Maleate Buffer Research Articles

Histochemical demonstration of enterokinase.

A new simple method for the histochemical demonstration of enterokinase (enteropeptidase, EC 3.4.4.8) was elaborated. Unfixed cold microtome sections adherent to the slides are covered with agar-gel medium containing trypsinogen (0.5–1 mg per 1 ml), Nα-benzoyl-DL-arginine-β-naphthylamide hydrochloride (2–4 mM), and Fast Blue B Salt (0.5 mg per 1 ml) in 0.05 M Tris maleate buffer pH 6.5 with 0.05% CaCl2. After the incubation in a wet chamber at 37° C the slides are chelated in 2% copper sulphate. The method enables a localization on the histological level. The evaluation of the overall staining obtained with this method in sections of the duodenum, jejunum and ileum of guinea pigs, rats, monkeys, dogs and humans reflects well the biochemical quantitative data on enterokinase activity obtained in homogenates of mucosal scrapings of the corresponding gut segments of the same animals. In all animals a proximodistal gradient was found. The highest activities were found in enterocytes covering apical parts of villi in the duodenum. According to the activities in the duodenum the investigated animals can be arranged in the following series: guinea pig (highest activity), monkey, man, rat and dog.

A kinetic method for determination of plasma renin activity using radioassay of Angiotensin I.

A kinetic method for determination of plasma renin activity using radioimmunoassay of angiotensin I as the indicator system is presented. Angiotensin I is generated at 37 C. in plasma with the pH adjusted to 6.0 with maleic acid and ammonium maleate buffer. Chelating agents prevent the degradation of the angiotensin I product. Plasma samples from a small series of subjects on unrestricted sodium diets were analyzed with three replicate determinations. The mean values of 2.40 m μ g. and 5.38 m μ g. of angiotensin I per ml. per hr. were obtained after recumbency overnight and following 4 hours of activity, respectively. Run-to-run coefficient of variation was approximately 10%.

Histochemical evidence and consequences of the occurrence of isoenzymes of acetylcholinesterase.

Frozen sections of unfixed cat stellate (StG) and ciliary (CG) ganglia and intercostal muscle (ICM) and mouse ICM were exposed to saline (0.9% NaCl) solution and to 0.3% Triton X-100 in saline solution, and then stained for acetylcholinesterase (AChE) by a modification of the standard copper-thiocholine method. In the StG and CG, treatment with saline solution resulted in marked diffusion of AChE, and Triton X-100 caused its marked loss from the CG and nearly complete loss from the StG. In ICM, in contrast, both treatments caused increased intensity of staining of AChE at the motor end plates (MEP's). All of the foregoing effects were reversed progressively, with concomitant reduction in AChE activity, by prior fixation in cold formaldehyde (4%)-sucrose (7.5%)-maleate buffer (pH 7.3) solution for 2-24 hr. Results are interpreted on the basis that the AChE of the ganglia and MEP's exists predominantly in different isoenzymatic forms, the possible natures of which are discussed.

Studies on a testosterone glucuronyltransferase from the cytosol fraction of human liver.

An enzyme that conjugates the 17beta-hydroxyl group of testosterone was found in the cytosol fraction of human liver. The same enzyme preparation also conjugates the 16alpha-hydroxyl group of oestriol. The enzymic activity could not be sedimented by centrifuging the cytosol fraction at 158000g(av.) for 120min. The testosterone-conjugating as well as the oestriol-conjugating activities were found in the precipitate obtained after 30% saturation of the cytosol fraction with ammonium sulphate. Filtration of the precipitate through Sephadex G-200 enriched the testosterone-conjugating enzyme 50-fold and the oestriol-conjugating enzyme 100-fold. No separation of the two activities was achieved. With labelled testosterone the product of the reaction, testosterone 17beta-glucuronide, was identified by paper chromatography and by crystallization to constant specific radioactivity. Testosterone 17beta-glucuronyltransferase was active between pH7.0 and 8.6 in tris-HCl and tris-maleate buffers. The apparent K(m) values for testosterone and UDP-glucuronic acid were 6.4 and 25mum respectively. The enzyme was active between 37 and 45 degrees C; the activation energy was calculated to be 5kcal/mol. Oestriol did not influence the glucuronidation of testosterone. Controlled heating as well as alternate freezing and thawing of the purified enzyme preparation led to an inactivation of both testosterone-conjugating and oestriol-conjugating activities at similar rates. Testosterone and oestriol, when incubated together, gave a reaction rate that was approximately equal to the sum of the rates when the two substrates were incubated separately. The present findings suggest that testosterone and oestriol are conjugated by two separate enzymes.

The Pyruvate Formate-lyase System of Streptococcus faecalis: I. Purification and Properties of the Formate-Pyruvate Exchange Enzyme

Abstract The enzyme exchanging formate with the carboxyl of pyruvate was purified 195-fold from extracts of Streptococcus faecalis by a procedure involving protamine treatment, dialysis, and gradient centrifugation. Centrifugation of protamine-treated extract in a 10 to 40% potassium tartrate gradient provided a 65-fold purification and separated the exchange enzyme from pyruvate decarboxylase, acetolactate synthetase, acetolactate decarboxylase, and phosphate-acetyltransferase. A second centrifugation in a 20 to 30% potassium tartrate gradient provided an additional 3-fold purification. The purified preparation contains diphosphothiamine but not coenzyme A, folate, lipoate, biotin, pyridoxal, pyridoxamine, Vitamin B12, or flavin. The enzyme has an average molecular weight of 268,000 to 288,000 as estimated by gradient centrifugation and by gel filtration. Purified preparations do not degrade pyruvate. S-Adenosyl-l-methionine has no effect on the purified enzyme; the apparent stimulation of activity with crude extracts apparently results from inhibition of competing reactions. The purified enzyme is rapidly oxidized and inactivated, but it can be stabilized in 30% potassium tartrate containing a reducing complex of 0.001 m FeSO4 and 0.003 m 2,3-dimercaptopropanol. Tris, maleate, and imidazole acetate buffers will substitute for phosphate buffer and phosphate ion is not required for the reaction. Optimal activity is observed over a range between pH 6.7 and 8.7. Purified extracts will exchange formate-14C with the carboxyl group of oxalacetate, α-ketoglutarate, and α-ketobutyrate. The velocities relative to the formate-pyruvate exchange rate of 100% were 100%, 9.9%, and 0.9%, respectively. Purified extracts give Michaelis constants of 0.02 m for pyruvate and 0.058 m for formate. Chloride ion produces an irreversible inhibition of exchange activity. Arsenite (3 x 10-5 m), p-chloromercuribenzoate (2 x 10-3 m), and sodium hypophosphite (6 x 10-6 m) inhibit exchange activity of the crude extract to the extent of 41%, 55%, and 47%, respectively.

Oligosaccharidases from Macracanthorhynchus hirudinaceus (Acanthocephala) from swine

1. 1. Enzymatic activities of cell-free extracts of Macracanthorhynchus hirudinaceus indicates that maltose, turanose and trehalose are hydrolyzed but with maltose being broken down four times as fast as any other disaccharide tested. 2. 2. M. hirudinaceus cell-free extracts readily hydrolyze dextrin but not: (1) the polysaccharide, inulin (2) the trisaccharides, raffinose and melizitose (3) disaccharides, sucrose, lactose and melibiose (4) methyl substituted aldohexoses, α- D-methyl glucopyranoside and α- D-methyl mannoside. 3. 3. One isozyme of maltase was observed using para-nitrophenyl-α- D glucopyranoside in starch gel electrophoresis. 4. 4. The pH optimum for maltase was 4·5 using 0·2 M Tris malate buffer, but with 0·2 M acetic acid-sodium acetate buffer, the pH optimum was 5·0. The optimum temperature in either case was 50°C. 5. 5. Trehalase had a pH optimum of 5·8 and an optimum temperature of 45°C using 0·2 M phosphate buffer.

Accelerating Effects of Indolederivatives and Metallic Ions on the Extension of Lamina Joints in Excised Rice Leaves

This paper reports the actions of indolederivatives, metallic ions, surfactant and chelating. agents on the cell elongation in lamina joints of rice. The angles increased at 5×10-7 or 10-6M of indoleacetic acid, at 10 M of indoleacetonitrile and indolebutyric acid and at 10-4M of indolepropionic acid. But no increment of the angle was obtained by tryptophan at any concentrations used. As regards to actions of metallic ions, a slight effect of about 15 per cent increment of control was presented at 5×10-6M of cupric ion, nevertheless any effect was not obtained at various concentrations of manganous, ferrous and cobaltous ions. The promotive effect of cupric ion was shown at presence of indoleacetic acid but not of 2, 4-dichlorophenoxyacetic acid. However a promotive effect of tween 20 was shown either absence or presence of indoleacetic acid at low concentrations. The effect of ethylenediaminetetraacetate upon the cell elongation was very excellent at absence of potassium maleate buffer but not the presence of the buffer solution. In addition, several problem upon the cell elongation in plants discussed in this paper on the action of the substances using in this experiment.

Iron Translocation I. Plant Culture, Exudate Sampling, Iron-Citrate Analysis

Plant culture, exudate sampling, and analytical methods designed to ascertain the form of iron translocated are presented.Restoration of iron to sunflower plants precultured at different Fe levels resulted in exudate iron concentrations ranging from 0.2 to 31 x 10(-5)m. Citrate was from 3 to 89 x 10(-5)m. Iron and citrate were highest in exudates from iron-deficient plants. Citrate/Fe ratios were between 1 and 3 for exudates of deficient plants. Exudate from normal plants gave a citrate/Fe ratio of 15.Malate, iron, and a fraction of the citrate in stem exudates migrated electrophoretically to similar positions in acetate buffer. Extracts of narrow bands from the iron-containing areas gave curves suggesting that citrate bound the iron. Citrate that was not combined with iron migrated in a slower band. The effect of iron on citrate migration was confirmed in several related experiments.The stability of Fe-citrate was demonstrated electrophoretically in malate buffer. Citrate retained iron against malate.Data given in this paper indicate that citrate binds iron in sunflower exudate. The data suggest that citrate carries iron in intact plants.

REDUCED LIVER GLUCOSE-6-PHOSPHATASE IN HUMAN LEUKEMIA.

During experiments concerned with rat liver glucose-6-phosphatase it became of interest to study human liver glucose-6-phos-phatase. Specimens of human liver obtained during post-mortem examinations were homogenized and assayed immediately. It was noted that each of the livers from children with acute lymphatic leukemia was very deficient in this enzyme activity. Some of the mechanisms responsible for this reduction in activity were investigated and form the basis of this report. Methods. At time of autopsy the patient's age, diagnosis, drug therapy, and hours since death were recorded. A 10 g specimen of liver was placed immediately in 0.12 M KC1 at 0°C, and was kept at 0°C during subsequent steps. A 10% homogenate was prepared using a glass-teflon homogenizer at 1000 rpm for 2 to 3 minutes. The homogenate was centrifuged at 600 × g for 10 minutes and the residue was discarded. The supernatant was centrifuged in a Servall refrigerated centrifuge at 37000 × g for 30 minutes and the residue was suspended in 0.12 M KC1 at a concentration 5 to 10 mg protein per ml. It had been previously demonstrated that rat liver glucose-6-phosphatase activity was purified 3-fold in this residue, when compared to liver homogenate. The activity of the 37000 Xg residue was determined by the method of Swanson(l), at pH 6.5 in malate buffer. Protein was determined by Lowry's(2) method. The effects of protein synthesis inhibitors on enzyme activity were determined in two ways. First, 6-mercaptopurine, methotrexate (4-amino-N-methylpteroyl glutamic acid), and cortisol were added during the assay of enzyme activity. Second, 6-mercaptopurine (1.0 mg/150 g rat/day), and methotrexate (0.1 mg/150 g rat/day) were fed in 2 groups of rats with their regular Purina Laboratory Chow diet, and other rats Received 100 μg actinomycin D by intraperitoneal injection daily for 4 days.

Prevention by 2,4-Dichlorophenoxyacetic Acid of an Increase in the Rate of Phosphate Utilization, caused by the Prolonged Treatment of Segments of Pea Stems in Maleate Buffer

WHILE examining the influence of 2,4-dichlorophenoxyacetic acid (2,4-D) on the absorption of ions by epicotyl segments of etiolated peas, it was established that if freshly cut segments are first placed in aerated, distilled water or 0.01 M maleate buffer (pH 5.2) for 12 h, their ability to absorb and esterify inorganic phosphate in a given time (1 h) is increased 12-fold. This marked increase in the rate of phosphate absorption necessitated further study before the effects of 2,4-D on the uptake of ions could be further elucidated. On the basis of this work, which has been reported elsewhere1, it is concluded that one of the basic changes occurring in the segments during the period in buffer or water is an increase in the rate of synthesis of protein, which in turn results in an increased rate of absorption of phosphate. An increased turnover of protein when pea segments were placed in water for 24 h was reported by Christiansen and Thimann2 in 1950. Recently, Click and Hackett3 have suggested that the increased respiratory rate and other metabolic changes occurring when potato slices are similarly treated depend on the synthesis of new RNA and protein.

UREOLYTIC RUMEN BACTERIA. II. EFFECT OF INORGANIC IONS OF UREASE ACTIVITY.

The urease activity of whole cell preparations of the mixed rumen microflora was reduced when the cells were washed with maleate buffer. Resuspension of the washed cells in rumen supernatant liquor (RSL) enhanced their urease activity owing to the presence of an inorganic urease-stimulating factor in RSL. When the whole cells were washed in buffer there was a progressive reduction in the urease activity of the cells as the number of washings increased. This effect was attributed to progressive removal from the cells of the urease-stimulating factor. The urease activity of preparations of whole cells washed three times in buffer was stimulated in the presence of Mn2+, Mg2+, Ca2+, Sr2+, and Ba2+, and inhibited in the presence of Na+, K+, and Co2+. These ions brought about similar effects on the urease activity of acetone-dried powders and cell-free extracts of washed rumen microorganisms. The activity of jack-bean urease was reduced in the presence of the ions which stimulated rumen urease.When inorganic ions were added individually to suspensions of washed rumen microorganisms there were time lags before the ions exerted their stimulating or inhibiting effects on urea hydrolysis. Once the stimulation or inhibition of activity by an ion had begun, however, the reaction proceeded at a constant rate which was characteristic of the ion tested. Modification of the permeability properties of washed cells of rumen microorganisms by acetone treatment modified but did not eliminate the time lags. When a cell-free extract of washed cells was tested, Mn2+, Mg2+, Ca2+, Co2+, and K+all influenced urease activity immediately upon addition to the extract, whereas Sr2+, Ba2+, and Na+were active only after a lag period.The urease produced by rumen bacteria is considered to be a metal-activated, intracellular enzyme. The urease-stimulating factor present in the cells was not bound to any appreciable extent intracellularly. The time lags observed with the whole cells were attributable in most cases to the time taken for the ions to penetrate the cells; with Sr2+, Ba2+, and Na+they were apparently due, at least in part, to a delay in the formation of an active complex between ion and enzyme.

Permeation of Amino Acids in Moniliformis and Macracanthorhynchus (Acanthocephala)

Both sexes of Moniliformis and Macracanthorhynchus concentrate methionine against a chemical difference. The uptake of methionine by both sexes of Moniliformis is inhibited by alanine, serine, leucine, and isoleucine-these inhibitions are reciprocal. The uptake of serine and alanine is not affected by methionine in female Macracanthorhynchus; however, methionine does inhibit the accumulation of those amino acids in males of Macracanthorhynchus. The effect of a mixture of four neutral amino acids on the uptake of methionine can be predicted by the generalized equation derived by Read et al. (1963, Ann. N. Y. Acad. Sci. 113 Art. 1: 154-205). Considerable information is available concerning the entrance of potential nutritional substances into cestodes (see the review by Read and Simmons, 1963, as well as Read, Rothman, and Simmons, 1963); however, there is a paucity of such data pertaining to that process in the Acanthocephala (Laurie, 1957, 1959). Like the cestodes, Acanthocephala have no gut and it is assumed that nutrients enter through the body wall. Read et al. presented exhaustive data concerning the initial rates of entry of a number of amino acids into Hymenolepis diminuta. In that study they demonstrated that amino acids were concentrated against a chemical gradient and that there was competitive inhibition between certain groups of amino acids. Those authors further demonstrated that there was inhibition of uptake of a given amino acid in the presence of a complex mixture of amino acids and that this competition could be predicted by an extension of the Lineweaver-Burk treatment of enzyme kinetics. The following study was undertaken: (1) to obtain information concerning the permeation of a selected group of neutral amino acids into two species of acanthocephalans and (2) to Received for publication 20 March 1964. * This research was supported in part by U. S. Public Health Service Grants AI-01384 and 2E-106. t Present address: California State College, Fullerton, California. further test the application of the generalized equation of Read et al. (vide supra). MATERIALS AND METHODS Acanthocephala used in this study were obtained from two sources. Moniliformis dubius were reared in male Sprague-Dawley rats (Holtzman Rat Co.). Cystacanths of Moniliformis were obtained from laboratory-reared and infected Periplaneta americana. Rats weighing 70 to 90 g were infected with 25 12-week-old cystacanths. The hosts were maintained in groups of ten in 24by 18by 12-inch wire cages in air-conditioned quarters: Food and water were provided ad lib. until the time of autopsy at 5 weeks. Rats were killed by a blow on the head and the small intestine removed to a container of saline composed of NaCl 120 mM, KCI 4.8 mM, CaCl2 2.6 mM, MgSO4 1.2 mM, and Tris (hydroxymethyl) aminomethane: maleate buffer 25 mM at pH 7.4. The intestine was opened with scissors and the worms gently removed from the mucosa. Macracanthorhynchus hirudinaceus were carefully dissected from swine small intestines at the Houston Packing Company and transported to the laboratory in the above saline in thermos containers at 36 to 39 C. Both species of parasites were separated according to sex and treated separately throughout the remainder of the experiments. The worms were preincubated in 30-ml beakers in the above saline for 60 min at 37.5 C. Following the preincubation period the worms were blotted and incubated in 30-ml beakers containing the amino acid solution (10.0 ml for Macracanthorhynchus, 5.0 ml for Moniliformis) for 3 min. The various C14 amino acids were used at a specific activity of 0.6 ,uc/,mole. After 3 min (-+5 sec) incubation, the worms were removed, rinsed rapidly in three changes of the saline (50 ml each), blotted, and placed in tubes containing a

HYDROLYTIC ENZYMES OF RABBIT MONONUCLEAR EXUDATE CELLS. I. QUANTITATIVE ASSAY AND PROPERTIES OF CERTAIN PROTEASES, NON-SPECIFIC ESTERASES, AND LIPASES OF MONONUCLEAR AND POLYMORPHONUCLEAR CELLS AND ERYTHROCYTES.

Oil-induced mononuclear phagocytes (MN) were quantitatively assayed for various hydrolases as unfractionated suspensions of frozen and thawed cells. They apparently contain two proteases. The first, measured with urea- or acid-denatured hemoglobin, was similar to purified Proteinase I of lung with respect to pH optimum (pH 4), stability, hydrolytic and polymerizing activities, and reactions to various inhibitors. The second protease resembled chymotrypsin in its hydrolysis of glycyl-L-phenylalanine amide, acetyl-L-tyrosine ethyl ester and N-benzoyl-DL-phenylalanine-beta-naphthol ester (BPN). With the latter, its pH optimum was between 5.0 and 5.8, and its action was inhibited by diisopropylphosphorofluoridate (DFP) and p-chloromercuribenzoate. When assayed under the above conditions, polymorphonuclear exudate cells (PMN) and red blood corpuscles (RBC) manifested little or no hydrolysis of either hemoglobin or BPN. MN also contained esterases that split methyl butyrate and beta-naphthyl acetate. The pH optimum with the latter was 7.4, and its hydrolysis was partially inhibited by DFP, fluoride, taurocholate, and eserine. PMN had low esterase activity; RBC had little or none. MN, but not PMN or RBC, contained a stable lipase with a pH optimum of 6.1 in maleate buffer. Protamine, NaCl, heat, p-chloromercuribenzoate, ethylenediamine tetraacetate, taurocholate, and DFP were inhibitory, but no appreciable activation occurred in the presence of heparin or serum. Thus it possessed some of the characteristics of Korn's lipoprotein lipase, but not others.

FORMATE-PYRUVATE EXCHANGE REACTION IN STREPTOCOCCUS FAECALIS. I. FACTOR REQUIREMENT FOR INTACT CELLS.

Wood, N. P. (A. & M. College of Texas, College Station), and D. J. O'Kane. Formate-pyruvate exchange reaction in Streptococcus faecalis. I. Factor requirement for whole cells. J. Bacteriol. 87:97-103. 1964.-A factor present in plant and animal sources was found necessary for the incorporation of formate-C(14) into pyruvate by Streptococcus faecalis 10Cl. Yeast extract produced a response linear in the range between 10 and 30 mg/ml of reaction mixture. Soy peptone, beef peptone, and Brain Heart Infusion replaced yeast extract, but various intermediates, cofactors, amino acids, purines, pyrimidines, and peptides did not stimulate the reaction. A lag occurred in the rate of formate incorporation that was not influenced by anaerobic conditions or growth of cells in a medium containing pyruvate and formate. Phosphate or maleate buffer permitted rapid exchange velocities but tris(hydroxymethyl)aminomethane or collidine buffer was inhibitory. Heating yeast extract at 121 C for 15 min in 3 n H(2)SO(4) produced 66% inactivation of the factor(s), whereas treatment with 3 n KOH produced 97% inactivation. The factor(s) was insoluble in butanol, benzene, ethyl acetate, or chloroform. The material adsorbed on Dowex-1 (OH(-)) and Amberlite IR-120 (H(+)) but not on Amberlite IR-4B (OH(-)). The active component(s) was highly polar, nonvolatile, dialyzable, and had amphoteric properties.

The manometric determination of thiamine pyrophosphate and the inhibition of the acid yeast phosphatase

Sodium molybdate is a powerful inhibitor of the acid yeast phosphatase in both fresh baker's yeast and dried brewer's yeast, provided that the yeast is suspended in a suitable buffer. It displays no action in citrate or phosphate buffers, but is active in acetate or maleate buffers, both at the optimum pH of 3·7 of the phosphatase and at pH 6.2. Depending upon the relative concentrations of yeast and phosphate ester substrate, either 0.1 m M or 1 m M molybdate gives adequate protection against hydrolysis. In the manometric determination of thiamine pyrophosphate molybdate can replace thiamine as inhibitor of acid phosphatase with full retention of sensitivity if the assay is performed in 0.1 M maleate (pH 6.2) instead of 0.1 M phosphate (pH 6.2). In studies of the phosphorylation of thiamine and its antagonists by ATP molybdate has also proved to be useful for inhibiting the acid phosphatase present in preparations of yeast thiaminokinase.

Sur l'oxydation de l'acide maléique et de l'acide fumarique en présence d'adrénochrome

Spectrographic and manometric study, at 37°C, of the oxidation reaction of adrenalin by oxygen (Cu ++ acting as catalyst), in the presence of potassium maleate buffer solution (pH 6.5), shows that, under these conditions, adrenalin is transformed into adrenochrome. However, adrenochrome thus formed undergoes very slowly oxidative transformation and the large consumption of oxygen measured depends on the oxidation of the maleate by adrenochrome. The control tests show that neither copper nor adrenalin catalyses the oxidation of maleate in an oxygen atmosphere; in a nitrogen atmosphere the adrenalin-copper system is equally without effect. During the oxidation of adrenalin in phosphate buffer solution, the maleate (or fumarate) added is susceptible to oxidation because of the intervention of adrenochrome, formed intermediately. However, under these conditions, adrenochrome undergoes a subsequent oxidative transformation nearly as rapid as if the reaction had taken place in pure phosphate buffer solution.

The Effects of Various Ions on Enamel Solubility

Several reports in the literature indicate that the solubility of enamel in acids can be changed by the action of various ions. Miller (1) using silver nitrate was unable to show retardation in enamel decalcification. The research work of Rickert (2) contradicted this and led him to believe that increased resistance to decalcification of silver nitrate-treated enamel was due to the clogging of tooth substance with reduced silver which prevented the penetration of acids. Hill and Arnold (3) studied the effects of silver nitrate on powdered enamel and found that after treatment, the solubility of enamel was significantly reduced. Difference in enamel solubility in acetate and maleate buffers was attributed by Benedict and Kanthak (4) to the formation of calcium maleate which acted as a protective coating over the enamel particles. Volker (5) and Bibby (6) investigated the effect of fluorides on enamel and concluded that the fluorine ion produced marked reduction in solubility. Because little or no consideration has been given to the effects of other ions on the solubility of enamel, it seemed to us worthwhile to investigate the matter. The choice of the ions used in the experiments was determined by a consideration of the mechanisms whereby the phenomenon of reduced solubility might occur. In order to better understand these, an examination of the chemical nature of enamel is necessary. This will be limited to facts relevant to the adequate understanding of certain assumptions which are made in this paper. Tooth enamel is a crystalline substance showing a characteristic x-ray diffraction pattern. Moreover, this pattern is practically identical with that given by bone, dentin and certain synthetic or natural inorganic materials, such as fluorapatite. On the basis of these x-ray findings these substances have been included in the general class of apatites. It should be remembered that this apatite classification refers only to a particular atomic pattern or arrangement in the crystal lattice and not to any constancy of chemical composition. For instance, McConnell (7, 8) has pointed out that a large number of substitutions are possible in the general apatite pattern. If X10(ZO4)6. (F20H2C12) represents the general formula for the apatites, then X may be calcium, lead, manganese, potassium, strontium and cesium or in lesser amounts yttrium, lanthanum, titanium, iron, aluminum and magnesium; Z may be chromium, phosphorous, arsenic, vanadium, silicon, and carbon. Further evidence of the complex and variable nature of the apatites is obtained from the work of Eisenberger, Lehrman and Turner (9). In their critical review of the literature on the system