Eur. J. Biochem. 214, 829-835 (1993)
`© FEBS 1993
`
`Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide,
`glucagon-like peptide-1(7-36)amide, peptide histidine methionine
`and is responsible for their degradation in human serum
`
`Rolf MENTLEIN', Baptist GALLWITZ 2 and Wolfgang E. SCHMIDT'
`' Anatomisches Institut and
`2 Abteilung Allgemeine Innere Medizin der Universitat Kiel, Germany
`
`(Received February 9/April16, 1993) - EJB 93 0215/3
`
`Peptides of the glucagon/vasoactive-intestinal-peptide (VIP) peptide family share a considerable
`sequence similarity at their N-tenninus. They either start with Tyr-Ala, His-Ala or His-Ser which
`might be in part potential targets for dipeptidyl-peptidase IV, a highly specialized aminopeptidase
`removing dipeptides only from peptides with N-terminal penultimate proline or alanine. Growth(cid:173)
`hormone-releasing factor(1-29)amide and gastric inhibitory peptide/glucose-dependent insulino(cid:173)
`tropic peptide (GIP) with terminal Tyr-Ala as well as glucagon-like peptide-1 (7- 36)amide/insulino(cid:173)
`tropin [GLP-1(7-36)amide] and peptide histidine methionine (PHM) with terminal His-Ala were
`hydrolysed to their des-Xaa- Ala derivatives by dipeptidyl-peptidase IV purified from human pla(cid:173)
`centa. VIP with terminal His-Ser was not significantly degraded by the peptidase. The kinetics of
`the hydrolysis of GIP, GLP-1 (7- 36)amide and PHM were analyzed in detail. For these pep tides Km
`values of 4-34 J.1M and Vm•x values of0.6-3.8 ).!IDOl· min-'· mg protein-' were determined for the
`purified peptidase which should allow their enzymic degradation also at physiological, nanomolar
`concentrations. When human serum was incubated with GIP or GLP-1 (7- 36)amide the same frag(cid:173)
`ments as with the purified dipeptidyl-peptidase IV, namely the des-Xaa- Ala peptides and Tyr-Ala
`in the case of GIP or His-Ala in the case of GLP-1 (7- 36)amide, were identified as the main
`degradation products of these peptide hormones. Incorporation of inhibitors specific for dipeptidyl(cid:173)
`peptidase IV, 1 mM Lys-pyrrolidide or 0.1 mM diprotin A (Ile-Pro-Ile), completely abolished the
`production of these fragments by serum. It is concluded that dipeptidyl-peptidase IV initiates the
`metabolism of GIP and GLP-1(7-36)amide in human serum. Since an intact N-terminus is obligate
`for the biological activity of the members of the glucagon!VIP peptide family [e. g. GIP(3-42) is
`known to be inactive to release insulin in the presence of glucose as does intact GIP], dipeptidyl(cid:173)
`peptidase-IV action inactivates these peptide hormones. The relevance of this finding for their
`inactivation and their determination by immunoassays is discussed.
`
`r
`
`Dipeptidyl-peptidase IV (DPP IV) is a highly specialized
`aminopeptidase removing dipeptides from bioactive peptides
`and synthetic peptide substrates provided that proline or ala(cid:173)
`nine are the penultimate N-terrninal residues (Mentlein,
`1988, for review). Small peptides or chromogenic substrates
`with proline in this position are far better hydrolysed than
`those with alanine (Heins et al., 1988). DPP IV occurs in
`human serum, as an ectoenzyme on the surface of capillary
`endothelial cells, at kidney brush-border membranes, on the
`
`Correspondence to R. Mentlein, Universitat Kiel, Anatomisches
`Institut, Olshausenstrasse 40-60, D-24118 Kiel, Germany
`Fax: +49 431 8801557.
`Abbrevifltions. DPP IV, dipeptidyl-peptidase IV; GIP, gastric in(cid:173)
`hibitory polypeptide or glucose-dependent insulinotropic polypep(cid:173)
`tide; GLP-1 (7- 36)amide, glucagon-like peptide-1 (7- 36)amide or
`insulinotropin or preproglucagon(78-107)amide; GLP-2, glucagon(cid:173)
`like peptide-2 or preproglucagon(126-159); GRF, growth-hor(cid:173)
`mone-releasing factor/hormone; PHI, peptide histidine isoleucine;
`PHM, peptide histidine methionine; VIP, vasoactive intestinal pep(cid:173)
`tide; PACAP, pituitary adenylate-cyclase-activating polypeptide.
`Enzyme. Dipeptidyl peptidase IV (EC 3.4.14.5).
`
`surface of hepatocytes (here termed also GP110 or OX-61
`antigen), on the surface of a subset of T-lymphocytes and
`thymocytes (here termed CD 26, or thymocyte-activating
`molecule) and other sites (Loijda, 1979; Nausch and Hey(cid:173)
`mann, 1985; Mentlein et al., 1984; McCaughan et al., 1990).
`The enzyme has been shown to be responsible for the degra(cid:173)
`dation and inactivation of circulating peptides with penulti(cid:173)
`mate proline, like substance P (Heymann and Mentlein,
`1978; Ahmad et al., 1992), but also for growth-hormone(cid:173)
`releasing factor (GRF) with penultimate alanine (Frohman et
`al., 1989; Kubiak, 1989; Boulanger et al., 1992).
`[Ala' 5]GRF(1-29)amide with penultimate Ala is even a
`comparably good substrate as a synthetic Pro2-containing de(cid:173)
`rivative for purified DPP IV (Bongers et al., 1992). This sug(cid:173)
`gests that the conformation or chain length may greatly influ(cid:173)
`ence the cleavage of peptides with penultimate proline/ala(cid:173)
`nine-residues by DPP IV.
`We therefore evaluated whether or not other peptide hor(cid:173)
`mones related to GRF might be substrates for DPP IV, and
`whether this probable proteolytic degradation might be of
`relevance in the circulation. GRF belongs to the glucagon/
`
`MPI EXHIBIT 1075 PAGE 1
`
`MPI EXHIBIT 1075 PAGE 1
`
`

`

`830
`
`:fir Ala Asp Ala lie
`Glu Gly Thr
`
`10
`Phe Thr Asn Ser Tyr
`-
`lie Ser Asp -
`
`... -29
`... -42
`
`h GRFI1-29)amide
`hGIP
`
`~- Glu Gly Thr
`~- - Gly Ser
`~- -
`Gly Val
`~- - Gly Val
`
`-
`-
`-
`
`Thr Ser Asp Val
`Ser Asp Glu Met
`-
`Ser Asp Phe
`Ser Asp -
`
`... -30
`... -34
`... -27
`.... -27
`
`h GLP-1 17 -36)amida
`hGLP-2
`hPHM-27
`r PHI-27
`
`-
`
`AspAsn-
`Ser Glu Leu
`Ser Asp-
`- Asp-
`-
`
`••• -28
`•.• -27
`... -29
`... -38
`
`hVIP
`h Secretfn
`hGiucagon
`hPACAP-38
`
`-
`
`-
`
`-
`Val
`~ Ser-
`~ Ser- Gly Thr
`~ Ser Gin Gly Thr
`~ Ser-:- Gly -
`Fig. I. N-terminal sequences of peptides related to growth hor(cid:173)
`mone-releasing factor (GRF). Penultimate alanine and serine resi(cid:173)
`dues are in bold; N-terminal tyrosine and histidine residues are un(cid:173)
`derlined; (-) identity to GRF. h, Human sequences; r, rat sequence.
`
`family
`secretin/vasoactive-intestinal-peptide(VIP) peptide
`(Fig. 1) which share N-tenninal sequences of considerable
`similarity. A number of them begin with Tyr-Ala, namely
`GRF and gastric inhibitory polypeptide/glucose-dependent
`insulinotropic peptide (GIP), or with His-Ala, namely gluca(cid:173)
`gon-like peptide-1(7- 36)amide/insulinotropin [GLP-1(7-
`36)amide], glucagon-like peptide-2 (GLP-2), peptide histi(cid:173)
`dine methionine (PHM) and peptide histidine isoleucine
`(PHI, the rat counterpart of human PHM), whereas others
`have terminal His-Ser (VIP and others). For biological activ(cid:173)
`ity the N-terrninal moiety is supposed to be the determinant
`for transducing the ligand message and the C-terrninal moi(cid:173)
`ety for playing the major role in specific binding (Christophe
`et al., 1989, for review). Thus, proteolytic truncation of the
`N-terminus of the members of the glucagon/VIP family by
`DPP IV should inactivate them.
`
`EXPER~ENTALPROCEDURES
`Peptides, inhibitors and enzymes
`Synthetic peptide hormones (human sequences) were ob(cid:173)
`tained from Saxon Biochemicals, dipeptides and diprotin A
`were purchased from Bachem. Purity of peptides was
`checked by HPLC; their amino acid compositions were ana(cid:173)
`lyzed by the manufacturer. Lys-pyrrolidide was a gift from
`Dr. Mike Schutkowski, Martin-Luther-Universitat Halle/
`Saale, Germany. Dipeptidyl-peptidase IV was purified from
`human placenta and free of contaminating proteases (Ptischel
`et al., 1982).
`
`Degradation assays with purified enzyme
`5 nmol of the peptides (5 111 of a 1 mM solution in water)
`were incubated at 37°C with 0.111g peptidase in 50 mM tri(cid:173)
`ethanolamine/HCl, pH 7.8, for 10-60 min in 500 J.!l (final
`peptide concentration 10 J.!M) or less (other peptide concen(cid:173)
`trations). Enzymic reactions were terminated by addition of
`5 J.!l 10% trifluoroacetic acid, and the mixtures applied onto
`a Vydac C, 8 widepore (30-nm pores, 5-J.LM particles)
`250 mm X 4.6 mm HPLC column and eluted at a flow rate of
`1 ml/min with gradients of acetonitrile in 0.1% trifluoro(cid:173)
`acetic acid. Either a linear gradient of 0-80% acetonitrile
`formed within 42 min (GIP degradation), or a stepwise linear
`gradient of 0-32% acetonitrile formed in 17 min followed
`by linear gradient of 32-48% acetonitrile formed in 30 min
`
`(other pep tides) were used for separations. In some HPLC
`separations, trifluoroacetic acid was replaced by heptafluoro(cid:173)
`butyric acid. Peptides and their degradation products were
`monitored by their absorbance at 220 nm (peptide bonds) or
`280 nm (aromatic amino acids). They were quantified by in(cid:173)
`tegration of their peak areas related to those of standards
`(synthetic Tyr-Ala or turncated peptides made by complete
`dipeptidyl-peptidase IV digestion). The concentrations of all
`peptide solutions were routinely calculated from their absor(cid:173)
`bance at 280 nm relative to their content of Trp and Tyr
`(using additively the known absorption coefficients) .
`Activities were determined from estimations with less
`than 10% substrate turnover. Catalytic constants were calcu~
`lated according to the statistical method of Wilkinson (1961).
`
`Degradation of peptides in serum
`200 J.!l serum of healthy males were incubated with 10 J . .tl
`1 mM peptide solution in water (final concentration 20 11M)
`for 60 min at 37°C. Inhibitors were added as 10 mM or
`100 mM stock solutions in water. Enzymic reactions were
`terminated by addition of 20 J.!l 10% trifluoroacetic acid.
`Samples were centrifuged (5 min 13000Xg), and the super(cid:173)
`natant liquids applied to a C, 8 reverse~phase Sep-Pak car(cid:173)
`tridge (Millipore-Waters) that had been previously activated
`and washed with 10 ml each of methanol, 80% acetonitrile
`in 0.1% trifluoroacetic acid and finally 0.1% trifluoroacetic
`acid. After washing of the serum-loaded cartridges with
`20 ml 0.1% trifluoroacetic acid, peptides were eluted with
`2 ml 80% acetonitrile in 0.1% trifluoroacetic acid. Acetoni(cid:173)
`trile eluates were lyophilized, dissolved in 100 J.!l 0.1% triflu(cid:173)
`oroacetic acid and analyzed as described above. Non-bound
`supernatants and washings were combined, lyophilized, re(cid:173)
`acted with 4-dimethylaminoazobenzene-4-sulphonyl chloride
`and separated by reverse-phase HPLC as described by
`Stocchi et al. (1985) for amino acids.
`
`Peptide chemistry and other assays
`Fragments separated by HPLC were collected and lyo(cid:173)
`philized for chemical determinations. Amino acid composi~
`tion was determined by acid hydrolysis (6 M HCl in vacuo
`at 100°C for 24 h) followed by lyophilisation, reaction with
`4-dimethylaminoazobenzene-sulphonyl-chloride and HPLC
`separation of derivatized amino acids (Stocchi et al., 1985).
`N-terminal amino acids were determined by manual microse(cid:173)
`quencing with 4-N,N~dimethy laminoazobenzene-4' -isothio(cid:173)
`cyanate (Wittrnann-Liebold et al., 1986).
`Hydrolysis of 0.5 mM Gly-Pro-4-nitranilide at pH 8.6
`and at 37 °C was monitored as described (Mentlein and
`Struckhoff, 1989).
`
`RESULTS
`Digestion of peptides by purified DPP IV
`DPP IV purified from human placenta liberated Tyr-Ala
`from GRF(1-29)amide and GIP, and His-Ala from GLP~
`1(7-36)amide and PHM (Table 1, Fig. 2). No further proteo(cid:173)
`lytic cleavage of these peptides was observed indicating the
`high specificity of the DPP IV for N-terminal Xaa-Ala (and
`Xaa-Pro) and the absence of contaminating proteases in the
`enzyme preparation. Liberated Tyr-Ala (Fig. 2) was iden(cid:173)
`tified by its retention time and co~chromatography with a
`synthetic standard. His-Ala was adsorbed to the C, 8 column
`
`MPI EXHIBIT 1075 PAGE 2
`
`MPI EXHIBIT 1075 PAGE 2
`
`

`

`

`

`832
`
`Table 2. Separation ofDPP IV cleavage products of gastric inhibitory polypeptide (GIP), glucagon-like peptide-1(7-36)amide [GLP-
`1(7-36)amide] and peptide histidine methionine (PHM) by reverse-phase HPLC on a C18 column. For conditions see Experimental
`Procedures, retention times varied ± 0.3 min. The first 20 min of gradients are identical.
`
`Peptide
`
`Retention time
`
`Gradient
`
`GIP
`GIP(3-42)
`Tyr-Ala
`His-Ala
`
`GLP-1(7-36)amide
`GLP-1(9-36)arnide
`PHM
`PHM(3-27)
`VIP
`His-Ala
`
`min
`27.4
`27.1
`14.3
`3.8
`18.2
`40.7
`41.7
`44.1
`44.8
`35.2
`3.8
`
`{
`
`0-3 min 0% + 3-45 min 0-80% acetonitrile
`in 0.1% trifluoroacetic acid
`
`0-3 min 0% + 3-45 min 0-80% acetonitrile in 0.1% heptafluorobutyric acid
`
`0-3 min 0% + 3-20 min 0-32% + 20-50 min 32-48% acetonitrile
`in 0.1% trifluoroacetic acid
`
`I
`
`Table 5. Catalytic constants for the degradation of bioactive peptides by human DPP IV. Assays were performed in 50 mM triethano(cid:173)
`lamine/HCl, pH 7.8, at 37°C. Values of kc., were calculated using a molecular mass of 120 kDa for one identical subunit of the human
`placental DPP IV dimer (Pi.ischel et al., 1982). GLP-1 (7- 36)arnide shows substrate inhibition above 50 J.tM, catalytic constants ( ± SD)
`were calculated from the linear ranges of Lineweaver-Burk plots.
`
`Peptide
`
`N-ter-
`minus
`
`So
`
`J.A.M
`
`No. of
`runs
`
`Km
`
`J.A.M
`
`Ymax
`
`JJ.mol· min-•
`. mg-•
`
`GIP
`GLP-t(7-36)-
`amide
`PHM
`[Ala"]GRF(l-29)-
`amide
`P-Casomorphin
`Substance P
`
`YA-E. ..
`
`1-100
`
`HA-E ...
`HA-D ...
`
`YA-D ...
`YP-F. ..
`RP-K. ..
`
`5-100
`5-100
`
`2-350
`20-500
`25-200
`
`7
`
`7
`6
`
`12
`
`34 ±3
`
`3.8 ± 0.2
`
`4.5 ± 0.6
`6.5 :+::: 0.5
`
`0.97 ± 0.05
`0.62 ± 0.03
`
`4.7 ± 0.3
`59
`22
`
`4.7
`90
`10
`
`:+::: 0.1
`
`kcat
`
`s-•
`
`7.6
`
`1.9
`1.2
`
`9.5
`180
`20
`
`kca/Km
`
`Reference
`
`M-1. s-1
`
`0.22. to•
`
`this study
`
`0.43. to•
`o.t9. to•
`
`this study
`this study
`
`. to•
`Bongers et al., 1992
`2.0
`. to• Nausch et al., 1990
`3.1
`o.91 . to• Nausch et al., 1990
`
`A220
`
`0.2
`
`GP+Serum
`
`0.1
`
`/
`
`___ _.-
`
`/
`
`GP
`_ ............ --.. -
`
`__ ,. .....
`
`50%
`
`/ /
`__ / GIP(3-42)
`I
`
`t
`
`~ ~
`li/j '2
`I v :g
`
`Tyr-Aia
`
`___. ----".---
`
`'
`l __ /
`
`_.-----
`.J
`______ .. --
`1:1
`~
`I
`BQ)
`1,!
`l
`/--
`II\, }
`!.~~~}.~) .... v
`U~"--f~uJ'V, V'11•-_r-,)tJ
`__ ,
`0
`___ .-----· ·-
`lj'\
`~ Serum
`1\,lli ~
`j
`J ~
`J l_,._n.JtJ '·,) ... ,,j\ _ _,.·''1 ~~~--··-··n,__.-\_.-.j
`~
`l Ill l
`I
`o
`
`n
`------· 1r
`1
`1:
`o ~------.:--/
`L - - - - ' -1 - - - - ' - - -11 '
`
`I
`I
`Time (min)
`Fig. 3. Reverse-phase HPLC analysis of an incubation assay of 20 pM gastric GIP with human serum (GIP + Serum) compared to
`a serum blank (Serum, inset). Positions of GIP and its degradation products Tyr-Ala and GIP(3 -42) are indicated. Experimental conditions
`as in Fig. 2.
`
`I
`
`I
`
`:::·21
`
`is metabolized by DPP IV activity of human serum mainly
`to Tyr-Ala and GIP(3 -42).
`Incubation of human serum with 20 ~M GLP-1(7-
`36)amide yielded one degradation product at the position of
`
`the des-His- Ala-peptide after reverse-phase HPLC (not
`shown). This fragment was identified by identical retention
`time with a standard (obtained with pure DPP IV, Table 2)
`and by determination of the N-terminal amino acid. His-Ala
`
`MPI EXHIBIT 1075 PAGE 4
`
`MPI EXHIBIT 1075 PAGE 4
`
`

`

`

`

`834
`
`Based on the identification of cleavage products and in(cid:173)
`fluence of specific inhibitors, DPP IV is the main degrada(cid:173)
`tion enzyme for GLP-1(7-36)amide in human serum. Buck(cid:173)
`ley and Lundquist (1992) have reported recently in an ab(cid:173)
`stract the formation of GLP-1(9-37) by human plasma, but
`did not identify the peptidase responsible for its generation.
`As outlined for GIP above, plasma-membrane-bound DPP
`IV of endothelial and other cells might be still more impor(cid:173)
`tant for the inactivation of GLP-1(7- 36)amide than the
`plasma activity.
`PHM (rat counterpart PHI) and VIP are processing prod(cid:173)
`ucts of a common precursor and are co-released from central
`and peripheral neurons. As far as is known, PHMIPHI have
`biological effects similar or identical to VIP. Since it is
`known that the biological actions of VIP critically depend on
`an intact N-terminus (Christophe et al., 1989; Robberecht et
`al., 1990), in analogy also PHMIPHI might be inactivated by
`cleavage of the N-terminal dipeptide by DPP IV. Since serum
`concentrations of PHM like VIP are low and in contrast to
`GIP and GLP-1(7-36)amide do not rise postprandially (Bo(cid:173)
`den and Shelmet, 1986), inactivation in serum is probably of
`minor importance and was not investigated. It can, however,
`be suspected that DPP IV cleaves the paracrine acting pep(cid:173)
`tides PHM/PHI in other tissues where it is present on the
`surface of various epithelial and endothelial cells.
`In conclusion, members of the glucagon!VIP peptide
`family with N-terminal 1)rr-Ala or His-Ala, namely GRF,
`GIP, GLP-1(7-36)amide and PHM, are inactivated by action
`of DPP IV in human serum. The truncated peptides could
`also be antagonists, because the binding specificity is di(cid:173)
`rected by the C-terminal parts of these peptide hormones
`(Christophe et al., 1989; Gallwitz et al., 1990). Since the
`cleavage by this peptidase removes only 2 of 29-42 total
`residues of the hormones, antisera against these peptides not
`directed specially to the N-terminus should cross-react also
`with the truncated peptides. Therefore, immunoassays for
`these hormones can be hampered by the measurement of bio(cid:173)
`logically inactive, des-Xaa- Ala forms beside the active pep(cid:173)
`tide, due a potential cross-reactivity of the antisera. Unless
`specific N-terminally directed antisera are available, serum
`samples should be stored for immunoassays at least in the
`presence of DPP-IV inhibitors (specific ones mentioned here
`or serine protease inhibitors like phenylmethanesulphonyl
`fluoride).
`DPP IV in human serum and at the surface of endothelial
`cells is known to be involved in the inactivation of other
`circulating bioactive pep tides: removal of the N-terminal
`tetrapeptide Arg-Pro-Lys-Pro of substance P (Heymann and
`Mentlein, 1978) inactivates only some biological actions of
`this neuropeptide (e. g. histamine release from mast cells),
`but renders the peptide possible for the complete degradation
`by aminopeptidase M (Ahmad et al., 1992). Several other
`bioactive peptide with N-terminal Xaa-Pro including gastrin(cid:173)
`releasing peptide, corticotrophin-like intermediate lobe pep(cid:173)
`tide and ,8-casomorphin are excellent substrates for the puri(cid:173)
`fied peptidase (Nausch et al., 1990).
`
`We thank Martina von Kolszynski for her expert technical assis(cid:173)
`tance. This work was supported by grants Me 758/2-3 and Ga 386/
`2-2 from the Deutsche Forschungsgemeinschaft.
`
`REFERENCES
`Ahmad, S., Wang, L. & Ward, E. (1992) Dipeptidyl(amino)peptidase
`IV and aminopeptidase M metabolize circulating substance P in
`vivo, 1. Pharm. Exp. Ther. 260, 1257-1261.
`
`Boden, G. & Shelmet, J. J. (1986) Gastrointestinal hormones and
`carcinoid tumors and syndrome in Endocrinology and metabo·
`lism (Felig, P., Baxter, J.D., Broadus, A. E. & Frohman, L. A.,
`eds) pp. 1629-1662, McGraw-Hill, New York.
`Bongers, J., Lambros, T., Ahmad, M. & Heimer, E. P. (1992) Kinet(cid:173)
`ics of dipeptidyl peptidase IV proteolysis of growth hormone(cid:173)
`releasing factor and analogs, Biochim. Biophys. Acta 1122, 147-
`153.
`Boulanger, L., Roughly, P. & Gaudreau, P. (1992) Catabolism of rat
`growth hormone-releasing factor(1-29)amide in rat serum and
`liver, Peptides (Elmsford) 13, 681-689.
`Buckley, D. I. & Lundquist, P. (1992) Analysis of the degradation
`of insulinotropin [GLP-1(7-37)] in human plasma and pro(cid:173)
`duction of degradation resistant analogs, Regul. Peptides 40, 117.
`Christophe, J., Svoboda, M., Dehaye, J.-P., Winand, J., Vander(cid:173)
`meers-Pire, M.-C., Vandermeers, A., Cauvin, A., Gourlet, P. &
`Robberecht, P. (1989) The VIP/PHI/secretin/heloderminlhelos(cid:173)
`pectin/GRF family: structure-function relationship of the natural
`peptides, their precursors and synthetic analogues as tested in
`vitro on receptors and adenylate cyclase in a panel of tissue
`membranes, in Peptide hormones as prohormones: processing,
`biological activity, pharmacology (Martinez, J., ed.) pp. 211-
`243, Ellis Horwood LTD, Chichester.
`Frohman, L.A., Downs, T. R., Heimer, E. P. & Felix, A.M. (1989)
`Dipeptidyl peptidase IV and trypsin-like enzymatic degradation
`of human growth hormone-releasing hormone in pla~ma, J. Clin.
`Invest. 83, 1533-1540.
`Gallwitz, B., Schmidt, W. E., Conlon, J. M. & Creutzfeldt, W. (1990)
`Glucagon-like peptide-1 (7- 36)amide: characterization of the
`domain responsible for binding to its receptor on rat insulinoma
`RINm5F cells, 1. Mol. Endocrinol. 5, 33-39.
`Gossrau, R. (1979) Zur Lokalisation der Dipeptidylpeptidase IV
`(DPP IV). Histochemische und biochemische Untersuchungen,
`Histochemistry 60, 231-248.
`Hartel, S., Gossrau, R., Hanski, C. & Reutter, W. (1988) Dipeptidyl
`peptidase IV in rat organs. Comparison of immunohistochem(cid:173)
`istry and activity histochemistry, Histochemistry 89, 151-161.
`Heins, J., Welker, P., Schonlein, C., Born, I., Hardtrodt, B., Neubert,
`K., Tsuru, D. & Barth, A. (1988) Mechanism of proline-specific
`proteinases: (I) substrate specificity of dipeptidyl peptidase N
`from pig kidney and proline-specific endopeptidase from Flavo(cid:173)
`bacterium meningosepticum, Biochim. Biophys. Acta 954, 161-
`169.
`Heymann, E. & Mentlein, R. (1978) Liver dipeptidyl peptidase IV
`hydrolyzes substance P, FEBS Lett. 91, 360-364.
`Jtimvall, H., Carlquist, M., Kwauk, S., Otte, S. C., Mclntosch, C.
`H. S., Brown, J. C. & Mutt, V. (1981) Amino acid sequence and
`heterogeneity of gastric inhibitory polypeptide (GIP), FEBS Lett.
`123, 205-210.
`Kubiak, T. M., Kelly, C. R. & Krabill, L. F. (1989) In vitro metabolic
`degradation of a bovine growth hormone-releasing factor analog
`Leu27-bGRF(1-29)NH2 in bovine and porcine plasma, Drug.
`Metab. Dispos. 17, 393-397.
`Loijda, Z. (1979) Studies on dipeptidyl(amino)peptidase IV. II.
`Blood vessels, Histochemistry 59, 153-166.
`McCaughan, G. W., Wickson, J. E., Cheswick, P. F. & Gorrell, M.
`D. (1990) Identification of the bile canicular cell surface mole(cid:173)
`cule GP110 as the ectopeptidase dipeptidyl peptidase IV: an
`analysis by tissue distribution, purification and N-terminal amino
`acid sequence, Hepatology 11, 534-544.
`Mentlein, R. (1988) Proline residues in the maturation and degrada(cid:173)
`tion of peptide hormones and neuropeptides, FEBS Lett. 234,
`251-256.
`Mentlein, R., Heymann, E., Scholz, W., Feller, A. C. & Flad, H.-D.
`(1984) Dipeptidyl peptidase IV as a new surface marker for a
`subpopulation of human T-lymphocytes, Cell. Immunol. 89, 11-
`19.
`Mentlein, R. & Struckhoff, G. (1989) Purification of two dipeptidyl
`aminopeptidases II from rat brain and their action on proline(cid:173)
`containing neuropeptides, J. Neurochem. 52, 1284-1293.
`Nausch, I. & Heymann, E. (1985) Substance Pin human plasma is
`degraded by dipeptidyl peptidase IV, not by cholinesterase, J.
`Neurochem. 44, 1354-1357.
`
`MPI EXHIBIT 1075 PAGE 6
`
`MPI EXHIBIT 1075 PAGE 6
`
`

`

`Nausch, I., Mentlein, R. & Heymann, E. (1990) The degradation of
`bioactive peptides and proteins by dipeptidyl peptidase IV from
`human placenta, Bioi. Chem. Hoppe-Seyler 371, 1113-1118.
`Ptischel, G., Mentlein, R. & Heymann, E. (1982) Isolation and char(cid:173)
`acterization of dipeptidyl peptidase IV from human placenta,
`Eur. J. Biochem. 126, 359-365.
`Rahfeld, J., Scbierhom, M., Hartrodt, B., Neubert, K. & Heins, J.
`(1991 a) Are diprotin A (lle-Pro-lle) and diprotin B (Val-Pro(cid:173)
`Leu) inhibitors or substrates for dipeptidyl peptidase IV? Bio(cid:173)
`chim. Biophys. Acta 1076, 314-316.
`Rabfeld, J., Schutkowski, M., Faust, J., Neubert, K., Barth, A. &
`Heins, J. (1991b) Extended investigation of the substrate speci(cid:173)
`ficity of dipeptidyl peptidase IV from pig kidney, Biol. Chem.
`Hoppe-Seyler 372, 313-318.
`Robberecht, P., Cauvin, A., Gourlet, P. & Christophe, J. (1990)
`Heterogeneity of VIP receptors, Arch. Int. Phannacodyn. 303,
`51-66.
`Robberecht, P., Gourlet, P., De Neef, P., Woussen-Colle, M.-C.,
`Vandermeers-Piret, M.-C., Vanderrneers, A. & Christophe, J.
`(1992) Structural requirements for the occupancy of pituitary ad(cid:173)
`enylate-cyclase-activating peptide (PACAP) receptors and ade(cid:173)
`nylate cyclase activation in human neuroblastoma NB-OK- 1 cell
`membranes, Eur. J. Biochem. 207, 239-246.
`Schmidt, W. E., Siegel, E. G., Kiinunel, H., Gallwitz, B. & Creutz(cid:173)
`feldt, W. (1987) Commercially available preparations of porcine
`glucose-dependent insulinotropic polypeptide (GIP) contain a
`biologically inactive GIP-fragment and cholecystokinin-33/-39,
`Endocrinology 120, 835-837.
`
`835
`
`Schmidt, W. E., Siegel, E. G., Ebert, R. & Creutzfeldt, W. (1986)
`N-terminal tyrosine-alanine is required for the insulin-releasing
`activity of glucose- dependent insulinotropic polypeptide (GIP),
`Eur. J. Clin. Invest. 16, A9.
`Schutkowski, M. (1991) Untersuchungen zur Substratspezijitiit pro(cid:173)
`linspezifischer Peptidasen, Thesis, Martin-Luther-Universitat
`Halle-Wittenberg.
`Stocchi, V., Cuccbiarini, L., Piccoli, G. & Magnai, M. (1985) Com(cid:173)
`plete high-performance liquid chromatographic separation of
`4-N,N-dimethylamino-azobenzene-4' -thiohydantoin and 4-di(cid:173)
`methylaminoazobenzene-4'-sulphonyl chloride amino acids
`using the same reverse-phase column at room temperature, J.
`Chromatogr. 319, 77-89.
`Unson, C. G., Gurzenda, E. M., Iwasa, K. & Merrifield, R. B. (1989)
`Glucagon antagonists: contribution to binding and activity of the
`amino-terminal sequence 1-5, position 12, and the putative a.(cid:173)
`helical segment 19-27, J. Bioi. Chem. 264, 789-794.
`Wilkinson, G. N. (1961) Statistical estimations in enzyme kinetics,
`Biochem J. 80, 324-332.
`Wittmann-Liebo\d, B., Hirano, H. & Kimura, M. (1986) Manual
`microsequence determination of proteins and peptides with the
`DABITIC/PITC method, in Advanced methods in protein micro(cid:173)
`sequence analysis (Wittmann-Liebold, B., ~alnikow, J. & Erd(cid:173)
`mann, V. A., eds) pp. 77-90, Springer-Verlag, Berlin Heidel(cid:173)
`berg.
`
`MPI EXHIBIT 1075 PAGE 7
`
`MPI EXHIBIT 1075 PAGE 7
`
`