A simple method for the isolation and purification of total lipides from animal tissues.

Lipid decomposition studies in frozen fish have led to the development of a simple and rapid method for the extraction and purification of lipids from biological materials. The entire procedure can...

A rapid method of total lipid extraction and purification.

Publisher Summary Studies of the cell surface binding, internalization, and metabolism of low-density lipoprotein (LDL) in cultured cells have provided useful information regarding the general aspects of receptor-mediated endocytosis. The study of the LDL receptor has been facilitated by analysis of mutant fibroblasts obtained from human subjects with disorders of cholesterol metabolism. The most informative cells, obtained from patients with familial hypercholesterolemia (FH), have defects in the gene encoding the LDL receptor. The existence of three classes of mutant alleles at the LDL receptor locus has been deduced on the basis of genetic and kinetic data. One of these alleles specifies a receptor that is unable to bind LDL. The second type of allele specifies a receptor that can bind small amounts of LDL; and the third type of allele specifies a receptor that can bind LDL but cannot be incorporated into coated pits and hence cannot carry the LDL into the cell. The first two alleles are common among FH patients, whereas the third allele is extremely rare.

Receptor-mediated endocytosis of low-density lipoprotein in cultured cells

Physiological and pathophysiological roles of excitatory amino acids during central nervous system development

Publisher Summary This chapter discusses the chemical analysis of microbial cells. The preparation of material for analysis is discussed, because changes in the chemical composition of cells may occur as a result of the washing and storage conditions used. Wet- and dry-weight determinations of bacterial samples and assay of total cell numbers are described, because analytical results must refer to one or other of these values. Selection of an analytical procedure is a subjective process, because the number of suitable methods is large and each will have different merits and defects. Primary considerations are sensitivity, specificity, reproducibility, and absolute accuracy. Automatic methods for performing biochemical analyses, already widely accepted in hospitals and in industry, are beginning to make their way into the research laboratory. All automatic analyzers developed so far may be classified as either “continuous-flow” or “discrete” types. All of them use colorimetric methods exclusively and contain some form of automatic colorimeter for final read-out. The first and best-known is the Technicon “AutoAnalyzer,” which is of the continuous-flow type.

Chapter III Chemical Analysis of Microbial Cells

Preparation of lipide extracts from brain tissue.

INTRODUCTION IT has been well established that meso-inositol exists in brain in both free and combined states. More than fifty years ago, THUDICHUM (1901) reported the presence of free inositol, and this observation has since been amply confirmed. More recent work (FOLCH and WOOLLEY, 1942) demonstrated the OcCuITence of inositol combined in brain lipides, and resulted in the isolation from brain of diphosphoinositide (FOLCH, 1949), a phosphatide which contains inositol metadiphosphate as a constituent. Results obtained in this laboratory in the course of work on the nature of neurokeratin have established the presence in brain tissue of a second form of combined inositol, namely, that of a phosphoinositide combined to protein. This paper reports these findings and some other observations bearing on the nature of neurokeratin. Preliminary reports of this work have already appeared (FOLCH and LEBARON, 195 1 , 1953). Neurokeratin is a fraction of brain proteins first isolated by EWALD and K u m (1877) which can be summarily described as a proteolytic enzyme-resistant protein residue obtained primarily from nerve tissue myelin, and which has traditionally been considered to be associated with the protein network of the myelin sheath. The classical procedure for preparing neurokeratin consists essentially of two major operations; namely, the quantitative removal of lipides with organic solvents, and the subsequent treatment of the proteins thus obtained, with digestive enzymes. The procedure takes several months, and involves Such drastic steps as continuous extraction with hot ethanol for four weeks, digestion with pepsin in dilute hydrochloric acid for three weeks, and, in some of its versions, treatment with hot alkali. The product is a protein residue insoluble in water and in dilute acids and bases, rich in sulphur, and resistant to the action of proteolytic enzymes. The preparations obtained by various workers differed somewhat in composition; and BLOCK (1951) concluded that neurokeratin is most likely an artifact, derived from a definite group of substances specific to nervous tissue. The work which led to the isolation of the protein fraction containing combined inositol was an attempt to develop a method of preparation of neurokeratin less

The isolation from brain tissue of a trypsin‐resistant protein fraction containing combined inositol, and its relation to neurokeratin*

A NUMBER of papers have recently appeared describing studies on various types of aqueous extracts of brain proteins, particularly with relation to fractionation of such extracts by electrophoresis. In these studies, the rate of extraction of proteins by physiological saline has been investigated (NAKAMURA, HAYASHI and TANAKA, 1954; HAYASHI, 1953) and the effect of the pH of the extracting solution on the subsequent electrophoretic patterns has been determined (KEUP, 1955; POLYAKOVA and GOTOVTSEVA, 1957). However, to our knowledge, no systematic study has been undertaken to determine the conditions necessary for extraction of the maximum amount of protein material from brain in aqueous medium. In connexion with our work on brain proteins and lipoproteins, these data were requisite for the development of satisfactory fractionation methods, so a survey of the effect of p H and ionic strength on the amount of material extracted in aqueous solution was carried out. To this end, we prepared extracts at pH’s from 4 to 11-6 and ionic strengths from 0 to 5 and determined the amounts of nitrogen and phosphorus which could be precipitated by trichloracetic acid from these extracts. The results of these experiments are reported in this paper as well as some chemical analyses and the results of fractionation studies on the extracts thus obtained. A preliminary report of this work has been published previously (LEBARON and FOLCH, 1958). The main reason why these data had not been obtained previously for brain tissue is that the high proportion and variety of lipids found in this tissue are bound to each other and to proteins in various types of complexes which easily form emulsions and suspensions in aqueous media that are difficult to resolve. We have found that these mixtures can be separated by the use of a relatively high centrifugal force, and waterclear extracts can be thus obtained. Using the method detailed below, maximum extraction occurs when the extracting solution has an ionic strength of from 2.5 to 3 and when the pH of the homogenate is between 6 and 9. The p H of homogenates made with unbuffered salt solutions is within the optimum range. About 20 per cent of white matter protein and 50 per cent of grey matter protein can thus be obtained in aqueous solution. Such extracts have been separated by dialysis into globulin and albumin fractions, the former containing about 25 per cent lipid, of which about onefifth is cholesterol.

The effect of pH and salt concentration on aqueous extraction of brain proteins and lipoproteins.

MANY investigators have studied the nature of brain proteins which are extracted with aqueous solvents in the region of neutral pH, but relatively few have investigated brain proteins extracted with dilute aqueous acid. The latter studies have been limited almost exclusively to basic proteins capable of inducing experimental allergic encephalomyelitis (EAE) and have resulted in the isolation of reasonably pure basic proteins (or polypeptides) having high EAE-inducing activity (EINSTEIN, ROBERTSON, DICAPRIO and MOORE, 1962; KIBLER, Fox and SHAPIRA, 1964; b s , 1965~; WOLFGRAM, 1965; CASPARY and FIELD, 1965; NAKAQ and ROBOZ-EINSTEIN, 1965). In work reported by HONEGGER (1965) the nature of the relatively acidic proteins extracted by dilute aqueous acid was investigated to a limited extent by means of gel filtration and starch gel electrophoresis. The present studies were undertaken to examine the constituents of a ‘viscous protein’ fraction iirst prepared by LEBARON and ROTHLEDER (1960). According to the method used by these authors for preparing phosphoinositide-protein complexes (phosphatidopeptides) from bovine brain white matter, the tissue was first depleted of a considerable amount of its protein and most of its lipid by successive treatments with CHC1,: CH,OH (2: 1, v/v), H,O, and 0.03 N-HCI. The HC1 extract, which appeared rather viscous, was accordingly designated by LEBARON and ROTHLEDER as the ‘viscous protein’ fraction. Attempts to fractionate the acid-extractable proteins comprising the ‘viscous protein’ fraction of bovine white matter led to a method whereby a glycine-rich basic protein can be readily obtained both in reasonable purity and high yield. This basic protein is highly active in inducing EAE in guinea pigs (KIES, ALVORD, MARTENSON and LEBARON, 1966). In addition, the procedure permits the preparation of a fraction of large molecular-size proteins rich in glutamic and aspartic acids. An abstract of this work has appeared elsewhere (MARTENSON and LEBARON, 1965).

F. N. LeBaron

Papers

Preparation of lipide extracts from brain tissue.

The isolation from brain tissue of a trypsin‐resistant protein fraction containing combined inositol, and its relation to neurokeratin*

The effect of pH and salt concentration on aqueous extraction of brain proteins and lipoproteins.

Studies on the acid‐extractable proteins of bovine brain white matter

Chemistry of myelin development.