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Biomedical subjects

A Linde

Publications and source records attributed to A Linde.

At least 289 records · Page 16Linked to original sources
PubMed1978

Purification fo cathepsin D by AH-sepharose affinity chromatography.

A rapid and reliable method for coupling the protease inhibitor pepstatin to AH-Sepharose 48 was developed. The matrix prepared was used to purify cathepsin D from rat liver. The enzyme was eluted in one fraction and proved to be pure by gel electrophoresis, two types of ion exchange chromatography, molecular sieve chromatography, and immunologically homogenous by immunoelectrophoresis. This method is more rapid and gives a higher yield than previous techniques. The possibility to use this technique for the purification of other enzymes inhibitable by pepstatin should be considered.

Cathepsins↗
PubMed1978

Odontoblast metabolism in rats deficient in vitamin D and calcium. III. Protein synthesis in vitro.

Dentinogenically active rat incisor odontoblasts were dissected out from animals fed a low calcium, vitamin D free diet (R 25). Protein synthesis by these cells was studied by means of short-time incubation in an in vitro system in the presence of the radioactive labeled precursors L-leucine, L-fucose and L-proline. A significantly increased leucine and proline incorporation into the protein synthesized was noted in the cells from rats fed the deficient R 25 diet compared with odontoblasts from rats fed an adequate control diet (R 47). No difference between the two groups was found when fucose was utilized as a precursor. The well known increase in predentin width when feeding a rachitogenic diet may thus be explained by an increase in organic matrix synthesis in addition to the possible negative direct effects of lowered serum Ca content. Prior to this study, the behavior of proline as a precursor in the in vitro system was studied. The possibility of separating leucine-labeled proteins synthesized in the in vitro system by means of SDS-polyacrylamide gel electrophoresis was also shown.

Animals↗
PubMed1978

Ultrastructural localization of alkaline phosphatases in rat incisor odontoblasts.

The localization of alkaline phosphatases in dentinogenically active rat incisor odontoblasts was studied by means of subcellular fractionation and electron microscopical histochemistry. Subcellular fractionation revealed the predominant phosphatase activity to be present in the microsome fraction and to a lesser extent in the mitochondrial fraction. Adenosine triphosphate degrading enzyme activity was determined in the presence or absence of (+/-)-6(m-bromophenyl)-5, 6-dihydroimidazo(le) (2,1-b) thiazole oxalate (R 8231). Before the histochemical study, the effects on phosphatase activities by aldehyde fixation were studied by biochemical assay. A method of fixation for optimal preservation of phosphatase activity is presented. Phosphatase electron microscopic histochemistry was performed by using ATP as a substrate and with or without addition of the inhibitor R 82319 Precipitates were seen in the membranes of vesicles present in the odontoblast process and the Golgi region. When there were signs of insufficient fixation, precipitates were also seen in the outer membranes of mitochondria. No phosphatase activity was seen in the cell membrane. ATP degrading enzyme activities mediated by nonspecific alkaline phosphatase (APase) and Ca2+ -adenosine triphosphatase thus have the same morphological localization. This close association is consistent with earlier biochemical studies.

Adenosine Triphosphatases↗
PubMed1978

Cathepsin D. Purification from rat liver and immunohistochemical demonstration in rat incisor.

Rat liver cathepsin D (EC 3.4.23.5) was purified using precipitation technique, ion exchange chromatography, molecular sieve chromatography and isoelectric focusing. Rabbit anti-rat cathepsin D IgG was prepared and rat incisor teeth were cross-sectioned in a cryostat. These sections were incubated with FITC-conjugated anti-rat cathepsin D IgG. Marked fluorescence, indicating the localization of cathepsin D, could be seen over the odontoblast and predentin area. No specific fluorescence could be dmonstrated in the pulp connective tissue proper nor in the dentin.

Animals↗
PubMed1978

Odontoblast alkaline phosphatases and Ca2+ transport.

The same isoenzyme of nonspecific alkaline phosphatase (APase), assayed with p-nitrophenylphosphate (p-NPP), was shown be present in different calcifying tissues, bone, calcifying cartilage, odontoblasts and enamel organ. Indications were also found that the enzymatic degradation of inorganic pyrophosphate (PPi) in calcifying tissues is mediated by APase. By using specific APase inhibitors, it was shown that two enzymes capable of degrading ATP exist. These were characterized in dentinogenically active odontoblasts, and it was concluded that one is the classical APase, the other is a Ca2+ and Mg2+ activated ATPase, named Ca2+-ATPase. The two phosphatases were solubilized from odontoblasts and separated. The localization of APase and Ca2+-ATPase in odontoblasts was investigated by subcellular fractionation and EM histochemistry. Routine methods for fixation were found to almost completely inactivate the enzymes. By using a mild fixation technique that preserved 80% of the enzyme activity, the main localization for both APase and Ca2+-ATPase was found to be in the membranes of intercellular vesicles located in the cell body and odontoblasts process. No activity was found in the cell membranes. It is concluded that there are at least two enzymes able to degrade phosphate compounds at alkaline pH in hard tissue forming cells. One is the nonspecific alkaline phosphatase (APase; EC 3. 1. 3. 1), which is active against p-NPP, PPi, glycerophosphates and ATP among other substrates. The other is a more specific Ca2+-ATPase (EC 3. 6. 1. 3). There seems to be an intimate relation between these two enzymes in the tissue. The function of APase in biological calcification is still obscure. In contrast, the finding of an ATP dependent, intravesicularly directed, transmembranous Ca2+-transport in vesicles derived from the microsomal fraction of odontoblasts may explain the role of Ca2+-ATPase.

Alkaline Phosphatase↗
PubMed1977

Cathepsin D activity in isolated odontoblasts.

The presence of an acid proteinase with a high activity has been demonstrated in isolated odontoblast-predentine material from dentinogenically active rat incisors. The enzyme was identified as cathepsin D (EC 3.4.23.5). The possible significance of the enzymatic degradation of proteoglycans and glycosaminoglycans in the course of the calcification process is discussed.

Animals↗
PubMed1977

A comparative study of alkaline phosphatase in calcifying cartilage, odontoblasts and the enamel organ.

The enzyme alkaline phosphatase (AP) (EC 3.1.3.1) in three different calcification areas was studied by means of a spectrophotometric micro method using p-nitrophenylphosphate as a substrate. Rat maxillary incisor odontoblasts and enamel organ from the zones of matrix formation and maturation and tissue from rabbit metatarsal cartilage were allowed to react with the substrate in glycine-NaOH buffer at room temperature. The reaction was found to be linear for a minimum of 20 min. The pH optima for AP from these tissues were in the pH range of 10.0-10.3. In order to compare AP from the four calcification areas different parameters were studied. Heating at 56 degrees C or 60 degrees C for varying times revealed that the enzymes were almost completely inactivated after 10 min. Mg2+ ions activated the enzymes by about 25% at concentrations of 2.5 mM (enamel organ 1.25 mM); while only higher concentrations of Mg2+ had an inactivating effect, Ca2+ and PO3-4 ions were inactivating at varying concentrations. F- ions showed no effect on AP activity at concentrations below 250 mM (enamel organ 125 mM) but caused inactivation of the enzymes at about 50% at 1 M. EDTA was found to be a very effective AP inactivator at concentrations above 0.06 mM, whereas urea did not noticeably affect the enzyme reactions at concentrations below 1 M. At higher concentrations, inactivation was observed. In order to determine AP localization in the epiphyseal plate successive 40-mum-thick, freeze-sectioned slices were analyzed. The activity was highest nearest the zone of cartilage calcification and decreased towards the reserve cell zone. It was concluded that the same AP isoenzyme is present in these quite different calcification loci.

Alkaline Phosphatase↗
PubMed1977

Odontoblast metabolism in rats deficient in vitamin D and calcium I: A histochemical survey.

Different rat diets, deficient in calcium and/or vitamin D, were tested for varying experimental periods to obtain changes in serum calcium values, body weight gain and odontoblast-predentine morphology. One diet, R 25, used during a 14-day period, was found to induce lowered serum calcium values and an increased predentin width in incisor teeth. Rats fed this diet demonstrated an increase in alkaline phosphatase and ATP-splitting enzyme activity in dentinogenically active incisor odontoblasts. No other metabolic changes in these cells were demonstrated by the histochemical methods employed.

Animals↗
PubMed1977

Odontoblast metabolism in rats deficient in vitamin D and calcium. II. Changes in activities of alkaline phosphatases.

Rats were fed a low calcium diet deficient in vitamin D for 14 days. Changes in alkaline phosphatase activities in odontoblasts dissected out from incisor teeth were studied biochemically. A strong increase in pNPP-ase, PPi-ase, total ATP-degradation and Ca2+- ATPase was observed in the deficient animals compared with animals fed a control diet.

Adenosine Triphosphatases↗
PubMed1977

ATP-ase activity in the odontoblastic layer of rat incisor. Determination with a radiochemical and a colorimetric method.

The ATP-splitting enzyme activity in odontoblasts isolated from rat incisors has been studied by means of a radiochemical and a colorimetric micromethod. The results with the two methods were virtually identical. The reaction was linear with time for at least 45 min. The pH optimum was found to be 9.8 independently of the ATP concentration. Maximal substrate saturation occurred at a total ATP concentration of 3 mM. Ca2+ and Mg2+ ions activated ATP degradation. F-ions did not affect the activity at low concentrations, whereas higher concentrations were inhibitory. Na+ and ions were slightly inhibitory. Urea inhibited the enzyme activity at concentrations above 1.5 M, while EDTA and EGTA were strong inhibitors at very low concentrations. When incubating in the presence of low concentrations of specific inhibitors for nonspecific alkaline phosphatase, levamisole and R8231, about 20% ATP degrading enzyme activity remained. In conclusion it is suggested that there are at least two ATP degrading phosphatases active at alkaline pH.

Adenosine Triphosphatases↗
PubMed1976

Ultrastructural localisation of proteoglycans in the odontoblast-predentin region of rat incisor.

The localization of proteoglycans in the predentin of the rat incisor was investigated by ultrastructural histochemistry. Ruthenium red stained the cell coat of the odontoblasts as well as intracellular vesicles. There was also a staining of the extracellular matrix, but not of collagen fibers in the predentin. Treatment with the enzyme hyaluronidase prior to staining with ruthenium red abolished the staining of the vesicles and the extracellular matrix but not that of the cell coat. Bismuth nitrate and phosphotungstic acid gave similar staining of odontoblast vesicles and extracellular matrix. It is likely that the stained structures contain proteoglycans. The importance of these proteoglycans and their ultrastructural localization are discussed in relation to intracellular transport and the calcification process.

Animals↗
PubMed1976

Acid hydrolases in the odontoblast-predentin region of dentinogenically active teeth.

The presence of hyaluronoglucosidase (EC 3.2.1.35; hyaluronidase), beta-N-acetylglucosaminidase (EC 3.2.1.30), exo-1,4-beta-xylosidase (EC 3.2.1.37), and arylsulfatase (EC 3.1.6.1) in dentinogenically active odontoblasts isolated from the rat incisor has been demonstrated by means of biochemical methods. The possible function of these enzymes in relation to the calcification process is discussed.

Acetylglucosaminidase↗
PubMed1976

A comparison of ATP-degrading enzyme activities in rat incisor odontoblasts.

In active odontoblasts from the rat incisor, used as a model system for biologic calcification, two distinguishable enzyme activities capable of degrading adenosine monophosphate (ATP) exist. Once can be inhibited ny 1-tetramisole, (+/-)-2,3,5,6,-tetrahydro-6-phenylimidazo (2.1B) THIAZOLE HYDROCHLORIDE (Levamisol) and (+/-)-6(m-bromophenyl)-5.6-dehydroimidazo (2.1-b) thiazole oxalate (R823) and is probably identical with nonspecific alkaline phosphatase (EC 3.1.3.1). The activity of the other enzyme, named Ca2+-ATPase, is dependent on the presence of Ca2+ or Mg2+ and is activated by these ions. The pH optimum of Ca2+-ATPase is 9.8. The Ca2+-ATPase is unaffected by Levamisole, R 8231, ouabain, ruthenium red, Na+ and K+ ions. Maximal activity was found against ATP, whereas adenosine diphosphate, guanosine triphosphate, inosine triphosphate and adensoine monophosphate were hydrolysed at lower rate. It may be speculated that the Ca2+-ATPase is concerned with the transmembranous transport of Ca2+ ions to the mineralization front.

Adenosine Triphosphatases↗