26 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (15) J. A. Swift and B. Bews, The chemistry of human hair cuticle. II. The isolation and amino acid analysis of the cell membranes and A-layer, J. Soc. Cosmet. Chem., 25, 355-366 (1974). (16) E. S. Cooperman and V. L. Johnsen, Penetration of protein hydrolysates into human hair strands, Cosmet. Perfum., 88, 19-22 (1973). (17) L. Lorand and P. Stenburg, "Endo-•/-Glutamine: e-Lysine Transferases: Enzymes Which Cross-Link Proteins," in Handbook of Biochemistry and Molecular Biology, Proteins, Vol. 2, G.D. Fasman Ed. (CRC Press, Cleveland, 1976), pp. 669-684. (18) L. Lorand, Fibrinoligase: The fibrin-stabilizing factor system of blood plasma, Ann. N.Y. Acad. Sci., 202, 6-30 (1972). (19) R. B. Credo, C. G. Curtis, and L. Lorand, Ca2+-related regulatory function offibrinogen, Proc. Natl. Acad. Sci. USA, 75, 4234-4237 (1978). (20) P. J. A. Davies, D. R. Davies, A. Levitzki, F. R. Maxfield, P. Milhaud, M. C. Willingham, and I. H. Pastan, Transglutaminase is essential in receptor-mediated endocytosis of ot2-macroglobulin and polypeptide hormones, Nature, 283, 162-167 (1980). (21) H. G. Williams-Ashman, A. C. Natides, S.S. Pabalan, and L. Lorand, Transamidase reactions involved in the enzymic coagulation of semen: Isolation of •/-glutaminyl-e-lysine dipeptide from clotted secretion protein of guinea pig seminal vesicle, Proc. Natl. Acad. Sci. USA, 69, 2322-2325 (1972). (22) G. E. Siefring, Jr., A. B. Apostol, P. T. Velasco, and L. Lorand, Enzymatic basis for the Ca 2 +- induced cross-linking of membrane proteins in intact human erythrocytes, Biochemistry, 17, 2598- 2604 (1978). (23) L. Lorand, L. K. H. Hsu, G. E. Seifring, Jr., and N. S. Rafferty, Lens transglutaminase and cataract formation, Proc. Natl. Acad. Sci. USA, 78, 1356-1360 (1981). (24) M. M. Buxman, C. J. Lobitz, and K. D. Wuepper, Epidermal transglutaminase, J. Biol. Chem., 255, 1200-1203 (1980). (25) J. E. Folk and J. S. Finlayson, Transglutaminases, Adv. Protein Chem., 31, 1-133 (1977). (26) J. M. Connellay, S. I. Chung, N. K. Whetzel, L. M. Bradley, and J. E. Folk, Structural properties of guinea pig liver transglutaminase, J. Biol. Chem., 246, 1093-1098 (1971). (27) J. E. Folk, J.P. Mullooly, and P. W. Cole, Mechanism of action of guinea pig liver transglutam- inase, J. Biol. Chem., 242, 1838-1844 (1967). (28) M. Gross, N. K. Whetzel, and J. E. Folk, Amine binding sites in acyl intermediates of transglu- taminases, J. Biol. Chem., 252, 3752-3759 (1977). (29) K. Mori, T. Miyamoto, and H. Nakayama (to Kanebo Co., Lt_d_.), Japan Patent Application 1-233519 (1991). (30) M. Satao, Y. Ohtakachi, H. Kamisaka, and M. Suzuki (to Pola Kasei Kogyo Co., Ltd.), Japan Patent Application 1-173275 (1991). (31) T. Miyamoto, (to Kanebo Co., Ltd.), Japan Patent Application 1-25198 (1990). (32) M. Ogawa, N. Ito, and K. Sekiguro (to Ajinomoto Co., Ltd.), Japan Patent Application 2-2771 (1990). (33) V. Iwanij, The use of liver transglutaminase for protein labeling, Eur. J. Blochem., 80, 359-368 (1977). (34) L. Lorand, K. N. Parameswaran, P. Stenberg, Y. S. Tong, P. T. Velasco, N. A. Jonsson, L. Mikiver, and P. Moses, Specificity of guinea pig liver transglutaminase for amine substrates, Bio- chemistry, 18, 1756-1765 (1979). (35) J. Schode and J. E. Folk, Transglutaminase-catalyzed cross-linking through diamines and polyamines, J. Biol. Chem., 253, 4837-4840 (1978). (36) J. E. Folk, M. H. Park, S. I. Chung, J. Schrode, E. P. Lester, and H. L. Cooper, Polyamines as physiological substrates for transglutaminases, J. Biol. Chem., 255, 3695-3700 (1980). (37) H. U. Bergmeyer and H. Beutler, "Ammonia," in Methods of Enzymatic Analysis, Vol. 8, 3rd ed., H. U. Bergmeyer Ed. (VCH, Weinheim 1985), pp. 454-461. (38) L. Lorand, L. K. Campbell-Wilkes, and L. Cooperstein, Anal. Blochem., 50, 623-631 (1972). (39) J. E. Folk and P. W. Cole, Transglutaminase: Mechanistic features of the active site as determined by kinetic and inhibitor studies, Biochim. Biophys. Acta, 122, 244-264 (1966). (40) J. E. Folk and S. I. Chung, Transglutaminases, Methods Enzymol., 113, 358-374 (1985). (41) L. Lorand and S. M. Conrad, Transglutaminases, Mol. Cell. Biochem. 58, 9-35 (1984). (42) P. J. Birckbichler, G. R. Orr, E. Conway, and N. K. Patterson, Transglutaminase activity in normal and transformed cells, Cancer Res., 37, 1340-1344 (1977).

TRANSGLUTAMINASE 2 7 (43) (44) (45) (46) (47) (48) (49) (50) G. Paonessa, S. Metafora, G. Tajana, P. Abrescia, A. De Santis, V. Gentile, and R. Porta, Trans- glutaminase-mediated modifications of the rat sperm surface in vitro, Science, 226, 852-855 (1984). L. A. Goldsmith, Human epidermal transglutaminase, J. Invest. Dermatol., 80, 39s-41s (1983). R. J. Ward, H. A. Willis, G. A. George, G. B. Guise, R. J. Denning, D. J. Evans, and R. D. Short, Surface analysis of wool by x-ray photoelectron spectroscopy and static secondary ion mass spectrometry, Textile Res., J., 63, 362-368 (1993). A. P. Negri, H. J. Cornell, and D. E. Rivett, Effects of processing on the bound and free fatty acid levels in wool, Textile Res. J,, 62, 381-387 (1992). A. P. Negri, H. J. Cornell, and D. E. Rivett, A model for the surface of keratin fibers, Textile Res. J., 63, 109-115 (1993). Ajinomoto U.S.A., Inc., Teaneck, New Jersey. M. Motoki and N. Tambi (to Ajinomoto Co., Ltd.), Japan Patent Application 60-12311 (1985). M. Nonaka, H. Tanaka, A. Okiyama, M. Motoki, H. Ando, K. Umeda, and A. Matsuura, Poly- merization of several proteins by Ca 2 +-independent transglutaminase derived from microorganisms, Agric. Biol. Chem., 53, 2619-2623 (1989). APPENDIX A Calculation for Comparison of Transglutaminase Activity With Rat Sperm Cells and Human Hair The rat sperm data used in this calculation were taken from reference 43. 1. Substrate surface area, where Cell head diameter 5 •m Ssphere = •D 2 No. of sperm cells 6.0E06 Exposed hair length 0. 125 m Scylinder = •rDL No. of hairs 10 Ref. 43: Hair: 6.0E06 sperm X •r (5.0E-06 m) 2 = 4.7E-04 = 4.7 cm 2 10 hairs X • (70E-06 m) (0. 125 m) = 2.7E-04 = 2.7 cm 2 The ratio of total surface area of hair and sperm in the respective incubations was roughly 1:2, using average values for hair and sperm cell diameters. If we assume that the number of active glutamine sites per unit area was comparable, then the total number of active sites available for reaction was 1:2. Therefore, rat sperm cell binding data will be used on a one-to-two basis to estimate binding expected with hair. 2. Amount of enzyme used, where Ref. 43: Hair: 10 •xg/0.5 ml = 20 •xg/ml 0.5 units/ml X 1 mg/1.9 units X 1E03 •xg/mg = 260 •xg/ml We used about ten times more enzyme than used in the incubation with rat sperm. Reference 43 showed that the asymptotic number of reactive glutamine sites was not reached after a one-hour incubation of sperm cells with [•4C]-spermidine. It will be assumed that our increased enzyme concentration allowed all hair glutamine sites to react during the one-hour incubation. Discounting effects of enzyme activity loss, an estimate of the asymptotic number of disintegrations per minute (dpm) that would have been observed for complete reaction with rat sperm is 7,500 dpm (from Figure 1A in reference 43). 3. Isotope activity difference, where Ref. 43: 0.5 ml of 2 IxM [•4C]-spermidine yielded 2E5 dpm A) 2E5 dpm x 0.67 (our counter efficiency) = 1.34E5 cpm B) 2E-06 mol/L X 0.5E-03L/1.34E5 cpm = 7.46E-15 = 7.5 fmol/cpm Hair: 50 IxCi @ 1 mmol/115 mCi activity in 2 ml a 10-lxl aliquot of [•4C]-putrescine solution yielded 3.74E5 cpm.