The distributions of some granule-associated enzymes in guinea-pig polymorphonuclear leucocytes
TL;DR: In this paper, homogenates of guinea-pig polymorphonuclear leucocytes were separated by differential centrifugation into six particulate fractions and a soluble fraction.
Abstract: 1. Homogenates of guinea-pig polymorphonuclear leucocytes were separated by differential centrifugation into six particulate fractions and a soluble fraction. 2. The distributions in these fractions of protein, DNA, succinate dehydrogenase, beta-glucuronidase, peroxidase, alkaline phosphatase, acid phosphatase (against p-nitrophenyl phosphate and beta-glycerophosphate), cathepsin, and catalase were compared. 3. Almost all of the DNA sedimented in the first two pellets, indicating that the nuclei were relatively intact. 4. The four hydrolases and peroxidase showed different distribution patterns, although these activities were previously reported to be localized mainly in the single ;granule' fraction isolated from leucocytes. 5. The particles containing peroxidase, acid phosphatase and alkaline phosphatase all exhibited latency. Maximum activity for each enzyme was obtained at roughly similar concentrations of Triton X-100. 6. The acid phosphatase of these cells was distributed between two populations of particles that differed in both sedimentation characteristics and density. The acid phosphatase(s) of the two populations showed slightly different substrate specificities. This bimodal distribution was not an artifact of the procedure used to elicit the cells. 7. Catalase was recovered almost entirely in the soluble fraction and showed no latency in freshly prepared homogenates. No urate oxidase was detected. 8. We conclude that the ;granule' fraction of the polymorphonuclear leucocyte, as isolated by previous workers, contains at least three, probably more, populations of particles with different enzyme contents, and that these cells probably do not contain peroxisomes.
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TL;DR: By isolating plasma membranes from the proper cell types, it should be possible to design experiments with more direct bearing on the functions of this organelle, and most studies on plasma membranes are concerned simply with their chemical and enzymatic compositions.
Abstract: The plasma membrane surrounding living cells \"plays a role in intracellular metabolism, but what distinguishes this structure from other organelles is that the plasma membrane also mediaces interactions between the cell and its external environment. The simplest of these interactions maintain a desirable intracellular milieu by bringing in needed substances and getting rid of waste products. The plasma membrane acts as a passive diffusion barrier to charged and large molecules, and in addition carries out facilitated diffusion, active transport, endocytosls (pinocytosis and phagocytosis), and exocytosis. Further, this membrane is involved in locomotion and chemotaxis, processes which could remove a cell from noxious surroundings or to a more nutritive medium. On a more complex level, cells must communicate with other cells; this is especially important in the formation and maintenance of multicellular organisms. The plasma membrane functions in the secretion and reception of hormones, conduction of nerve impulses, and direct cellular interactions such as adhesion and contact inhibition. Finally, on yet another level the plasma membrane riaay be involved in such sophisticated processes as immunological defense and information storage and retrieval. The \"isolate-and-characterize\" approach was first applied to plasma membranes around 1960. Unfortunately, our present state of knowledge in this field is not as great as might have been anticipated at that time. This slow progress is partially due to the considerable complexities inherent in working with membranes of any sort; but there are other factors as well. For example, many investigators have not been rigorous enough in their choice of plasma membrane markers and have not thoroughly analyzed their plasma membrane fractions for contamination by other subcellular particles; consequently, the true identity of many so-called plasma membrane preparations is uncertain. The vast majority of plasma membrane isolations have been preparative rather than analytical. Although the preparative approach is potentially a very valuable short-cut to an understanding of the entity being isolated, it can also lead to conclusions that are wrong unless really rigorous criteria are applied. Finally, most studies on plasma membranes are concerned simply with their chemical and enzymatic compositions; by isolating plasma membranes from the proper cell types, it should be possible to design experiments with more direct bearing on the functions of this organelle. Isolation of plasma membranes may conveniently be divided into five sequential steps--choice of the tissue to be used, selection of markers, disruption of the tissue, fractionation, and analysis. The following discussion combines some general comments about each of these steps with a review of the literature. The literature review extends through November 1971 and is limited to studies with mammalian cells. Of course, the information derived from mammalian cells should ultimately
402 citations
TL;DR: A suspension of intact guinea pig polymorphonuclear leukocytes hydrolyzed added ATP, AMP, and p-nitrophenyl phosphate under physiologically appropriate conditions, leaving open the possibility that an ecto-enzyme is the only protein in polymorphon nuclear leukocyte capable of hydrolyzing AMP.
342 citations
TL;DR: Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60 and this model is a promising model for myeloid development.
Abstract: Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.
291 citations
TL;DR: Gel electrophoresis revealed that a protein with a molecular weight of 110,000 daltons present in the particulate fraction of normal neutrophils was absent from the patient's cells, and that its levels were below normal in cells from his mother and sister but normal in neutrophil from his father and brother.
Abstract: Neutrophils from a five-year-old boy with recurrent bacterial infections failed to spread on surfaces, leading to a severe defect in chemotaxis and a mild impairment in phagocytosis. Failu...
288 citations
TL;DR: Polymorphonuclear leukocytes, one of the main sources of enzymes responsible for tissue damage in inflammatory processes, store all four classes of tissue proteinases, carboxyl, thiol and serine proteinase in the azurophil granules, and metallo proteinases in the specific granules.
Abstract: Polymorphonuclear leukocytes (PMNs) are one of the main sources of enzymes responsible for tissue damage in inflammatory processes. These enzymes are stored in two types of cytoplasmic granules. Azurophil granules contain lysosomal hydrolases, neutral serine proteinases, and bactericidal elements (myeloperoxidase and lysozyme). Specific granules contain collagenase, lysozyme and lactoferrin but lack lysosomal hydrolases. PMNs store all four classes of tissue proteinases, carboxyl, thiol and serine proteinases in the azurophil granules, and metallo proteinases in the specific granules. Three serine proteinases have been identified, elastase, cathepsin G and a third enzyme, which together account for a large proportion of the protein of the azurophil granules. In the course of phagocytic events, all these enzymes are released extracellularly. The neutral proteinases degrade proteoglycans and collagen. In vitro, they stimulate B-lymphocytes, which suggests that they may have immuno-potentiating activity when they are released at sites of chronic inflammation.
229 citations
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TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
289,852 citations
TL;DR: The present study arose from the observation that a more intense colour was sometimes produced if, instead of being heated at 1000 for 10 min., the reaction mixture was allowed to stand overnight at room temperature.
Abstract: Of the colour reactions available for the determination and identification of deoxyribonucleic acid (DNA), the reaction with diphenylamine in a mixture of acetic and sulphuric acids at 1000 (Dische, 1930) has been perhaps the most widely used. The present study arose from the observation that a more intense colour was sometimes produced if, instead of being heated at 1000 for 10 min., the reaction mixture was allowed to stand overnight at room temperature. As a result of this observation the procedure has been modified, principally by adding acetaldehyde to the reagents and by allowing the solution to stand for about 17 hr. at 30° instead of heating it at 1000. The modified method is 3-5 times as sensitive as Dische's original procedure, and several substances which interfere in the original method do not do so in the modified procedure. Some observations on the mechanism of the reaction have been made; in particular it was discovered that there is a liberation of inorganic orthophosphate from DNA during the early stages of the reaction. This finding has a bearing on the structure of DNA. The modified method has already been used in an investigation of nucleic acid metabolism during bacteriophage multiplication (Burton, 1955).
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TL;DR: The discovery of a muscular dystrophy in a strain of mice has facilitated the search for biochemical alterations in myopathy and it seems probable that many, at least, of the secondary biochemical changes may be common to various types of muscle disease.
Abstract: Little is known about biochemical abnormalities which accompany or are responsible for the morphological changes which occur in the primary myopathies. A number of such diseases have been characterized in humans (Walton & Nattrass, 1954). The discovery (Michelson, Russell & Harman, 1955) of a muscular dystrophy in a strain of mice, inherited by an autosomal recessive gene, has facilitated the search for biochemical alterations in myopathy. Although this condition may not be identical with any of the types of human muscular dystrophy, its investigation may help to throw light on the biochemistry ofhuman muscular dystrophies. It seems probable that many, at least, of the secondary biochemical changes may be common to various types of muscle disease. A few workers (e.g. Weinstock, Epstein & Milhorat, 1958; Hazzard & Leonard, 1959; White, 1959) have reported altered concentrations of certain muscle enzymes in the mouse myopathy. In view of reported morphological abnormalities in mitochondria of dystrophic mouse muscle (Dr G. W. Pearce, unpublished work; Ross, Pappas & Har-
980 citations