John S. Brand

Assay of picomole amounts of ATP, ADP, and AMP using the luciferase enzyme system.

Procedural modifications of the luciferase method for ATP assay in conjunction with enzymatic conversion of AMP and ADP allow the assay of all three adenine nucleotides in quantities ranging from 4 to 20 pmoles. An unmodified Beckman scintillation detector at ambient temperature and in a coincidence mode of operation serves as a suitable instrument for quantitating light emitted by the enzyme preparation. The most significant modifications include use of Ca3(PO4) activated crude arsenate extracts of desiccated firefly lanterns, low arsenate concentrations during the assay, and an assay pH of 8.0. Extracts handled in this manner exhibit approximately fivefold higher activity than nonactivated extracts employed at pH 7.4 and 50 mm arsenate. Stability of activated extracts is also somewhat greater than for nonactivated preparations. ADP can be 95% enzymatically converted to ATP by treatment with phosphoenolpyruvate and pyruvate kinase under the conditions described. If myokinase is included, approximately 90% of sample AMP can be converted to ATP. Follwing the appropriate enzymatic treatment, the nucleotides are assayed as ATP and amounts calculated by comparison to curves established for known nucleotide standards. The method is appropriate for perchloric acid extracts of biological tissue and certain considerations necessary for application to experimental situations are described.

Effect of transforming growth factor-β on DNA synthesis by growth plate chondrocytes: Modulation by factors present in serum

SummaryChondrocytes in the growth plate undergo rapid proliferation during the process of enchondral ossification. The factors that regulate this proliferative activity have not been defined although several local autocrine or paracrine growth factors have recently been discovered in cartilage. Transforming growth factor-β1 (TGF-β) is an important regulator of cell metabolism and growth and is present in cartilage. The present study was designed to examine the influence of TGF-β on DNA synthesis in chick epiphyseal chondrocytes. Chondrocytes were plated in serum-free (BSA-supplemented) medium or medium containing 10% FBS, and after 24 hours in monolayer culture, were treated with TGF-β caused a biphasic dose-dependent increase in thymidine incorporation. The effect was greatly increased in serum-containing cultures where a maximal 20-fold increase in thymidine incorporation occurred compared with the 21/2-fold maximal increase obtained in serum-free cultures. The effect was present by 12 hours and maximal between 0.3 and 1.0 ng/ml of TGF-β. Higher concentrations of TGF-β were less stimulatory. The serum enhancement was dependent upon the simultaneous presence of TGF-β and serum factors and was abolished when chondrocytes were plated and exposed to TGF-β in medium containing dialyzed FBS (12–14 kD MW membrane). The nature of the uptake and incorporation of thymidine into DNA by individual chondrocytes appeared to be the same in both TGF-β-treated and control cultures as the apparent Kms were similar. The present study indicates that TGF-β increases DNA synthesis by growth plate chondrocytes. The effect is enhanced by factors present in serum.

Fine structure of fetal rat calvarium; provisional identification of preosteoclasts

SummaryThis is a study of the fine structure of cells of the 20-day fetal rat calvarium. Special attention is given to identifying and characterizing preosteoclasts. These cells are relatively common and located largely, but not exclusively, at the endocranial bone surface. The preosteoclasts are characterized by abundant mitochondria, an incomplete perinuclear Golgi apparatus, and variable-shaped dense granules. The dense granules are unique in appearance in that they contain an internal dense matrix surrounded by a clear halo. Most granules are circular in shape but some are elongate or tubular in form. Granules with identical appearance are observed in osteoclasts. The preosteoclasts are mononucleate, or occasionally binucleate. It is suggested that because preosteoclasts are morphologically distinctive and relatively abundant, it should be feasible to separate these cells from a heterogeneous cell isolate.

Enzymatic isolation of cells from neonatal calvaria using two purified enzymes from Clostridium histolyticum

The enzymatic isolation of cells with bacterial collagenase has proved to be a powerful technique for the study of a wide variety of tissues. Unfortunately, for some applications such as the isolation of cells from membranous bone, the cellular damage that results from the exposure of the cells to cytotoxic contaminants of bacterial collagenase has limited the usefulness of this approach. The use of chromatographically purified collagenase alone is often ineffective or very slow to release cells from tissue. We have found that two enzymes are necessary and sufficient to isolate cells from neonatal mouse calvaria: purified collagenase and neutral protease. These two enzymes can be chromatographically purified on a preparative scale to yield 100 mg amounts of each enzyme. The purified enzymes can be recombined in amounts which will digest calvaria at the same rate as the crude bacterial collagenase from which they were derived. The cells that are isolated using the purified enzymes are undamaged, as indicated by the measurement of their equilibrium density on gradients of Ficoll and sodium metrizoate. Cells isolated with crude collagenase never reach an equilibrium density upon isopyknic centrifugation, whereas cells isolated with the purified enzymes reach an equilibrium density of 1.074 g/ml in 90 min.

The Effect of Bone Cell Stimulatory Factors Can be Measured with Thymidine Incorporation Only Under Specific Conditions

SummaryThis study examines the efficacy of using radiolabeled thymidine as a measure of bone cell DNA synthesis. The effect of a bone-active peptide on cell proliferation is assessed under different labeling conditions and it is shown that the apparent stimulation in DNA synthesis is due to an increase in participating cells and not to labeling artifacts. In another series of experiments we demonstrate how the use of carrier-free thymidine can cause erroneous results. From these data it is shown that only 10–28% of the cells in culture are participating in DNA synthesis. Therefore, under specific conditions, a 100% stimulation in thymidine incorporation by a mitogenic factor can be caused by as little as a 10% increase in the number of DNA synthesizing cells.

Collagenolytic activity from isolated bone cells

A true collagenase (EC 3.4.24.3) which had been discovered previously in bone culture fluids and extracts of whole bone has now been localized to the cellular component of bone. The cellular enzyme bears the same characteristics as that of bone collagenases described earlier. Moreover, it is directly extractable in relatively large quantities. Attempts to demonstrate the presence of a bone cell procollagease were unsuccessful. It was also observed that the cells secrete significant amounts of collagenase in vitro. With increasing incubation time the extracellular collagenase levels rise and the intracellular collagenase levels drop.

In vitro use of vitamin D3 metabolites: Culture conditions determine cell uptake

SummaryThis report demonstrates that routine variations in cell culture conditions dramatically affect the amount of vitamin D3 metabolites to which cultured cells have access. Increasing the concentration of a metabolite in the medium increases the amount of the metabolite in the cell compartment. Increasing the volume of medium in the culture dishes (while maintaining a constant metabolite concentration) also increases the amount of metabolite in the cell compartment. Moreover, daily changes of the medium containing fresh metabolite increase the amount of the metabolite in the cell compartment as well. These variables may explain the inability of different laboratories to duplicate dose-response curves.

The reaction of 3-diazonium-1,2,4-triazole with tryptophan at acid pH: A scintillation method for tryptophan determination in proteins

Unlike conventional aryl diazonium reagents which are usually reactive at alkaline pH, 3-diazonium-1,2,4-triazole (3-DT) has been found to react with a variety of proteins at acid pH. Using indole, phenol, and imidazole as models of aromatic amino acid side chains, the rate of 3-DT coupling to the indole nucleus has been shown to increase exponentially from m −1 min−1 at pH 6 to > 2400 m −1 min−1 at pH 2.5. Coupling rates with phenol and imidazole show the conventional alkaline reactivity, with no evidence of reaction at acid pH. In parallel experiments using trypsin, 3-DT was found to cause loss in enzymatic activity at acidic as well as alkaline pH. Furthermore, amino acid analysis has demonstrated that the acidic reaction with trypsin resulted in the modification only of tryptophan. Based on the acidic specificity of 3-DT seen in these studies, a scintillation method for determining the tryptophan content of proteins has been developed. Using 3-diazonium-1,2,4-[5-14C]triazole, accurate tryptophan contents for trypsin, chymotrypsin, lysozyme, pepsin, insulin, and fibrinogen have been measured.

Isolation and Characterization of Epithelial and Connective Tissue Cells from Rat Palate

Epithelial and connective tissue cells were isolated from rat palate by sequential enzymatic digestion. Differences between the two populations were noted with respect to proline uptake and incorporation, % collagen synthesized, effects of parathyroid hormone and metabolism of D-valine. From these studies it can be concluded that the cell populations are viable and distinct with respect to the biochemical parameters examined.

Application of Light Sensitive Chemicals to Probe Protein Structure and Function

These results suggest that important information regarding the conformation of complementary binding sites and the behavior of various charged states of inhibitors can be obtained from studies of the pH dependence of photoaffinity labeling experiments. This information may assist in identifying the pKas of key residues controlling the binding interaction. This technique should prove very useful in probing binding site structure on nonfunctioning targets, such as enzyme zymogens, as well as complicated receptor interactions.