N. Stagni

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.

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.

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.

Glycosaminoglycans and endochondral calcification.

Present controversy about endochondral calcification and ossification is concerned with changes in glycosaminoglycans. Some authors report a rise in amounts of glycosaminoglycans while others a fall. Cartilage was carefully sliced under microscopic control to provide samples of material from different functional zones of the developing tissues. The following zones were studied histochemically and analyzed for their content of total nitrogen, hydroxyproline, total hexosamines, uronic acid and phosphorus: the resting zone; the zone of proliferating and maturing cells; the calcifying zone, characterized by degenerating hypertrophic cells and early mineral deposition; the ossifying region, where early bone formation takes place. Serial analyses provided evidence that glycosaminoglycans increases before calcification starts. Afterwards, part of the glycosaminoglycan content is removed. This biphasic process appears to occur during the calcification of other tissues too, such as secondary bone and dentine.

Influence of calcium depletion on medullary bone of laying hens

SummaryMedullary bone of birds maintained on a low-calcium diet represents a good model to study modifications of matrix composition in calcified tissue undergoing intense formation and resorption. The composition of the bone matrix during the low-calcium diet has been analyzed by both chemical and histological techniques. Sixty White Leghorn pullets 1 year old were used for the experiment. Fifteen birds served as controls and were killed on day zero; the remaining birds were placed on a calcium-deficient diet (0.13% calcium) and sacrificed after 4, 7, and 12 days of treatment in groups of 15. Serum levels of calcium, PTH, and estrogens were also measured. Chemical analysis of the samples were made for total nitrogen, hydroxyproline, hexosamine, hexoses, calcium, and phosphorus. Collagen and proteoglycans of the matrix of medullary bone of the egg-laying hens were found to be affected by the low-calcium diet. They either increased or decreased during the experiment but never in parallel. The increment of serum PTH is considered responsible for the variations in the amount of collagen. The effects of this hormone are magnified by the fall of serum estrogens as shown also by variations in the amounts of noncollagenous protein. In the late phase of the diet the matrix is represented by poorly calcified osteoid tissue rich in noncollagenous protein, i.e., proteoglycans and glycoproteins.

A possible role for polyamines in cartilage in the mechanism of calcification

The role of polyamines in cartilage is not known: they may be somehow related to the mechanism of calcification. In epiphyseal cartilage from calf scapulas, they are more concentrated in the ossifying area, where calcification takes place, than in the resting region. Spermidine is present in greater amounts than spermine and putrescine. Since ornithine decarboxylase (EC 4.1.1.17) is measurable only in the resting region of the tissue, it is in this area that polyamine biosynthesis occurs, while they accumulate in the ossifying area. Immunohistochemical evidence is obtained that only in the ossifying zone is spermidine extracellular. It is at this level that the matrix is rearranged to become calcified, and proteoglycans are dissociated and partially removed. The effect of polyamines on solutions of proteoglycan subunits has been studied in vitro by following variations of turbidity and viscosity. While in the presence of putrescine the specific viscosity decreases to asymptotic values, in the presence of either 30 mM spermidine or 2.5-10 mM spermine, the decrement is more marked. At the same concentrations, increase of the turbidity of proteoglycan subunit solutions was observed. Only spermidine showed the capacity of displacing proteoglycan subunits from a column of Sepharose 4B-type II collagen: at 15 mM concentration, about 90% of proteoglycans were removed from the column. Alkaline phosphatase activity, which plays an important role in calcification, is enhanced by spermidine and spermine. These results obtained in vitro support the hypothesis that polyamines may be related to calcification of preosseous cartilage.

In vitro biosynthesis by articular chondrocytes of a specific low molecular size proteoglycan pool

Proteoglycans synthesized by articular and epiphyseal chondrocytes in culture were compared. Proteoglycans extruded by the two types of cells into the culture medium are of identical M r. On the other hand, the proteoglycans of cells or pericellular matrix synthesized by the articular chondrocytes are characterized by an heterogeneous fraction of low‐M r which is not present in the material derived from epiphyseal chondrocytes. There are at least two components in this fraction: the first seems to be a precursor of aggregated proteoglycans, the other may represent a component of cell coat. Stimulation of the cell cultures with vitamin D metabolites and somatomedin enhances proteoglycan biosynthesis but no modification is observed in the proteoglycan M r.

Solubility properties of alkaline phosphatase from matrix vesicles

Alkaline phosphatase has been extracted from matrix vesicles of a calcifying cartilage with 0.15 M KCl, 0.4 M guanidinium chloride and 0.05 M deoxycholate/50% butanol mixture. The catalytic properties of the three extracts have been compared. Although the highest amount of enzyme activity is extracted with the latter reagent (55%), some of it is also extracted with KCl (11%) and guanidinium (7%). By submitting isolated matrix vesicles to a short time sonication the distribution pattern of the alkaline phosphatase activity in the extracts is clearly modified, as the amount extracted with KCl increases from 14 to 50% and the portion extracted with deoxycholate decreases from 55 to 27% of the total enzyme activity of matrix vesicles. The enzymatic preparations were comparable on the basis of specific activities, affinity for the substrates (p-nitrophenylphosphate, ATP), thermostability, sensitivity to inhibitors and activators. By electrofocusing a value of pI = 4.15 was found for the alkaline phosphatase of matrix vesicles independently of the extraction medium. These results contradict the concept that alkaline phosphatase is exclusively an intrinsic membrane protein.

Evidence in vitro for an enzymatic synthesis of phosphocitrate

A biological synthesis of phosphocitrate is described from precursors, citrate and adenosinetriphosphate reacting in the presence of rat liver homogenate. Identity of the newly formed product was examined by enzymatic digestion of reactions mixtures, HPLC chromatography and 1H-NMR spectra. Authenticity of product was established by comparison to chemically synthesized phosphocitrate. Recognition of the existence of a biologically synthetic pathway adds credence to the known presence of phosphocitrate in mitochondria and a postulated role to control calcium phosphate deposition in that organelle.