THEODORE ROSETT, PAMELA S. GARNER, and LOUIS P. GANGAROSA
Department of Biochemistry, Temple University School of Dentistry, Philadelphia, Pennsylvania, 19140, USA and Department of Oral Biology, Medical College of Georgia, Augusta, Georgia
J Dent Res May-June 1972, Vol 51 No. 3
Previous studies (RoSETT ET AL, J Dent Res 50:161, 1971; GANGAROSA and ROSETT, J Dent Res 50:163, 1971) and the present one aim to determine physiologic and metabolic processes in human gingival tissue to discover how these processes are altered during the induction of periodontal disease. We chose cattle gingival epithelium to have a readily available supply of tissues.
In this study we investigated the normal levels of some enzymes involved in glucose metabolism within bovine attached gingival epithelium. In determining the relative levels of the enzyme in any particular series of metabolic pathways, it is emphasized that we are only reporting the potential for conversion of substrate to product. The existence of these pathways in tissues and their quantitation do not necessarily indicate their relative importance in normal metabolism, because of such variables as compartmentalization of various enzymes within the cell and the effect of control mechanisms such as hormonal or negative feedback. Since the problems of importance of pathways and control mechanisms can only be studied after the actual potential of the pathways are shown, we selected five enzymes involved in the preliminary steps of glucose metabolism. These studies involve disruption of gingival epithelium to release soluble enzymes and to render suspendable those enzymes that are classified as particulate. These enzymes then are assayed for their relative activity in micromoles per minute by addition of large excesses of commercially available enzymes and cofactors that lead to oxidation of TPNH or DPNH or reduction of DPN or TPN. Such reactions may be followed with the recording spectrofluorometer by using an activating wavelength of 340 nm and an emission wavelength of 460 nm. A spectrofluorometer* with a foursample changer and output recorder was used. In general the following approach in the study of each enzyme was followed. With the use of published values (BERGMEYER, Methods of Enzymatic Analysis, 1963, pp 697, 983, 992, 993, 1036; COLOWICK and KAPLAN, Methods in Enzymology, Vol I, 1955, pp 209, 294, 304, 323, 328, Vol V, pp 226; HALPRIN and OHKAWARA, J Invest Derm 46:43, 1966 and 46: 51, 1966) of additions of substrate, cofactors, or various coupling enzymes, we made a dilution of commercial enzyme sufficient to obtain a rate between 0.01 and 0.04 matmoles per minute. Then, using the same additions, dilutions of gingiva were made to obtain the same rate. After this, each addition was varied in turn to obtain maximal enzymatic velocity with the diluted gingiva. With these newly obtained values, commercial enzyme rates were obtained and then commercial enzyme and gingiva were tested together to ensure additive rates, ie, to ensure that no inhibitor was present.
The four-sample changer permitted controlled observation of each enzymatic conversion. The first cuvette contained either TPNH or DPNH in a known concentration so that the 100% span on the recorder could be set; the second cuvette contained a no-enzyme control; the third contained a no-substrate control; and the fourth was the experimental cuvette. The reactions were carried out at 30 C. (All commercial enzymes, substrates, and cofactors were obtained from the Sigma Co.)
The table shows that the largest potential for metabolism lies in the direction of the Embden-Meyerhoff pathway. Halprin and Ohkawara studied carbohydrate metabolism in human skin and the alteration in enzyme activities in psoriatic human skin by these methods. They found the highest potential for glucose metabolism in normal skin in the Embden- Meyerhoff pathway; in psoriasis there was a large shift toward the hexose monophosphate shunt pathway.
This work was supported, in part, by Grants GRS- 535-951-08, GM-16973, and DE-03-022 from the National Institutes of Health, Bethesda, Md and Grant DAHC-19-69-D-007 from the Army Research Office. Additional information available on request to authors. Received for publication June 11, 1971.