Subcellular distribution of diacylglycerol lipase in rat and mouse brain

Rat Brain has a lipase which hydrolyzes diacylglycerol at an optimal pH of 4.8 (1). The subcellular distribution of this acid diacylglycerol lipase was studied in brain tissue of rats and mice; in the latter case neurological mutants and their normal controls were used. Several other acidic hydrolases were employed as normal controls were used. Several other acidic hydrolases were employed as lysosomal markers. In mouse brain, the specific activity which is about 50-100 times lower than in rat brain, was greatest in the lysosomal fraction. In contrast, no enrichment of DG-lipase was observed in any subcellular fraction of the active enzyme of rat brain. Activities were about equally distributed in the microsomal, myelin-synaptosomal and lysosomal fractions.     

Evidence for NAD nucleosidase in rabbit-liver lysosomes.

A method is described for the isolation of secondary lysosomes from homogenates of rabbit liver; The uptake of Triton WR-1339 by rabbit-liver lysosomes when administered by intraperitoneal injection was used to decrease the density of secondary lysosomes. Lysosomal fractions prepared by this method contain an NAD nucleosidase (NAD glycohydrolase, EC 3;2.25), an enzyme which has previously been considered to be associated with other subcellular fractions. The enzyme has maximum activity at pH 6 and cleaves both NAD and NADP. It is inhibited by nicotinamide (Ki equals 4.5 mM) and by HgCl2. Both nucleosidase and 2'-nucleotidase show in-vitro latency typical of lysosomal acid hydrolases. Rabbit-liver plasma-membrane fractions were isolated which contained most 5'-nucleotidase but relatively little nucleosidase, whereas rabbit liver lysosomes contain both 5'-nucleotidase and nucleosidase enzymes but little adenyl cyclase.     

Acid phosphatase localization in individual neurons by a quantitative histochemical method

—By an adaptation of the fluorometric method of Campbell and Moss (1961), the activity of α-naphthyl acid phosphatase was measured in individual neurons of monkey and human spinal cord and found to be many times higher in nerve cell bodies than in the surrounding neuropil. It was also measured in cerebellar cortex and found most concentrated in the granular (neuronal) layer. As this distribution is distinctive and paralleled by two other acid hydrolases, β-galactosidase and β-glucuronidase, it is considered to offer additional support for the lysosomal concept in nervous tissue and to indicate that nerve cell perikarya are much richer in lysosomes than are axons, dendrites or glial cells.     

Lysosomal enzymes in cells separated from rat testis

Fractions enriched with Sertoli cell (S), germ cells (G), and interstitial cells (I) were separated from rat testis after enzymic treatment and double filtration through nylon meshes. The fractions were analysed for protein content and for enzymic activity of 4 acid hydrolases known to be of lysosomal nature in other tissues. Acid phosphatase activity was preferentially recovered in Fraction G, the highest activity of beta-glucuronidase was found in Fraction I while the activity of aryl sulphatase and beta-N-acetyl-D-glucosaminidase was prominent in Fraction S. With the exception of acid phosphatase, the enzymes were mostly recovered in a subcellular fraction of whole testis homogenate separated between 600 and 27 000 g. The results may reflect the peculiar enzyme composition of the lysosomal apparatus of each cell type.     

Lysosomal phospholipase A activities of rat ovarian tissue.

1.1. Lysosome-enriched fractions were prepared by differential centrifugation of homogenates of luteinized rats ovaries. Acid phospholipase A activities were characterized with [U-14C]diacyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-[9,10-3H]- or [1-14C]oleoyl-sn-glycero-3-phosphocholine as substrates. Acid phospholipase A1 activity had properties similar to other hydrolases of lysosomal origin; subcellular distribution, latency and acidic pH optimum. Acid phospholipase A2 activity with similar characteristics was also tentatively identified. We were unable to exclude the possibility that the combined action of phospholipase A1 and lysophospholipase contributed to the release of acyl moieties from the 2-position of the synthetic substrates. 2. Lysophospholipase activity was present in the lysosome-enriched fractions. This activity had an alkaline pH optimum. 3. Phospholipase A1 and A2 activities solubilized from lysosome fractions by freeze-thawing were inhibited by Ca2+ and slightly activated by EDTA. A Ca2+- stimulated phospholipase A2 activity, with an alkaline pH optimum, remained in the particulate residue of freeze-thawed lysosome preparations. This activity is believed to represent mitochondrial contamination. 4. Activities of acid phospholipase A, as well as other acid hydrolases, increased approx. 1.5-fold between 1 and 4 days following induction of luteinizatin, suggesting a hormonal influence on lysosomal enzyme activities.     

Localization of arylsulphatase in neurons

Abstract— Arylsulphatase activity, with 4-methylumbelliferone sulphate as substrate, was measured by a quantitative histochemical method in individual anterior horn nerve cell bodies and adjacent neuropil of man and monkey; and in molecular and granular layers and subjacent white matter of cerebellum of monkey, rat and guinea pig. The activity was much higher in neuronal perikarya than in neuropil, and higher in the granular layer of cerebellum than in the molecular or white matter, thus resembling the distinctive distribution, reported in monkey, of three other lysosomal enzymes, β-galactosidase, β-glucuronidase and α-naphthyl acid phosphatase. One exception was encountered: the white matter of guinea pig cerebellum had more arysulphatase activity than the granular layer. For comparison, other lysosomal enzymes also were measured in rat and guinea pig cerebellum; in these species, α-naphthyl acid phosphatase distribution was found to differ from that of β-galactosidase and arysulphatase, and from the pattern common to four lysosomal enzymes in the monkey.     

Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney.

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r     

The quantitative histochemistry of acid proteinase in the nervous system: localization in neurons

A new, fluorometric method was used to assay acid proteinase in tissue samples at great sensitivity. Digestion of hemoglobin at pH 3.8 (in brain mostly due to cathepsin D) was measured in individual nerve cell bodies and neuropil from the anterior horn of the spinal cord, and in the molecular and granular layers and while matter of cerebellum, in man and in monkey. Anterior horn cell perikarya were about 25 times more active than neuropil, and the granular layer of cerebellum had about three times the activity of the molecular layer. This predominantly neuronal localization resembles the distribution of other lysosomal hydrolases. The high capacity for protein breakdown found in neurons is in accord with their known high rates of protein synthesis and turnover.     

Lysosomal hydrolases in neuronal, astroglial, and olidodendroglial enriched fractions of rabbit and beef brain.

Arylsulfatases A, B, and C, beta-galactosidase, and acid phosphatase were assayed in neuronal, astroglial, and oligodendroglial fractions isolated from adult rabbit and beef brains. The specific activities of all acid hydrolases were lower in beef cells compared to rabbit cells. The lysosomal enzymes of the rabbit neuronal fraction showed 10--25 time higher activities than the oligodendroglial fraction and 5-fold higher activities than the astroglial fraction. In beef brain, the specific activities of these enzymes were similar in oligodendroglia and astrocytes but 4--10 times lower than in neurons. The low activity of arylsulfatase A and beta-galactosidase in oligodendroglial cells may suggest that the low turnover of cerebroside and sulfatide in myelin may be regulated in part by the enzymes that catalyze their degradation.     

Physicochemical modifications of lysosomal hydrolases during intracellular transport

1. The following fractions were prepared from rat kidney and characterized ultrastructurally, biochemically and enzymically: (a) an ordinary rough microsomal (RM(1)) fraction; (b) a special rough microsomal (RM(2)) fraction enriched seven- to nine-fold in acid hydrolases over the homogenate; (c) a smooth microsomal (SM) fraction; (d) a Golgi (GM) fraction enriched 2.5-fold in acid hydrolases and 10-, 15- and 20-fold in sialyltransferase, N-acetyl-lactosamine synthetase and galactosyltransferase respectively; (e) a lysosomal (L) fraction enriched 15- to 23-fold in acid hydrolases. The frequency of Golgi sacs and tubules seen in the electron microscope and the specific activity of the three glycosyltransferases in these fractions increased in the order: RM(2)相似文献   

Study of intracellular localization of the proteolytic enzyme complex and its protein inhibitors in bombyx grain

Intracellular localization of serine, cysteine and aspartate proteases, as well as their protein inhibitors, in bombyx grain in the postdiapause period of embryogenesis has been studied. Proteolytic activity of aspartate and cysteine proteases was found in lysosomal, mitochondrial, and nuclear fractions of grains. Serine protease activity was not observed in subcellular fractions of grains of the fourth day of postdiapause development. It has been shown that activities of protein inhibitors and certain peptide hydrolases in subcellular fractions provide consistent functioning and fine regulation of the proteolytic enzyme complex.     

Isolation of Kupffer cell lysosomes, with observations on their chemical and enzymic composition

The purpose of the present investigation was twofold: The isolation of Kupffer cell lysosomes by changing their density in vivo through uptake of colloidal silver iodide (Neosilvol^R), and the characterization of the isolated fraction. No significant changes in the activities or distribution of acid phosphatase, aryl sulphatase, and cathepsin D were found after the injection of Neosilvol^R. A method is presented for the isolation of silver-loaded lysosomes from rat liver Kupffer cells by means of ultracentrifugation in sucrose gradients. Morphological and biochemical data indicate that the lysosomal fraction was contaminated with other subcellular organelles only to a minor degree. The lysosomal fraction showed non-parallel enrichment of various acid hydrolases, with the highest degree of purification found for aryl sulphatase and the lowest for acid phosphatase. The lysosomal enzyme activity pattern was similar to that found in Kupffer cell preparations.     

Rabbit β-glucuronidase. Subcellular distribution and immunochemical properties

1. The subcellular distribution of beta-glucuronidase and other hydrolases in rabbit liver was investigated. beta-Glucuronidase was found in both microsomal and lysosomal fractions. 2. Multiple forms of beta-glucuronidase were present in extracts of microsomal and lysosomal fractions. All forms were common to both fractions. 3. A specific antiserum against beta-glucuronidase was raised, and characterized by immunoprecipitation and affinity-chromatography procedures. 4. The immunological identity of the multiple forms in the pure beta-glucuronidase preparation, and the immunological identity of the beta-glucuronidase complement of lysosomal extracts with that of microsomal extracts, were demonstrated by means of the antiserum. The presence of inactive enzyme in various enzyme preparations was shown.     

Hyaluronidase activity in lysosomes of bone tissue

1. The distribution pattern of hyaluronidase in subcellular fractions of bone-tissue homogenates is closely similar to that reported by Vaes & Jacques (1965b) for the other acid hydrolases of this tissue. The highest specific activity of hyaluronidase is also found in the light-mitochondrial fraction. 2. In cytoplasmic extracts of bone, about 60% of the activity of hyaluronidase is latent, and is unmasked by a number of treatments (digitonin, low osmotic pressure, freezing and thawing, Waring Blendor) that unmask the lysosomal β-glucuronidase in a closely parallel manner. Low concentrations of Triton X-100 render a larger proportion of β-glucuronidase than of hyaluronidase accessible to external substrates, but release the same proportion of both enzymes in unsedimentable form. 3. These results support the concept of an association of hyaluronidase with lysosomes in bone.     

Effect of freezing and thawing on the distribution of lysosomal hydrolases in leaves of Solanum tuberosum L.

Density-gradient ultracentrifugation techniques showed that freezing and thawing of potato leaves resulted in a change in the density of subcellular particles containing acid phosphatase and acid ribonuclease (RNase). Gel filtration experiments were used to characterise the molecular forms of the hydrolases associated with the various cell fractions. Freezing and thawing promoted a release of a portion of the complement of acid phosphatase and RNase from the lysosomes to the supernatant fluid fraction of cell homogenates. The freezing treatment appeared to activate latent lysosomal RNase.     

Immunocytochemical localization of lysosomal acid phosphatase in normal and "I-cell" fibroblasts

This study represents the first example of immunological localization of lysosomal acid phosphatase. The intracellular localization of lysosomal acid phosphatase was investigated with immunocytochemical methods at the light and electron microscopical level in cultured fibroblasts obtained from normal subjects and from a patient with I-cell disease. Double-labeling studies using fluorescence microscopy showed that acid phosphatase is present in the same organelles as other hydrolases. At the electron microscopic level in control fibroblasts acid phosphatase was found in the rough endoplasmic reticulum, lysosomes, at the plasma membrane, in vesicles just below the plasma membrane and in multivesicular bodies. This localization was comparable with that of other lysosomal enzymes tested (acid alpha-glucosidase, N-acetyl-beta-hexosaminidase, beta-galactosidase). Acid phosphatase labeling was mainly found in association with the lysosomal membrane and with membranous material present within the lysosome. In I-cell fibroblasts the label was present in the same subcellular organelles but always associated with membranous structures. We suggest that the association of acid phosphatase with membranes might explain the normal enzyme activity found in I-cell fibroblasts.     

INCORPORATION OF AMINO ACIDS INTO PROTEINS IN NEURONAL AND NEUROPIL FRACTIONS OF RAT CEREBRAL CORTEX: PRESENCE OF A RAPIDLY LABELLING NEURONAL FRACTION

Abstract— The time course of incorporation of intraperitoneally injected [³H]lysine and [¹⁴C]phenylalanine into neuronal and neuropil proteins has been followed for up to 8 days. At short times after injection (<2 h) the specific activity of the neuronal fraction was higher than that of the neuropil. At longer time intervals, although the total brain specific activity continued to rise, neuronal perikaryal specific activity fell below that of neuropil. Thus the neuronal/neuropil incorporation ratio with [³H]lysine as substrate was 1·5 at 1 h, but by 4 h had fallen to 0·4, a ratio which was maintained for up to 8 days. A similar reversal occurred with phenylalanine as substrate. These changes were interpreted as evidence for the presence of a rapidly-labelling protein fraction in the neurons which is subsequently transported out. Subcellular fractionation showed that over the 4 h period the rapidly labelling fraction was not transported to the synaptosomes. Incubation of prelabelled cortex slices followed by cell fractionation showed that a differential transport of protein of higher than average specific activity from both neurons and neuropil fractions occurred; there is a tendency for preformed highly labelled protein to accumulate during the in vitro incubation in Fraction D, a pellet enriched in red cells, some large neuronal perikarya and cell nuclei. When cell fractions were prepared after in vitro incubation, the distribution of the material down the gradient differed from that when fresh tissue was fractionated, as demonstrated by microscopic examination and the distribution of β-galactosidase, a neuronal marker. Double-label experiments showed that this redistribution could not account for the preferential loss and accumulation of prelabelled protein. It was noted that in vivo incorporation into the rapidly labelling neuronal protein is suppressed under certain changed environmental conditions, such as dark rearing. This is interpreted as lending support to the concept of the state-dependence of neuronal and neuropil protein synthesis and their inter-relations.     

SOME PROPERTIES OF ISOLATED NEURONAL CELL FRACTIONS

Abstract— 1. Histochemical evidence was presented illustrative of the composition of neuronal and neuropil ('glial') fractions isolated according to a previously published procedure. The neuropil refers to all cortical tissue other than neuronal perikarya.
2. On the basis of cell counts and of DNA content, an average cell mass of 100-110 pg was calculated for cells in the neuronal fraction. Eight per cent of the total DNA was recovered in the neuronal fraction.
3. Both fractions synthesized ATP in vitro. Concentrations after 60 min incubation with glucose were: neuropil, 7–36 μmoles/mg protein; neuronal, 12–31 μmoles/mg protein.
4. Osmotic shock or homogenization resulted in changes in turbidity of the cell fractions which were interpreted as indicative of loss of cell structure. The free pool amino acids glutamate, glutamine, GABA, aspartate and alanine were retained in the precipitable material through several washes with isotonic solutions. Homogenization released 72 per cent of the neuronal and 68 per cent of the neuropil amino acids into the supernatant, but only 37 per cent and 19 per cent respectively of the protein.
5. By contrast with earlier reports, K⁺ accumulation has now been demonstrated in both neuronal and neuropil fractions. After incubation with glucose, K⁺ level were calculated as being 80 per cent of slice in the neuronal, and 65 per cent in the neuropil fraction. These results, and those of the osmotic shock experiments, were taken as indicative of the retention of some cell structure.
6. By comparison, cell fractions prepared by other procedures, using acetone-glycerol-water or tetraphenylboron for tissue disaggregation, produced preparations with limited metabolic capabilities; oxygen uptake, CO₂ and lactate production were all lowered substantially.     

Retinyl ester hydrolases in retinal pigment epithelium

In bovine retinal pigment epithelium membranes we have found three hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.     

The activator of cerebroside sulphatase. Lysosomal localization.

1) An activator protein necessary for the enzymic hydrolysis of cerebroside sulphate could be partially purified from unfractionated rat liver. This activator, which is similar to that of human origin, proved to be a heat-stable, non-dialyzable, low molecular weight protein with an isoelectric point of 4.1. Its activity could be destroyed by pronase. 2) For elucidation of the subcellular localization of the activator, rat liver was fractionated by differential centrifugation. The intracellular distribution of the cerebroside sulphatase activator was compared to the distribution patterns of marker enzymes for different cell organelles and found to coincide with the lysosomal arylsulphatase, thus indicating a lysosomal localization. 3) This was confirmed using highly purified secondary, i.e. iron-loaded, lysosomes. After disruption by osmotic shock, these organelles hydrolyzed cerebroside sulphate when incubations were performed under physiological conditions with endogenous as well as exogenous sulphatase A as enzyme. 4) After subfractionation of the disrupted secondary lysosomes into membrane and lysosol fractions by high speed centrifugation, it was found that the activator protein was exclusively associated with the lysosol, whereas the acid hydrolases were distributed differently between the two fractions. 5) The lysosol was further fractionated by semi-preparative electrophoresis on polyacrylamide gels. Two protein fractions were obtained: a high molecular weight fraction, containing the activator-free acid hydrolases, and a low molecular weight fraction, containing the enzyme-free activator of cerebroside sulphatase. 6) The significance of these findings for the hydrolysis of sphingolipids in the lysosomes is discussed.