types, H and L, with M r s of about 21,000 and 19,000, respectively. Isoferritins differ metabolically and perhaps functionally. L-rich ferritins predominate in tissues that store substantial amounts of iron such as liver, spleen, and bone marrow. H-rich ferritins are found in organs with no major iron storage function, such as heart, pancreas, and kidney, and in rapidly proliferating tissues. The large differences in ferritin concentrations and in the relative amounts of H and L subunits in different tissues are determined primarily by differential gene transcription. This seems the case in malignant and nonmalignant lymphocytes, where large differences in the amounts of ferritin and in the relative amounts of H and L subunits correspond closely to differences in the levels of H and L mRNAs. Other evidence for transcriptional control comes from studies with HL60 cells, a promyelocytic leukemia cell line.

Human ferritin H and L sequences lie on ten different chromosomes

SummaryIn humans, the H (heavy) and L (light) chains of the iron-storage protein ferritin, are derived from multigene families. We have examined the chromosomal distribution of these H and L sequences by Southern analysis of hybrid cell DNA and by chrosomal in situ hybridization. Our results show that human ferritin H genes and related sequences are found on at least seven different chromosomes while L genes and related sequences are on at least three different chromosomes. Further, we have mapped the chromosomal location of expressed genes for human H and L ferritin chains and have found an H sequence which may be a useful marker for idiopathic hemochromatosis.

Recent staining artifacts with LKB “Ampholines” on gel isoelectric-focusing

Abstract Certain batches of LKB “Ampholines” gave multiple bands when stained after gel isoelectric-focusing with commonly used direct staining procedures. The stainable species are most prominent in the pH range 6–9. By contrast, ampholytes prepared in the laboratory gave no stainable species in this pH range.

In vitro stimulation of apoferritin synthesis by iron.

The apparent induction of apoferritin synthesis by iron has been examined in cell-free systems from rat and rabbit liver. Both systems allowed the complete synthesis de novo of apoferritin isolated by chromatographic or immunological means. Addition of iron at levels of 0.2--1 mM specifically stimulated incorporation of radioactive amino acids into apoferritin purified after classical heat extraction. The effect was also observed when iron was added at the end of the incubation period in the absence of continuing protein synthesis. Further, iron addition had no effect on the amount of newly synthesised apoferritin subunits as estimated by direct immunological precipitation from the reaction mixture. These results suggest that iron acts at some stage subsequent to translation in stimulating apoferritin biosynthesis.