Mechanism of Inhibition of Collagen Crosslinking by Penicillamine by Dr Marcel E Nimni (Departments ofBiochemistry and Medicine, Univetsity ofSouthern California, School of Medicine, Los Angeles, California, 90033)
Introduction Collagen fibres represent the primary structural element of connective tissue. They are responsible for the functional integrity of such tissues as bone, teeth, skin and tendon, where they account for the overwhelming majority of the proteins present, and for the structural integrity of many other structures such as blood vessels and most organs, to which they contribute a framework within which the tissue functions. A variety of human conditions, normal and pathological, involve the ability of tissues to repair and regenerate this collagenous framework. Some are characterized by excessive deposition of collagen (liver cirrhosis, scleroderma, keloid, glomerular nephritis). Following trauma or surgery, abnormal deposition of collagen may impair function (adhesions following repair of long tendons, scar formation during healing, &c.). In addition, many disabling conditions result from changes in the nature and organization of collagen (heart valve lesions, osteoarthritis, rheumatoid arthritis and congenital collagen diseases such as Marfan's syndrome and EhlersDanlos syndrome, osteogenesis imperfecta, &c.). Our interest in penicillamine and its potential involvement in the metabolism of collagen was prompted by two factors: (1) its resistance to metabolic degradation, and its strong capacity to chelate metals observed by Walshe (1962), which caused him to use it in Wilson's disease; and (2) the fact that penicillamine administration was shown by Scheinberg to be responsible for a number of side reactions which affected the connective tissue structures (Scheinberg 1964).
Molecular Structure and Crosslinking of Collagen Collagen is an extracellular macromolecule with unique characteristics which are most suited to the structural function it performs. It is a long asymmetric molecule approximately 3000 A (300 nm) long and 15 A (1.5 nm) in diameter, made up from three coiled polypeptide chains known as alpha chains. The coil arrangement of the amino acids which make up the collagen molecule is shown schematically in Fig 1. Every
third residue is glycine. Proline and hydroxyproline follow each other relatively frequently, and the gly-pro-hypro sequence makes up about 10 % of the molecule. The process of biosynthesis of this molecule is quite complex and requires a series of post-ribosomal modifications before the intracellular completion of the collagen molecule. Since it is known that the mode of action of penicillamine and other lathyrogenic agents is interference with the extracellular phase of collagen assembly which leads to the formation of crosslinks, these are summarized in Fig 2.
Specific lysine and hydroxylysine residues in collagen are oxidatively deaminated to give rise to the corresponding peptide-bound aldehydes. This modification is mediated by an enzyme, lysyl oxidase, which requires Cu+ +. Because this enzyme continues to act after the collagen molecules have been extruded, we tend to place it extracellularly. Formation of intramolecular crosslinks occurs soon after the lysines located near the N-terminal region have been converted to aldehydes. This crosslink involves an aldol condensation product that gives rise to an a-fl-unsaturated aldehyde. This is a spontaneous, nonenzymatic reaction that seems to occur more readily when the collagen molecules are packed into fibres. New aldehydes appear on the helical region of some collagens, particularly in those that are involved in extensive crosslinking, such as in skin and bone.
Intermolecular crosslinks form between peptide-bound aldehydes and unmodified- -NH2 groups of other lysine and OH-lysine residues. This reaction is of the Schiff base type. Further stabilization involving reduction or addition to the double bond seems to be necessary to render these crosslinks heat- and acid-stable. These intermolecular crosslinks are physiologically the most important ones. The collagen fibres found in the extracellular matrix of connective tissue vary in diameter and distribution. These differences in appearance seem to reflect some intrinsic structural characteristics, as well as the composition of the extrafibrillar matrix (proteoglycans, glycoproteins,
&c.). As becomes apparent, the key step in the formation of crosslinks involves the enzyme lysyl oxidase which is able to convert a specific lysine and hydroxylysine residues in collagen to peptide bound aldehydes. This reaction is shown schematically in Fig 3. The oxidative deamination catalyzed by this lysyl oxidase is inhibited
66 Proc. roy. Soc. Med. Volume 70 1977 Supplement 3 86 A
LYCINE * PREDOMINANTLY IMINO ACIDS
Fig I Schematic drawing showing the collagen triple helix. The individual a chains are left-handed helices with approximately three residues per turn. The a chains are in turn coiled around each other following a right-handed twist. The hydrogen bonds (which are not shown) form between residues of the different chains (interpeptide hydrogen bonding)
Fig 2 Extracellular events leading tofibreformuation. (1) Biosynthesis ofaldehydes near the N-terminal region. (2) Aldol condensation leading toformation of intramolecular crosslinks. (3) Appearance ofaldehydes in the helical region of collagen. (4) Formation of intermolecular crosslinks (Schiff bases). (5) Staggering of collagen molecules within afibre to generate the 680-A periodicity (640-A in the dry state). Arrows
;z :5 1-
4 4 PERVOM
CaO &-(CH, J -NH, NiH
indicate the potential sites for intermolecular crosslinking (for rat skin collagen)
C-O NH H-(CH3-C-0
c-AMINOADIP/C 8-SEMIALDEHYDE PEPTIDE BOUND LYSINE Fig 3 Oxidative deamination ofpeptide-bound lysine by the enzyme lysyl oxidase generates the aldehydes associated with the collagen molecule
Penicillamine irreversibly by fi-aminoproprionitrile (J6APN). This enzyme has been extracted from a variety of connective tissues and has recently been obtained in a highly purified form by Siegel (1977).
chelate heavy metals, are of primary significance in this connection. The former property manifests itself over all the effective dose range studied, whereas the latter seems to occur only at very high dosages, far beyond that ever administered to humans. The mechanism by which penicillamine inhibits crosslinking of newly formed collagen and that by which it depolymerizes incompletely crosslinked insoluble collagen will be discussed separately.
The cofactors required for the oxidative deamination of lysine have not been completely elucidated. Chelating agents abolish this activity, which can be restored by Cu+ + (Siegel et al. 1970, Deshmukh et al. 1971). This activity is absent from copper-deficient chicks and this elicits a state similar to that seen in osteolathyrism. Inhibition of Crosslinking The requirements for pyridoxal or other such The ability of penicillamine to increase the cofactors frequently associated with other amine amounts of soluble collagen (0.5 M NaCl fraction) oxidases have not been established. The inhibition in the skin of the rat is summarized in Table 1. of this enzyme by ,BAPN has proved to be of In addition, the effects of other analogues and major value in attempting to elucidate the compounds with lathyrogenic activity are also mechanism of crosslinking of collagen, since tabulated for comparison. experimental lathyrism induced by the administraThe collagen extracted from tissues of animals tion of ,BAPN or feeding the flowering sweet pea Lathyrus odoratus (Levene & Gross 1959) results treated with D-penicillamine is able to form stable in a collagen deficient in aldehydes and unable to fibres in vitro and is not deficient in aldehydes form crosslinks (Rojkind & Juarez 1966, Desh- like that from ,APN-treated animals. As a matter of fact, its aldehyde content is even higher than mukh & Nimni 1968, 1969). normal. This suggested that the mechanism of action of ,BAPN and penicillamine had to be Thiolism, a Defect Induced different. by Penicillamine Administration of penicillamine to animals and Using the method of equilibrium dialysis, it humans causes an accumulation of neutral saltsoluble collagen in various tissues (Nimni 1965, was observed that cysteine, penicillamine and Nimni & Bavetta 1965, Nimni 1968, Harris & other analogues bind specifically to free aldehydes Sjoerdsma 1966). The unusual features of on collagen (Deshmukh & Nimni 1969). Both the penicillamine posed an interesting question as to free amino and sulphydryl groups are necessary for binding to occur. The collagen-mercaptohow crosslinking of collagen could be impaired. As will become apparent later, two of the more ethylamine product is in equilibrium with its characteristic properties of penicillamine, namely constituents and can be completely dissociated by the ability to trap carbonyl compounds and to exhaustive dialysis. The plot in Fig 4 shows how,
Table I Collagen fractions extracted from the dorsal skin of young rats treated with various lathyrogens, D-penicillamine and related analogues (Nimni et al. 1972) Collagenfractions (mg/100 mng wet skin) Dose
Group Control Dithioglycolic acid
Dimercaptopropanol (BAL) D-penicillamine
(mg/100 g body wt)
200 40 70 70
3.0 6.4 3.5
5k 40 50 50
Procainamide Oxyalyl dihydrazide e-amino-caproic acid ,5APN fumarate
AET (2-amino-ethyl isothiouromium bromide hydrobromide)
(gld-) 6.3 2.8 3.0 5.8 2.0 6.1 6.3 6.4 5.6 4.0
65 100 150
Body wt gain
Insoluble NaCI 2.15±0.15 10.90+1.2 4.77±0.32
3.84±0.30 8.90+0.9 2.54+0.18 10.14±0.9 3.26±0.22 13.00+2.1 8.60+0.35 1.90+0.28 2.20+0.15 11.00+0.70 2.10±0.14 11.20±1.4 2.19±0.14 10.20±1.3 4.02+0.35 7.20±2.2 2.49±0.25 9.20+1.3 6.30±0.31 4.20±0.9 1.74±0.15 10.80±1.03 1.95 ±0.28 10.90±1.25
*The results are expressed as mean ± s.e. EAverage change in body weight over the experimental period (14 days); initial weight 70-80 g AInjected subcutaneously; all other compounds given orally
68 Proc. roy. Soc. Med. Volume 70 1977 Supplement 3 at equilibrium, the binding of cysteine to collagen (or gelatin) is proportional to the number of aldehydes on the protein.
Further studies using model compounds aided us in further understanding the mechanism of these interactions (Nimni 1973). Addition of Dpenicillamine to a solution of pyridoxal-5phosphate causes a decrease in absorbance at 390 ,um and the appearance of a new peak at 330 ,tm. The spectral changes seen after 10 min
§ 20 320
Fig 5 Change in the spectrum ofpyridoxal-5-phosphate (PLP) after its reaction with an 8-fold molar excess of D-penicillamine (spectral changes are noted after 10 min and 1 h ofreaction). This spectral change was used to monitor the nature and degree of interaction of various penicillamine analogues with aldehydes while attempting to correlate thisproperty with the pharmacological effects ofsuch compounds (reprodiucedfrom Nimni et al. 1969 by kind permission)
440 360 400 WAVE LENGTH (Mur)
ALVENYVERES/DUfS PER COLLAGEN MOLECLL
Fig 4 Binding of 14C-a-amino-thiol (cysteine) to collagens containing various amounts ofaldehyde residues. T>e lowest aldehyde-containing specimen was obtained by reduction with NaBH4 and the highest byperiodate oxidation. Specimens in between correspond to native collagens with various aldehyde contents. The affinity of the a-amino-thiol structure for peptide-bound aldehydes clearly reflects the mode of action ofpenicillamine (reproducedfrom Deshmukh & Nimni 1969 by kindpermission)
pH 7.8 and 6.3 (Mackay 1963). The N-acetyl derivative of penicillamine, as well as the alkylated analogue, S-carboxymethyl penicillamine, failed to show formation of a thiazolidine structure. These findings agree with the fact that D-penicillamine, L-cysteine, cysteamine or other a-amino thiols inhibit the formation of stable crosslinks 0.5
E 0.4 _
CYSTEAMINE pHs9.0 P.ENICILLAMINE
z 0.3 0
and at 1 h of incubation at room temperature are illustrated in Fig 5. Thiazolidine complexes prepared by mixing sulphur-containing amino acids and aldehydes absorb strongly at 330 ,um (Fig 6). Studying the rate of interaction of pyridoxal-5-phosphate with various amino thiols, it was reported that cysteamine, the amino thiol with the simplest structure capable of forming a thiazolidine complex, exhibited a second order rate constant of 7.5 x 10-4 mol-1 sec-1 at pH 9.0 and 20°C (Buell & Hansen 1960). If the negative charge was removed, as occurs in cysteine ethyl ester, the pH for maximal reaction decreased to 6.4 and the rate constant increased to 19.3 x 10-4 mol-1 sec-1. The second order rate constant for the reaction with DL-penicillamine was found to be slightly higher than that of L-cysteine between
z 49 w
4 6 TIME (MINUTES)
Fig 6 The increase in absorbance at 332 um reflects the affinity ofaldehydesfor a-amino-thiols. ,BAPNas well as compounds where the amino or the sulphydryl groups were blockedfailed to react with pyridoxal (reproducedfrom Nimni et al. 1969 by kind permission)
VJ / PENICILLAMINIE IN vIvo" OR IN VITRO
HS- C C
PE NICILLAM INE
Fig 7 Schematic drawing summarizing the mode of action ofpenicillamine. This compound in vivo as well as in vitro will interact with aldehydes on collagen, preventing their subsequent participation in the formation of intramolecular and intermolecular crosslinks. The reversibility of the collagen defect seen when penicillamine therapy is discontinued can be explained by the instability of the thiazolidine complexformed between penicillamine and the peptide-bound aldehydes
in vitro, while ,APN, N-acetyl or S-carboxymethyl derivatives of cysteine or penicillamine fail to cause such an inhibition (Deshmukh & Nimni 1968, Nimni et al. 1969), (Fig 7).
The relationship between the dose of penicillamine and the aldehyde content of collagen can be seen in Fig 8. Control rats at this stage of development show an average of 0.87 residues of aldehydes per 1000 amino acid residue. The lower doses of D-penicillamine caused the concentration of aldehydes to increase, reaching a maximum at 200 mg/kg of body weight/day. As the dose increased, the aldehyde concentration declined until at the highest dose it resembled lathyrism induced by ,BAPN.
Although all collagen preparations are able to form native collagen fibres with native 640 A periodicity, only those containing significant amounts of aldehydes form stable crosslinks. The collagen from the animals treated with high levels of penicillamine is incapable of forming stable crosslinks when reconstituted in vitro, and in this connection behaves like lathyritic collagen (Gross 1963). One can conclude from these experiments that penicillamine has the ability both to block aldehydes and to inhibit lysyl oxidase activity. At lower dosage, it acts primarily by blocking the aldehyde residues present on the collagen molecule, whereas at the higher dose it also affects the activity of lysyl oxidase (Nimni et al. 1972). This latter observation agrees with the fact that 10-4 M penicillamine partially blocked lysyl oxidase activity in vitro while 10-2 M inhibited it completely (Deshmukh et al. 1971). Although the mechanism operating under these circumstances
MG D-PENICILLAMINE/KG OF BODY WEIGHT/DAY
Fig 8 Variations of aldehyde content of neutral salt soluble collagen extractedfrom the skin ofyoung rats t-eated with varying dosages ofD-penicillamine for two weeks (reproducedfrom Nimni et al. 1972 by kind permission)
has not been defined, it may be related to the ability of penicillamine to chelate divalent cations or to interact with other cofactors.
Depolymerization oJ Incompletely Crosslinked Insoluble Collagen Fractionation of collagen according to its solubility has proved of value in revealing the process of collagen maturation and crosslinking. After rat skin has been extracted exhaustively with 0.45 M NaCl, an additional amount of collagen can be dispersed either by dilute organic acids (citric, acetic) or by compounds such as cysteamine or penicillamine (Nimni 1966). In rats less than 60 days old, practically all the skin collagen can be dispersed by amino-thiols to yield a collagen soluble in neutral salt. The intrinsic viscosity of this material, 20-25 dl/g, (tropocollagen 13 dl/g) reflects a mixture of tropocollagen with polymeric aggregates. This collagen will withstand centrifugation at forces greater than 100 000 g for several hours without appreciable precipitation (Nimni et al. 1967). In older animals, the amount of collagen soluble in dilute organic acids and amino-thiols becomes progressively less. In addition to preventing the crosslinking of newly synthesized collagen, penicillamine seems to be able to enhance the solubility in neutral salt solutions of a collagen fraction which otherwise would remain insoluble. This particular collagen
70 Proc. roy. Soc. Med. Volume 70 1977 Supplement 3
fraction rendered soluble by administration of penicillamine seems to be equivalent to that able to be dissolved in vitro by amino-thiols and by 0.5 M acetic acid. The fact that both in vivo and in vitro one is able to generate a collagen enriched in aldehydes suggests that penicillamine enhances the solubility of collagen by labilizing Schiff base type crosslinks (Rojkind & Juarez 1966, Nimni et al. 1967). The size of this degradable pool is inversely proportional to the age of the animal. Penicillamine given to young animals exhibits maximum effect. Because of the rapid rate of collagen synthesis and turnover, the pool of thiol-degradable material is extremely large at this time (Nimni et al. 1967). By using total skin mass as an index of absolute changes caused by penicillamine, a decline in the amount of total insoluble collagen during the first two weeks of treatment was observed. Continued administration of Dpenicillamine failed to affect the remaining insoluble collagen (see Fig 9). The soluble collagen that continues to accumulate during treatment may be a part of a rapidly-turning-over
PEN/CILLAM/NE rpEArMENr (I
AGE (WEEKS) ------o----- OSJM N/C/ SOLUDLE COLLAGEN -
Fig 9 Ten-week-old rats were maintained on the standard stock dietfor two more weeks and then administered D-penicillamine orallyforfour weeks. Animals were sacrificed at weekly intervals and their total skin oluble and insoluble collagen fractions were measured. Accumulation ofsoluble collagen results from inhibition of crosslinking, and probably some depolymerization ofinsoluble collagen following treatment reflects block of synthesis of crosslinked collagen in the presence oJ normal turnover together with a depolymerization of incompletely crosslinked insoluble collagen (reproducedfrom Nimni et al. 1969 by kind permission)
z 4 o 0 0
400 440 WAVE LENGTH (pJm)
Fig 10 Comparison ofthe absorption spectrum of pyridoxal-S phosphate (PLP) with that of the Schiff base it forms with y-amino butyric acid. This model Schiffbase was used to monitor in vitro the ability of various compounds to cleave such a product (reproducedfrom Nimni 1973 by kindpermission)
pool or may correspond to the more recently synthesized material which has not been allowed to mature (Nimni & Bavetta 1964, Klein 1969, Zika & Klein 1971, Klein & Nowacek 1969). Taking into account the variability in rates of collagen synthesis with age, it is possible to estimate that the half-life of this form of collagen containing labile Schiff bases is around 10 days in 2-month-old rats (Nimni et al. 1969). This period reflects the rate at which the Schiff base form of the crosslink is transformed into a more stable form of a covalent crosslink by reduction, addition to double bond or other means. The ability of various agents to affect the stability of Schiff bases using the model compounds has been investigated in an attempt to correlate this property with the in vivo depolymerizing activity (Nimni 1973). The spectral shift observed during the Schiff base formation between pyridoxal phosphate and y-amino butyric acid is recorded in Fig 10. A decrease in absorbance at 430 ,um was found most useful for monitoring Schiff base cleavage. This decrease in absorbance parallels the formation of thiazolidine complexes with amino-thiols. Fig 11 summarizes the changes observed at the pH ofmaximum reactivity of the various compounds involved. ,BAPN and cysteamine caused a rapid initial cleavage of the Schiff base, followed by formation of mixed Schiff bases after maximum cleavage was accomplished. This reversible effect seen with cysteamine at pH 9.0 reflects the instability of this particular thiazolidine. Schiff bases formed between pyridoxal phosphate and y-amino butyric acid (GABA) are quite unstable at acid pH. The first order rate
constants for the dissociation of such Schiff bases as a function of pH, at 20°C, are shown in Fig 12. This instability of Schiff bases at acid pH can explain the solubilizing effects of dilute organic acids on certain forms of polymeric collagen. On the other hand, base catalyzes the reaction of amino-thiols with a Schiff base made from pyridoxal phosphate and GABA. D-penicillamine showed a slight increase in reactivity as the pH was raised from pH 7 to 9, while reactions of Lcysteine and cysteamine were markedly accelerated by a rise in the pH of the media. This can explain our earlier observation that the effectiveness of cysteamine to solubilize insoluble skin collagen markedly increased over the pH range 6.0 to 9.0 (Nimni 1966).
Recent studies by Siegel (1977) have shed additional light on the mechanism of action of penicillamine on the inhibition of collagen crosslinking. One of the puzzling observations pH associated with the mode of action of penicilthe cleavage of 12 First order rate constantsfor Fig lamine related to the fact that this compound the pyridoxalphosphate y-amino butyric acid Shiff seemed selectively to inhibit the crosslinking of base at various pHs. Initial concentration 0.1 smol/ml, soft-tissue collagen and was much less effective temperature 20°C. The lability of the Schiff base the ability of than the ,-aminoproprionitrile lathyrogens in compound at acidpH correlatesinwith fibres to depolymerize dilute organic affecting bone collagen. This collagen, with a collagen acids (reproducedfrom Nimni 1973 by kind permission) high hydroxylysine content, is relatively unaffected in experimental animals fed penicilSiegel observed that the synthesis of bifunclamine, although increased N6: 6'-dehydro-5, 5'-dihydroxylysinonorleucine is found (Siegel tional crosslinks involving hydroxylysine increased in those animals treated with penicil1977). lamine, contrary to expectations. In the case of hydroxylysine derived crosslinks, the thiazolidine 0.5 _ interactions may not be sufficiently strong to ring E block the synthesis of the bifunctional crosslinks 0 involving these intermediates or those favoured on t 0.4 by the geometry of the fibril. If this were the case, findings would explain why the effects of these CYSTEAMINE pH*9.0 i penicillamine are significantly greater on soft 0 tissues than on bone, since the former involve aldehyde intermediates, which will lysine-derived zQ2t more readily form thiazolidine structures. -
P~~~~1APN pH a 9.0
Fig 11 Schiffbase cleavage by D-penicillamine, cysteamine, cysteine and PAPN measured by the decrease in absorbance at 430 sm. The curves were plotted at the pH where the reaction mixture reflected the maximum reactivity (reproducedfrom Nimni 1973 by kind permission)
On the other hand, Mechanic et al. (1977), looking at the periarticular collagen from immobilized rabbits treated with penicillamine to decrease contractures, found no significant crosslink formation in the collagen fibrils. The untreated animals synthesized normal crosslinks (hydroxylysinonorleucine, dihydroxylysinonorleucine and histidinomerodesmosine). It is therefore now apparent why the action of penicillamine varies from tissue to tissue. Its ability to block crosslinking depends on the types of crosslinks that stabilize that particular collagen fibre.
72 Proc. roy. Soc. Med. Volume 70 1977 Supplement 3 In addition, the state of maturation of these crosslinks, the number of crosslinking precursors (peptide-bound aldehydes) and the types of collagen present (1, II, III or IV) (Nimni 1974), may prove to be important factors which will influence the response to penicillamine therapy. REFERENCES Buell iN & Hansen R E (1960) Journal ofthe American Chemical Society 82, 6042 Deshmukh A, Deshmukh K & Nimni M E (1971) Biochemistry 10, 2337 Deshmukh K & Nimni M E ( 1968) Biochimica et biophysica acta 154, 258 ( 1969) Journal of Biological Chemistry 244, 1787 Gross J (1963) Biochimica et biophysica acta 71, 250 Harris E D jr & Sjoerdsma A (1966) Lancet ii, 996 Klein L(1969) Proceedings ofthe National Academy ofSciences ofthe United States of America 62, 920 Klein L & Nowacek C J (1969) Biochimica et biophysica acta 194, 504 Levene C & Gross J (1959) Journal of Experimental Medicine 110, 771 Mackay D (1963) Biochimica et biophysica acta 73,445
Dr 1 H Scheinberg (Chairmani): Dr Nimni is too modest. His studies have not only explained the pathogenesis of epidermolysis bullosa or the dermatopathy of penicillamine, but also why, in copper deficiency, there are severe abnormalities in connective tissues; aortas, for example, in experimental animals. I did not know until now that he started this work after hearing about Wilson's disease. Starting from a totally different point of view and approach, he has added immensely to our knowledge of the metabolism of copper and its complexity.
Dr E J Moynahan: What Dr Nimni has described is what happens in one of the seven forms of the Ehlers-Danlos syndrome, in one of which there is a deficiency of the enzyme in question. The Ehlers-Danlos syndrome is very different from epidermolysis bullosa. As a dermatologist who has seen these skin reactions, may I say that they do not resemble dystrophic epidermolysis bullosa in the slightest, nor are they like Ehlers-Danlos syndrome. They are not met with in children, but in adults in whom the turnover of collagen in the dermis is almost certainly reduced, when maximal stature has been attained. The ultrastructural study of the
MIechanic G L, Amiel D & Akeson W (1977) Connective Tissue Research (in press) NimniME (1965) Biochimica et biophysica acta 111, 576 (1966) Biochemical and Biophysical Research Communications 25,434 (1968) Journal of Biological Chemistry 243, 1457 (1973) Journal of Oral Pathology 2, 175 (1974) Seminars in Arthritis and Rheumatism 4, 95 Nimni M E & Bavetta L A (1964) Proceedings of the Societyfor Experimental Biology and Medicine 117, 618 ( 1965) Science 150, 905 Nimni M E, Deshmukh K & Bavetta L A (1967) Archives of Biochemistry and Biophysics 122, 292 Nimni M E, Deshmukh K & Gerth N (1972) Nature (London) 240,220 Nimni M E, Deshmukh K, Gerth N & Bavetta L A (1969) Biochemical Pharmacology 18, 707 Rojkind M & Juarez H (1966) Biochemical and Biophysical Research Communications 25,481 Scheinberg I H (1964) Journal of Chronic Diseases 17, 293 Siegel R C (1977) Journal of Biological Chemistry 252, 254 Siegel R C, Pinnell S R & Martin G R (1970) Biochemistry 9, 4486 Walshe J M (1962) American Journal of Medicine 201, 487 Zika J M & Klein L (1971) Biochimica et biophysica acta 229, 509
lesions is essential to establish both site and nature of the defect which produces the blistering. It might well be that penicillamine is inhibiting desmosome formation between epidermal cells in some patients, but it is much more likely that it disturbs dermal collagen and mimics the inherited disorder. Dr Nimni: My suspicion, as it relates to the bullous lesion, is that penicillamine may be working through its free sulphydryl group, rather than through crosslinking of collagen and the binding of penicillamine to any of these components. The basement membrane is known to be composed of a special kind of collagen with disulphide crosslinks holding things together. We know too that the attachment of the epidermal cells to basal lamina and the epithelial cells to each other may involve a desmosome-like crosslink which is extremely sensitive to sulphydryl agents. That kind of bullous lesion can be induced by interdermal injection of sulphydryl groups into the interphase between epithelium and mesenchymal cells. Therefore, I think that it may be the sulphydryl property of penicillamine which can cause that kind of lesion, not necessarily the lathyritic or the copper-chelating properties that have been discussed.