UCLA FORUM IN MEDICAL SCIENCES
`NU�1BER 5
`
`GASTRIN
`Proceedings of a Conference held in September, 1964
`Sponsored by the Scl1ool of lVIedicine, University of California, Los Angeles
`
`EDITOR
`
`MORTON I. GROSSMAN
`
`UNIVERSITY OF CALIFORNIA PRESS
`BERhELEY AND LOS ANGELES
`
`1966
`
`Bausch Health Ireland Exhibit 2041, Page 1 of 23
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`CITATION FORM
`
`
`
`
`Grossman, M. I. (Ed.), UCLA Forum Med. Sci. No. 5, Univcrsitv of
`Gastrin.
`
`
`California Press, Los Angeles, 196G.
`
`University of California Press
`
`
`Berkeley and Los Angelt:s, California
`
`© 19/l(l by The Regents of the University of California
`
`
`
`
`
`
`Library of Congress Catalog Card Number: GG-25(i14
`Printed in the United States of Ameriea
`
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`CHARACTERIZATION OF A PURE GASTRIN
`
`STUART D. TAUBER
`University of Texas
`Dallas
`
`As described by Dr. Gregory, studies over the past fifty years have indi­
`cated that there is an anb:al hormone, gastrin, capable of stimulating hy­
`drochloric acid secretion from the stomach. More Tecently, gastrin-like ac­
`tivity has been obtained from the antral mucosa of several species, and
`from human pancrnatic islet cell tumors. Not only may gashin play an im­
`portant physiological role in the humoral control of gastric acid secretion,
`but it may also be causally linked to the symptom complex: which charac­
`terizes the Zollinger-Ellison syndrome.
`The studies I shall describe were performed to isolate and cha:racterize
`this hormone, both physicochemically and physiologically. In addition, I
`shall relate experimental evidence suggesting a biochemical similarity be­
`tween gastrin and the gastric stimulatory activity extractable from tumor
`tissue of patients with ZoJlinger-Ellison syndrome.
`For the purpose of testing gastrin activity during the isolation procedure,
`bioassays were performed in unanesthetized trained female dogs with
`chronic gastric fistulae. The test animal was placed in a Pavlov stand, and
`gastric secretions were collected by gasu:ic cannula at 15-minute intervals
`during a control period when isotonic saline was being infused at a rate of
`LO ml/min. After several control collections, the sample to be assayed was
`infused at the same rate for 30 minutes. The 30-minute period beginning
`15 minutes after the onset of increased lwdrochlolic acid secretion was used
`for quantitating the response of samples ·being assared. The total amount of
`hydrochloric acid from collected samples was estimated by titration to a
`phenolphthalein endpoint with 0.1 N sodium hydroxide.
`Maximum histamine responses were determined in all dogs for compari­
`so? with the activity of the gastrin extracts. In these studies the unit of gas­
`h·m activity (U) is de-6necl as that amount of administered hormone which
`p�oduces a secretory response equal to one-half the maximal histamine
`stimulation.
`The starting material for the purification of the honnone was an ether
`extract of acetone-dried powder from porcine antral mucosa, obtained by
`
`27
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`28
`
`GASTRIN
`
`TABLE 2
`PuRJFH;ATION Of' PonCINE GASTRYN
`
`
`
`
`
`
`Sbrting lvfa.tcrbl: Acotono-Driod Po�·dc1·, Spocific Activity •15 m U /mg
`
`Stop
`
`Chromatography
`
`
`
`Elution with
`
`
`
`Specific Activitr
`
`G-25 Scphadcx
`
`() . 001 M phosphritr. buff or, 100 mU/mg
`
`
`I
`pH 7.0
`II
`
`
`Calcium phosphate, brushito
`form
`pH 7.0
`Ill
`DEAE Scphadcx
`1-160 mU/mg
`0.32 M Nt.1.Cl
`IV
`G-75 Soph:.1dnx
`() 2 M ammonium ,Lcctn.tc -1120 mU/mg
`huffcr, pH -1.. 7
`
`0. 005 M phosphn.k buff or, -17(, mU/mg
`
`11- CALCIUM PHOSPHATE
`
`fO 10 30 40 50
`
`IV. G-75 SEPHADEX
`
`SEPHADEX
`
`3
`
`2
`
`3
`
`0 2
`
`q
`
`0
`0
`2
`
`t20
`60
`
`Ill. DEAE
`
`SEPHADEX
`
`1
`
`Q
`0
`
`0
`
`0
`
`.4
`
`.3
`
`.2
`
`.1
`
`40 80 f20
`Tube Number
`
`Figure 10. Chromatographic
`steps in the ptuificntion of gastrin extracted
`
`
`
`
`
`
`from porcine antral mucosa. Gaslrin activity indicated by shaded areas.
`
`See text. (Modified from Tauber & Madison, IO.)
`
`&..
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`CHARACTElUZATJON OF A PURE GASTRIN 29
`the .tJICtbod of Gi-egory & Tracy (2). The subsequent isolation procedure
`developed in our laboratory consisted of the following steps, as shown in
`'fable 2. [n Step 1, the crude material, with a specific activity of 45
`J1l u /mg, was ch.romatograpJ1ecl 011 G-25 Sephadex and elated with 0.001
`M phosphate bu.ff er, pH 7; the specific activity was then 100 m U /mg. Fur­
`ther fractionation of this ncth·e peak was carried out in Step IT on a column
`of calcium phosphate in the brushite form; the biologically active material
`eluted with 0.005 M phosphate buffer, pH 7, had a specific activity of 476
`111 u / 11-1g. Th.is product was then subjected to anion exchange chromatogra-
`1:,by ou DEAE Sephadcx (Step Ill); the gastrin, eluted b�· addition of 0.32
`M sodium chloride, had a specific acth·ity of 1460 m U /mg. The final cluo-
`111atographic step (IV) was carried out on G -75 Sephadcx; elution with 0.2
`M ammonium acetate buffer resulted in reco"-ery of a peak of gastrin activ­
`ity which had a specific activity of --!120 mU /mg, representing a ninetyfold
`purification over tl1e startiJ.1g material. Figure 10 depicts the chromato-
`
`ELECTROPHORETIC COMPOSITION
`
`OF GASTRIN
`
`ON POLYMERIZED ACRYLAMIDE.
`
`
`TRIS-GLYCINE BUFFER. pH 9.5
`
`Step 11 Step Ill Step IV
`
`+
`
`*
`
`*
`
`*dye-protein marker
`rjgure 11. Disk gel electrophoresis
`of gastrin :1t various stages of pmification.
`
`
`tep Il, calcium
`
`
`
`phosphate chromatography; Step III, DEAE-Sephadex chroma­
`
`
`
`the The dotted line locates tog�a_phy; Step IV, SeJlhadex G-75 chromatography.
`P0s1t1on
`
`
`
`of gastrin at Stages Il and Ill with reference to the puri6ed ho1mone
`
`at Stage IV. (From Tauber & Madison, 10.)
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`-
`
`30
`
`...-
`
`'T
`
`G A STRIN
`
`-_A_ -A
`IL - -
`L-- 1 •
`
`.,.-.
`
`...
`_A.
`lit .. - .
`Figure 12. Ultraoentrifugal sedimentation pattern of purified gastrin in a synthetic
`boundary cell; rotor speed: 52,640 rpm. Photographs were taken at two-minute inter­
`vals. (From Tauber & Madison, 10.)
`graphic pattern of each step in the separation sequence.
`Gastrin activity is indicated by the shaded areas in the figure. As shown
`in the upper left-hand corner, chromatography on G-25 Sephadex sepa­
`rated the crude material into two protein peaks; gastrin activity was pres­
`ent only in the first peak This fraction was rechromatographed on calcium
`phosphate and yielded two protein peaks with the biological activity now
`localized iJ1 the second peak, as shown in the upper right diagram. When
`this material was chromatograpbed in Step III on DEAE Sephadex (lower
`left) two further protein fractions were obtained; the gastrin activity was
`present in the second fraction. Lastly, chromatography of the active frac­
`tion on G-75 Sephadex yielded one major peak, containing the gastrin ac­
`tivity, and a small, poorly denned &action which was inactive in stimu-
`lating acid production.
`The composition of the active fractions of Steps II, Ill, and IV was then
`investigated by high resolution electrophoresis on polymerized acrylamide.
`As seen in Figure 11, the active fraction ootained in Step II contained ten
`electrophoretic components. By Step in thkse were reduced to foUl· major
`bands, and by Step IV only one component was identified.· The dotted line
`indicates the presence or location of gastrin in Steps Il and ill with ref­
`erence to the purified honnone seen in Step IV. The finding of a single elec­
`trophoretic component strongly suggested a homogeneous material.
`The homogeneity of the final product was further tested by ultracentrif­
`ugal analysis, using a syntl1etic boundary cell in a Model E Spinco ultra­
`centrifuge. As seen in Figure 12, only one symmetrical peak was evident,
`and there was no obvious sign of heterogeneity. From these data the sedi­
`mentation coefficient of gastrin was calculated to be 0.97.
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`C H A R A C T E R ! Z A T I O N O F A P U R E G A S T R I N
`
`3 1
`
`In order to assess the molecular weight of gastrin, its amino acid com­
`position was determined following acid h drolysis. As shown in Tabl 3,
`gastrin was composed of 17 di:fferent amino acids, comprising 107 amino
`acid residues. From these data the minimum molecular
`eight of gasb·in
`was calculated to be 12,514.
`Porcine gastrin was found to be free of histamine by the spectrophoto­
`metric method of Shore (9). The data from N-terminal amino acid analysis
`indicate that the N-terminal amino acid of gastrin is lysine. The amino
`acid analysis reveals that the purified hormone contains a high concentra­
`tion of glutamic and aspartic acids. The high concentration of ammonia
`found suggests that some of the free carboxyl residues of these amino acids
`are combined with ammonia in amide form. However, since gastrin be­
`haves as an acidic protein during electrophoresis, it is probable that the
`major portion of the ammonia was contributed by buffer contamination;
`accordingly, ammonia has been omitted from the molecular weight estima­
`tion until more is known of the amide content of gash-in. It should be
`noted that only one cystine residue is found in gastrin; it is not yet known
`whether the hormone contains two polypeptide chains connected by the
`
`TABLE 3
`AMINO AcID CoMPOSITION OF PURIFIE D GASTRIN
`
`Amino A cid
`
`Aspartic Acid
`
`Threonine
`Serine
`
`Glutamic Acid
`Proline
`Glycine
`Alanine
`Cystine
`Valine
`Methionine
`lsoleucine
`Leucine
`Tyrosine
`Phenylalanine
`Lysine
`Histidine
`Arginine
`Ammonia
`
`Total amino acid residues
`
`
`Minimal molecular weight
`
`
`
`Residues per Molecule
`
`Grams amino acid Calculated residue
`
`
`
`per 100 g gastrin per molecule of gastrin
`
`1 2. 51
`4 . 27
`3 .2 1
`1 3. 26
`5 . 74
`2 . 83
`2 . 26
`1 . G3
`3 . 48
`2 . 85
`8 . 28
`7 . 99
`10. 05
`4 . 99
`7 . 22
`6 . 77
`2 . 67
`(1. 17)
`
`14
`5
`5
`1 3
`7
`
`6
`
`4
`1
`
`4
`3
`9
`9
`8
`
`4
`7
`6
`2
`
`-
`
`107
`12,514
`
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`32
`
`G A S T R I N
`
`12
`
`•'
`
`10 ;· •
`
`8
`
`···································}
`
`
`········································· Jf �:t;:::/;:e
`
`Maximal
`
`4 ··················· .......... .
`
`2
`
`10 20 30 4 0
`p�GASTRIN/k<;
`Figm-e 13. Quantitative effects of purified gastrin on HCl secretion:
`dose-response relationships compared with maximal histamine responses
`of dogs. Sec text.
`
`disulfide bridge of cystine, or only a single chain \'\11th two cysteine residues.
`The latter possibility is supported by the nndings of a single N-terminal ly­
`sine, although two separate chains with this same N-termioal residue woulcl
`give the same result. Isolation of the di-DNP lysine has not be.en sufficientl.Y
`quantitative to rule out two such residues per molecule.
`111e physiological properties of the purified hom10ne were next investi­
`gated. In these experiments, the dose-response relationship was deter­
`mined, and the response with gastrin cornpare<l to that with maximum his­
`tamine stimulation. These results are shown in Figure 13, which gives hy­
`drochloric acid secretion in milliequivalents per thirty-minute response pe­
`riod, plotted against micrograms of gastrin administered. The maximal his­
`tamine response of three dogs used in these studies is shown between the
`bracketed lines. Doses of gastrin in excess of 2500 rnU, or 20.8 pg/kg, re.·
`sulted in HCl secretion greater than that produced by maximal histamine
`stimulation. Despite administration of doses of gastrin as large as 4200 mtJ,
`or 37.0 pg/kg, resulting in acid secretion two- to threefold that of the max­
`imal histamine response, the linear dose-response relationship held b·uc.
`I would like now to turn to the relationship between gastrin and the
`clinical state characterized by tumors of the pancreas and recurrent ulcer­
`ation of the upper gastrointestinal tract-the Zollinger-Ellison syndrome.
`
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`C H A R A C T E R I Z A T I O N O F A
`
`P U R E
`
`G A S T R I N
`
`33
`
`2
`
`11 CAICIUM PHOSPHATE
`
`111 ClfA!
`
`SfPHACltX
`
`3
`
`2
`
`pORCINE 0
`(0
`
`MUCOSA
`
`Q
`
`3 '<'
`� I G,JS
`"< SEPHADEX
`2 jg
`�
`AHTRAt N �
`?Ji
`0
`�
`"'
`:a· � "" �
`0 "
`0 60 120
`
`OL,,
`0 to 20 30 40 50
`
`0.8
`
`0.6
`
`08
`
`06
`
`lOWNGEII-
`
`Ell/SON QN 0.4
`
`I
`
`0.4
`
`TVMOII
`
`0.2 \J
`
`0.2
`
`0
`\,._
`0 40 80
`024
`
`Q20
`
`0.16
`0.12
`
`008
`
`0.04
`
`o ic-
`
`0
`
`10 20
`Tube Numbgr
`
`0
`10 20
`0 10 20
`TuheNumber Tube Number
`
`Figure 14. Cbromatographs showing purification of an ether extract of Zollinger-Ellison
`tumor (bottom) compared with that of gastrin extracted from hog antral mucosa as
`shown in Figure 10. The shaded areas indicate gastrin activity.
`
`Patients with this syndrome are known to secrete massive amounts of hy­
`drochloric acid and this, in turn, is associated with ulcer formation. It has
`been suggested that gastrin might play a role in the increased acid produc­
`tion found in these patients. To date, the tumors and metastases of 17 cases
`of this syndrome have been extracted and assayed for gash-in-like activity.
`Although gastiic secretory stimulating activity free of serious histamine
`contamination has been found, there has been n o further biochemical char­
`acterization of the etiologic agent.
`To test whether the gastrin-like activity present in these tumors was bio­
`chemically similar to porcine gastrin, tumor tissue (kindly supplied by Dr.
`Grant Liddle) from a patient with the Zollinger-Ellison syndrome, was
`treated according to the purification procedure described for gastrin. Bioas­
`say of thic; material at the acetone-dried powder stage showed the 30 g wet
`weight sample to contain 1920 mU of gastrin activity. The specific activity
`of 124 mU/mg of protein, when compared to that of gastrin at this stage
`of purification, indicates that the tumor tissue contained three times more
`hormone than antral mucosa. Further purification of the ether extract of
`this powder was accomplished as shown in Figure 14, in which the chro-
`
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`GAS TRIN
`34
`matographs shown in the upper panel ar� of porcine antral mucosa and
`those shown in the lower panel are from the tumor; gastrin activity is indi­
`cated by the shaded areas. Fractionation of the crude material on G-2$ Se­
`phadex in Step I yielded two protein peaks. When the protein, represented
`by the first peak, was fractionated on calcium phosphate brushite in Step
`II, two proteins were obtained which were similar to the gastrin separa­
`tions. Chromatography of the second peak on DEAE Sephadex in Step III
`yielded two components, as did porcine antral mucosa at this stage.
`The tumor fractions corresponding to the active peaks from porcine an­
`tral mucosa were then investigated by electrophoresis on polymerized
`acrylamide. As shown in Figure 15, four components were identified in Step
`II, and in Step III only one compo_nent was detected. The single electro­
`phoretic band found in the protein eluted from DEAE Sephadex migrated
`rapidly toward the anode, as does purified gastrin.
`Thus, the chromatographic patterns and electrophoretic composition of
`the tumor preparations closely agree with the results obtained for purified
`porcine gastrin. These data strongly suggest that the gasb"ic stimulatory ac­
`tivity extractable from tumors of patients with the Zollinger-Ellison syn­
`drome is due to the presence of the hormone gastrin.
`In summary, these studies indicate that gastrin, purified by this method,
`is a single protein with a molecular weight of 12,514, possessing a single N-
`
`Step 1 1 S tep 1 1 1
`
`-
`
`.,
`
`+
`
`* dye-protein
`marker
`Figure 15. Electrophoresis of Zollinger-Ellison tumor fractions
`con-esponding to the active peaks from porcine antral mucosa
`(shown in Flgure 14), on polymerized acryl::unide, tris-glycine
`buffer, pH 9.5. The single clcctrophoretic component found in
`Step Ill migrnted rapidly toward tne anode, as does purified
`gastrin.
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`C H A R A C T E R I Z A T I O N O F A P U R E G A S T R I N
`
`35
`terminal lysine . The purified hom1one was ninetyfold more biologically ac­
`tive on a weight basis than the original gastric antral extracts. The magni­
`tude of the h) drocbloric acid secretion induced by this hormone surpasses
`tl1at of maximal histamine stimulation. And, finally, the purification and
`subsequent chromatographic and electrophoretic analysis of tumor tissue
`from a patient with the Zolhnger-Ellison syndrome suggests that the hy­
`drochloric acid-stimulating activity of these tumors i due to the pr sence
`of the hormone gastrin.
`
`Discussion
`
`Grossman: It is obvious there is much room for discussion here becaus�
`we have heard two quite different views of this subject.
`Smith: Dr. Tauber, I would like to ask you a couple of questions. One
`concerns the amino acid analysis. There was no mention of tryptophan in
`the molecule; I wonder whether this was because it was not deter�ined or
`was destroyed on acid hydrolysis, or because it is absent from the molecule.
`Certainly, one of the striking featmes of Dr. Gregory's peptide is the pres­
`ence of two tryptophan res idues, and one of th m is in the biologically ac­
`tive terminal tetrapeptide sequence. The presence of tryptophan becomes
`crucial if one is to attempt to relate the small peptide to the larger one.
`lectrophoresis of
`The other question I wanted to ask pertains to the gel
`the tumor material as compared to the mucosal material. I could not be
`certain at first glance whether these were mn under identi�al conditions, be­
`cause it seemed to me the tumor material had a greater migration rate. I
`would like to know whether the times, pH conditions, ionic strength, etc�,
`were identical.
`Tattber: First, with regard to tryptophan, it was looked for but not
`found. It was clear to me also that this was the single amino acid that dis­
`tinguished our preparation from. that of Dr. Gregory.
`'Wi th reference to the electrophoresis of the tumor preparations, the
`analyses were performed at the same pH, with the same amount of current,
`and for the same length of time. Although electrophoresis in pol) acr )am­
`ide gel is a powerful analytical tool, mobility of proteins in this s ·stem is a
`function of molecular size as well as of electrochemical charge. Thus small
`differences in electrophoretic mobility may reflect subtle changes in either
`or both of these factors.
`I thiuk the suggestion made by Dr. Smith that the use of various extrac­
`tion methods may lead to different final products is pertinent to this discus­
`sion. A well-known example of this phenomenon can be seen in the family
`of molecular sizes of parathyroid hormone obtained depending upon the
`solvent used to extract the glands.
`Smith: May I ask another question which I am sure my physiological
`friends plan to ask? One of the things which is lacking in terms of any at-
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`G A S T R I N
`36
`tempt to compare these different materials is a strict comparison of biologi­
`cal activity not on a milligram basis-because we cannot compare some­
`thing of 12,000 molecular weight with something of 1200 molecular weight
`-but strictlv on a micromolar or molar basis. I wonder whether Dr. Tauber
`or Dr. Gregory has made this comparison. If not, would they put their
`heads together and tell us to what degree these things differ in activity?
`This, again, may help us to understand what we are dealing with.
`Taube1·: We have attempted to quantitate the secretory response to gas­
`trin by defining an arbitrary unit of hormonal activity. One can then calcu­
`late a specific activity for any gasb'in preparation under consideration. Un­
`fortunately, I have not been able to convert Dr. Gregory's data into these
`terms in order to make any meaningful comparisons. In relation to hista­
`mine, the unit of gastrin activity is defined as that amount of hormone ca­
`pable of producing a gastric secretory response equal to one-half maximal
`histamine stimulation.
`Gregory: I do not think we can make the valuable comparison that Dr.
`Smith has suggested from existing data because of the different conditions
`of assay. We compared the activity of our material with that of histamine
`on the basis of the total acid output in response to single subcutaneous in­
`jections, whereas I understand that Dr. Tauber compared his material with
`histamine on the basis of sustained responses produced by continuous in­
`travenous infusions. It would certainly be very valuable to make a compari­
`son by the same method, whichever of the two it might be.
`Grossman: There is one other point I want to address to Dr. Smith. When
`he was referring to the comparison of material from porcine antral mucosa
`with the human tumor material, I thought he would certainly take the op­
`portunity to remark on the possible differences in the peptide structures
`that might be found between the species. What might one anticipate the
`difference between porcine and human gastrin to be? Will you predict for
`us, Dr. Smith?
`I cannot predict; I can only tell you what is known about other
`Smith:
`proteins and polypeptides. I am sure that all of you are aware that the
`structures of a number of polypeptides of vertebrate origin have now been
`determined, and some comparisons have been made. For any given protein,
`there is no question that the homologous protein differs minimally when
`the two species are closely related. It may differ by a considerable amount
`as one examines proteins from species of wider evolutionary divergence.
`For instance, human and equine insulin differ in three residues out of 51.
`For cytochrome C the difference between the human and equine proteins is
`12 residues out of 104. Yet it would be difficult to distinguish these two
`molecules by their biological properties.
`Amino acid replacement is inevitable in evolution, and this certainly is a
`possible difference between human and porcine hormones. However. one
`would not expect them to differ by 10,000 in molecular weight. All of the
`
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`C H A R A C T E R I Z A T I O N O F A P U R E G A S T R I N
`
`37
`
`mammalian insulins have 51 residues. All of the vertebrate cytochromes
`which have been examined, going from tuna fish to man, possess 104 resi­
`dues. It is only when we go as far back as yeast and an invertebrate that
`we find that there are 108 or 109 residues. A few residues have been lost
`along the way during the two billion years of evolution. Such differences
`may be expected.
`Grossman: If I may press the point one step further, let us assume that
`Dr. Gregory has indeed isolated the active center of the molecule, the te­
`trapeptide. What may we expect to be the difference in this along the evo­
`lutionary scale?
`Smith: Please recognize that this is speculation based on what we know
`about other peptides and proteins. First of all, let us be clear that the ter­
`minal amide group must be the C-terminal end of the molecule. There is
`no known method of simple boiling, alkaline treatment, or anything of that
`sort, to obtain a breakdown of an amino acid causing it to leave an amide
`group.
`The other point is concerned with replacement of an amino acid residue
`without changing function. It is helpful to consider two kinds of structural
`changes in protein molecules: if one considers a long polypeptide chain
`such as insulin, cytochrome C, or the oth rs which have been studied, one
`encounters two types of substitutions. There are certain spots in the mole­
`cule where one finds a large number of different amino acids that can re­
`place the one found in the first species investigated. Let us say, if the first
`one happened to be alanine, the next species might have lysine or glutamic
`acid or arginine. Then one can be fairly sure that this particular residue
`plays no important role in the molecule-i.e., that it is not present at the
`a ti e site or sites, nor is it important in determining the overall confmma­
`that drastic chai1ges in hydrophilic character will permit
`tion. It is likel
`this kind of ariation and still have full biological activit . That is one type
`of change; we have referred to such substitutions as radical. The other type
`of change can be considered conservative. Conservative chang s in a mole­
`cule of cytochrome C, insulin, or ribonuclease involv substitutions of amino
`acids that are closely related in structure. In these cases similar, but not
`identical, side chains have be n replaced without Joss of activit) .
`If we then consider various amino acids, what can we expect? vV" have
`a very limited number of residues. In the ca of gl) cine it is ver eas ; there
`is nothing that can replace glycine and still lack a side cha.in. One of the
`striking things to emphasize is that, in the case of cytochrome C, where we
`now kno"' the sequences of som 14 different species from yeast to man,
`is the one type of residue that has remained almost constant, and. it
`glycin
`clearly is not a part of the active site. Glycine plays an important role in
`permitting the bends and interactions of parts of peptide chains, which no
`other amino acid would allow because the side chains interfere. One can­
`not replace glycine if its role in conformation is important. We used to
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`G A S T R I N
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`think that glycine played a minor role in proteins; it may tum out to play
`one of the most important of all. As for tryptophan, the only amino acid
`replacement I can recall, which preserves function, is in the case of cyto­
`chrome C where there is a histidine-tryptophan interchange. We have no
`otl,er information regarding tryptophan substitutions in other pl'Oteins.
`Neither ribonuclease nor insulin contains tryptophan. In the case of methi­
`onine, the residue that most resembles it from the viewpoint of size and hy­
`drophobic characte1· is leucine; whether other residues can substitute for
`methionine, we simply do not know. In the case of aspartic acid, the obvi­
`ous replacement is glutamic acid or, where the acid function is not impor­
`tant, the con-esponding amide may be found; these are certainly the only
`really conservative substitutions that would be possible. In the case of phe­
`nylalanine, the obvious conservative substitution would be a tyrosine resi­
`due.
`These are the types of substitution which would appear to be most likely
`if we assume that every one of the residues is absolutely essential for func­
`tion. Keep in mind, ]1owever, that I am making an overstatement when I
`speak here of conservatiYe substitution, because it may be tbat no substitu­
`tions at all are possible where sometl1ing is .fitted phys:iologically for an
`exact and precise interaction. For example, in the many enzymes in which
`there is a reactive serine residue, one never finds threonine. Serine is obvi­
`ously unique in those circumstances.
`Perhaps, if one thinks of a protein in its complex three-dimensional con­
`fmmation, one can say that there js a specific part of the molecule whose
`structure is absolutely essential and that no substitutions whatsoever are pos­
`sible. On the other hand, there are parts of the molecule where extensive
`radical substitutions may occur. One may find that at a bend something is
`quite crncial, such as a glycine residue or a proline residue; again, no sub­
`stitutions may be found. With something as small as gastrin, I would not ex­
`pect an extensive set of possible substih1tions. I am sure that Professor
`Kenner is already making the tyrosine and the glutamic acid derivatives.
`Gregory: Dr. Tauber, if I understood correctly, you spoke of starti11g
`with crude material obtained by our earli.er method. ,vhat precisely was
`this material?
`Tauber: The starti11g material was the mucosa which had been extracted
`with acetone-trichloroacetic acid mixture, had undergone salt and isoelec-
`h'ic precipitations, and was :finally air-dried.
`In the polyacrylamide gel runs where you were showing the
`Gregory:
`progressive purification to isolation, did the active material move from the
`origin? And what dye was used? Is it possible that what you stained was
`not gastrin? I thillk oUJ· peptides, for instance, would not be stained. My
`point is tJ,at there might bave been other components present which did not
`stain.
`Ta!-'ber: The sample was applied at the cathode and migrated a consider-
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`able distance toward the anode. The gel columns were fixed and stained
`with 1 per cent amido black in 7 per cent acetic acid.
`Gillespie: I was interested in your results, Dr. Tauber. The response to
`some of the larger doses of the gastrin you used exceeded your maximal
`histamine response by a considerable amount. I would like to confirm that
`the preparation was simply a gastric fistula as outlined. How did you deter­
`mine the maximal histamine response?
`Tauber: The dose we used to produce maximal histamine stimulation
`was one recommended by Marks, Komarov & Shay (7): histamine base, 40
`µg/kg administered intravenously over a 30-minute period in the same
`war as the gastrin extracts.
`Grossman: That is the total dose for 30 minutes. Your maximal response
`to histamine on an absolute basis was of the order of 5 mEq per 30 min­
`utes. In our experience, that would be approximately one-third of the aver­
`age maximal histamine response of a simple gastric fistula dog, and that
`would make our average maximal histamfoe response equal to your average
`maximal gastrin response. thus elimiuating the discrepancy you found. Do
`you think it possible tl1at the source of the discrepancy might be, as Dr.
`Gillespie suggested, that you had not in fact achieved a maximal histamine
`response?
`Tauber: Certainly, it might be possible. On the other hand, that average
`value of 5 mEq per half-hom is in fairly close agreement with double the
`Marks, Komarov & Shay figme of 3.5 mEq for 15 minutes. \-Ve have not
`studied responses to other doses of histamine.
`Grossman: Those authors found that the dose of histamine required to
`produce maximal stimulation fo dogs was about 2 pg of histamine base per
`kilogram of body weight per minute {7). They emphasized that this dose
`had to be continued for at least 90 minutes to achieve maximal rates of se­
`cretion. The maximal rates of secretion tl1ey obserYed in five dogs varied
`from 7.2 to 21.0 mEq per 30 minutes (twice their 15-minute values), and
`their mean ,·alue was 14.0 mEq per 30 minutes. This agrees with the mean
`value we observed for maximal histamine response in dogs with gasb'ic
`fistulas, namely 17.0 mEq per 30 minutes {8). It seems therefore likely that
`the dose of histamine used by Dr. Tauber was not large enough to produce
`a maximal response, that the dose was not continued for a long enough
`time to achie,-e maximal response, and that the values he reports for maxi­
`mal histamine responses are lower tlrnn those reported by Marks and his
`colleagues and br us. His statement that his responses to large doses of
`gastrin were larger than maximal responses to histamine must therefore be
`considered in light of this evidence that the responses he obtained to hista­
`mine were not maximal.
`Uvniis: I "vould like to ask the specialists here whether a polypeptide of
`the molecular weight of Dr. Tauber's, for example, if fractionated would
`lose its specificity and show a wider stimulation pattern than the original
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`G A S T R I N
`molecule. Dr. Tauber, how have you tested your preparation? Has it any
`other effects than just to stimulate hydrochloric acid secretion? Have you
`tried it on pepsin secretion or pancreatic secret