B. de Bernard

Lysosomal enzyme activity in rat and beef skeletal muscle

Abstract 1. 1. Acid hydrolases (β-glucuronidase EC 3.2.31; cathepsin; β-galactosidase EC 3.2.1.23; ribonuclease EC 2.7.7.17) of rat and beef skeletal muscle are associated with cytoplasmic particles and are poorly reactive towards external substrates; however, their activity is enhanced or even fully displayed by injuring the particles with a variety of treatments (osmotic shock, sonication, alternate freezing and thawing, addition of Trition X-100). 2. 2. By gradually increasing the concentration of Triton X-100 in the enzyme assays, acid hydrolases are gradually liberated from the particles, more homogeneously from those of rat than of beef muscle. 3. 3. Osmotic and thermal treatments of beef skeletal lysosomes reveal a higher stability of the particles as compared to those from liver and kidney. 4. 4. The 4 hydrolytic enzymes display in the sedimented of fractions a very similar distribution pattern, different from that of cytochrome c oxidase and NAD glycohydrolase. Highest relative specific activity of lysosomal enzymes was found associated to a post-mitochondrial fraction. 5. 5. By isopicnic centrifugation of a post-mitochondrial fraction, both from rat an beef muscle, acid hydrolases were broadley distributed throught the water surcrose gradient (d = 1.10−1.20); cytochrome c oxidase was in a narrow band and NAD glycohydrolase showed a bimodal distribution. 6. 6. On the basis of sedimentation coefficient and equilibrium density acid hydrolases of skeletal muscle are associated to particles different from mitochondria. and microsomes. The homogenous properties of these enzymes suggest that they are associated with a single functional form of lysosome-like particles.

A glycoprotein located in the intermembrane space of rat liver mitochondria

Recently much has been focused on the glycoprotein content of biological membranes and in particular of mitochondria [ I1 1 ] . In a previous paper on this subject [ 121, we have reported the distribution of glycoprotein components between inner and outer mitochondrial membranes. An interesting observation in that paper was that a large portion of sialic acid and hexosamine-containing material was released during the separation of the two membranes. No information was however available about the original intramitochondrial location of the released glycoprotein. We found therefore of interest to compare the supernatant obtained from mitochondria upon swelling and contraction with that obtained by sonication from pre-swollen and contracted mitochondria. In this paper we report an electrophoretic study of the proteins released in the soluble fraction by different procedures. A technique is also described for the isolation of the glycoprotein, released by swelling, in an electrophoretically homogenous form.

Lysosomes in heart tissue

Abstract 1. 1. The acid hydrolase (β-glucuronidase, EC 3.2.1.31; cathepsin; β-galactosidase, EC 3.2.1.23; acid ribonuclease, EC 2.7.7.17 and acid deoxyribonuclease, EC 3.1.4.6) of beef-heart muscle are associated with cytoplasmic particles and are poorly reactive towards external substrates; however, their activity is enhanced or even fully displayed by injuring the particles with a variety of treatments (osmotic shock, prolonged homogenization, alternate freezing and thawing, addition of Triton X-100). 2. 2. By gradually increasing the concentration of Triton X-100 in the enzyme assays, the acid hydrolases are gradually liberated from the particles, although not in equal proportions. 3. 3. When particles in suspension are submitted to the action of various “protein” or “lipid” reagents, it appears that both types of reagent are able to increase the availability of the hydrolases for their substrates, but that a high solubilization of the enzymes is achieved only by attacking the membrane phospholipids. 4. 4. Preincubation either in hypo- or hypertonic sucrose solutions produces an increase in the availability of the hydrolases; under the same conditions, a partial solubilization, albeit to a different extent for each enzyme, is also observed. 5. 5. Each of the enzymes considered displays a peculiar intracellular distribution pattern, but none of them is strictly the same as that of succinoxidase. By isopicnic centrifugation of a mitochondrial fraction it is possible to isolate a particulate fraction ( d = 1.174) showing a high concentration of latent hydrolytic enzymes and a comparatively low concentration of cytochrome c oxidase. 6. 6. The acid hydrolases of beef heart do not seem, therefore, to belong to mitochondria but to exhibit the main features of lysosomal enzymes. A comparison between the properties of beef-heart and rat-liver lysosomes is also made and the heterogeneity of the former is discussed.

Alkaline phosphatase binds to collagen; a hypothesis on the mechanism of extravesicular mineralization in epiphyseal cartilage

Affinity chromatography on Sepharose 4B-collagen gels was used to test the affinity of alkaline phosphatase for collagen. Results indicate that 1) alkaline phosphatase of preosseous cartilage binds to collagen probably by electrostatic interactions, 2) this interaction is inhibited by proteoglycan subunits. These results suggest that, in vivo, the formation of a collagen-alkaline phosphatase complex may be a step of the process leading to cartilage calcification.

The calcium binding properties of a glycoprotein isolated from pre-osseous cartilage

Abstract The Ca ++ binding properties of a glycoprotein purified from preosseous cartilage have been investigated. This protein of M.W. 200,000 has two classes of Ca ++ binding sites, with dissociation constants of 10 −7 M and 10 −4 M respectively. About 10 nmoles of Ca ++ may be bound per mg of protein at the high and 3,000 at the low affinity sites. The Ca ++ binding ability is pH dependent and it is maximum at pH 8.3. Lanthanum and butacaine have no inhibitory effect on Ca ++ binding, whereas ruthenium red strongly inhibits both the high and low affinity sites. Possible functions of this glycoprotein in cartilage matrix are discussed.

Enzymatic properties of the Ca2+-Binding glycoprotein isolated from preosseous cartilage

SummaryThe Ca2+-binding glycoprotein isolated from preosseous cartilage shows also alkaline phosphatase activity. The purification procedure indicates that the enzyme is inhibited in crude extract and conceivably in the intact tissue; the activity may be controlled by the proteoglycans present in the matrix. Other substrates are hydrolyzed by the purified enzyme in addition top-nitrophenylphosphate; the highest specific activity was measured with ATP and pyrophosphate (PPi) at pH 7.5 and 9.0 Mg2+ induces an activation of ATP and PPi hydrolysis; Ca2+ activates hydrolysis of ATP but inhibits that of PPi. The glycoprotein shows also transphosphorylase activity,l-serine being the best phosphate acceptor. The release or transfer of Pi catalyzed by the glycoprotein can be an important step in calcium phosphate precipitation.

Glycoprotein components, sialic acid and hexosamines, bound to inner and outer mitochondrial membranes.

Much attention has been focused in the past on protein and phospholipid constituents of biological membranes. More recently, however, carbohydratecontaining materials have become subject of interest as important components of plasma membrane. These macromolecules have been recognized as responsibte for a number of phenomena including contact-inhibition, transport and antigenic properties (for review see [ 11). Intracellular membrane structures have also been shown to possess a glycoprotein complement [2-81 and an active synthesis of such components has been reported to occur in vitro in isolated mitochondria and microsomes [9111. The availability of a technique for the separation of inner and outer membranes [ 121 from isolated mitochondri~i makes it possible to investigate the intramitochondrial distribution of glycoproteins. In this paper we report the distribution between mitochondrial membranes of two typical constituents of glycoproteins such as sialic acid and hexosamines.

Some properties of proteoglycans of pre-osseous cartilage

Abstract Protein—polysaccharide complex has been extracted with 4 M guanidinium chloride from resting and transforming-ossifying regions of calf scapula cartilage. The extract has been purified by equilibrium density gradient sedimentation and/or by polyacrylamide gel electrophoresis in different experimental conditions. The following information has been obtained so far: 1. 1.|Transforming-ossifying regions contain more protein—polysaccharide complex than the resting zone. 2. 2.|Proteoglycan subunits and a glycoprotein are the main components of the protein—polysaccharide complex. 3. 3.|The analytical composition of the protein—polysaccharide complex from the two zones is virtually identical but different types of interaction seem to exist among the components as indicated by the viscosity data and the centrifugation pattern in CsCl density gradients. 4. 4.|A highly purified preparation of the glycoprotein component from the two zones has been analyzed; the most interesting result is that only glycoprotein from the ossifying regions of cartilage reacts with murexide, showing Ca 2+ binding properties.

A high molecular weight form of NADH-cytochrome b5 reductase from ox liver microsomes

Abstract We report here the solubilisation and purification of NADH-cytochrome b 5 reductase as a high molecular weight species of the order of 400000. As in the procedure reported by Ito and Sato for the isolation of polymer cytochrome b 5 , the solubilisation of the enzyme has been performed by means of Triton X-100 and sodium deoxycholate. The purification was then performed through (NH 4 ) 2 SO 4 precipitation between 25 and 45% saturation and gel filtration on Sephadex G-100 and G-50. The last traces of hemoproteins were easily removed by ion exchange chromatography on DEAE-Sephadex A-50. Removal of the phospholipids and detergents is achieved by precipitation with 90% cold acetone. The enzyme is finally purified by gel filtration on Sephadex G-200. The procedure results in a 208-fold purified enzyme with a 19% yield. Disc electrophoresis on 3% polyacrylamide gel of the preparation reveals the presence of a single component which stains positively with periodic acid-Schiff reagent and gives a metachromatic response to toluidine blue. These findings indicate that the enzyme contains carbohydrates. The band does not show positive reaction with dyes specific for lipids. Only upon treatment with urea or sodium dodecyl sulphate is the enzyme split into three subunits.

Ca2+-Binding Glycoprotein in Avian Bone Induced by Estrogen

The discovery in calcifying cartilage of a glycoprotein, endowed with high calcium affinity and alkaline phosphatase activity, has prompted the investigation of the presence of this compound in other calcified tissues. From medullary bone, a tissue which is highly mineralized under estrogen stimulus, a glycoprotein has been extracted which had the properties described. Besides the high calcium affinity (KD = 10(-7)M), this protein shows phosphatase activity and rate of hydrolysis of ATP, GTP and pyrophosphate was measured. Analysis of the chemical composition of the matrix of the medullary bone indicates that proteoglycans are present in large amounts. The calcium binding glycoprotein appears to be a compound present in different calcified tissues.