r
Biochem. Physiol., 1975, Vol. 50A, pp. 113 to 120. Pergamon Press. Printed in Great Britain
KINETICS OF METHIONINE INFLUX INTO VARIOUS REGIONS OF CHICKEN INTESTINE JOSEPH LERNER~ PEGGY SATTELMEYERAND ROBERT ~USH Department of Biochemistry, University of Maine, Orono, Maine 04473, U.S.A. (Received 1 October 1973)
Abstract--1. The intestinal absorption of methionine has been studied by an 9~ vitro tissueaccumulation t~fi'hiqu~,ill ~he chicken. 2. Carrier-mediated transport for this amino acid was found in the duodenum, jejunum, ileum and colon in week-old chicks; the affinity and capacity of the small intestine for methionine absorption are relatively constant from proximal duodenum through the ileum. 3. The colon in hens can absorb methionine by a saturable process, suggesting that the large intestine functions in the adult, 4. The caecum in day-old chicks transports methionine by way of two kinetically distinct processes, one with high affinity and low capacity, and another with low affinity and high capacity. 5. By the end of the first week of life, the caecum no longer possesses the ability to transport methionine or alanine by a carrier mechanism. 6. A comparison of the specificity limits of methionine uptake at low substrate concentration in the colon and caecum revealed a contr~tng order of maximal inhibitions brought about by a series of amino acids. This information, along with the kinetic findings, suggests the presence of different mechanisms for methionine absorption in the caecum and colon.
INTRODUCTION
WE r l m e reported in depth on the various aspects of the in vitro intestinM absorption of amino acids in the chicken (Miller et al., 1973). As a matter of convenience all former studies employed sections of intestine from the region about the yolk stalk, which is to be found midway in the bowel, In contrast, in the present investigation we were concerned with elucidating kinetic characteristics of methionine influx at ~ number of different levels in the gut. In particular, we have attempted to answer questions relating to whether (a) methionine is absorbed by a carrier mechanism at every level of the intestine; (b) there is a region of focus of absorption sites; (c) the affinity of the carrier varies with location; (d) the kinetic characteristics of methionine influx are the same in select regions of chick gut and the bowel of the hen; and (e) the caecum and large intestine possess carrier sites for methionine. MATERIALS AND METHODS Male chicks (Gallus domesticus) either day-old or "week-old" (7-15 days) and female chickens 90-92 weeks old were used as a source of intestinal tissue. The tissue handling and manipulations involved in the preparation and incubation of the intestinal segments have been previously described (Miller et aL, 1973). A portion of the appropriate region of the intestine 5-15 mm in length was excised and immersed in previously gassed (O2-CO2, 95 : 5 by volume) physiological saline enriched
with 0.3~ glucose. This solution was maintained at 41~ The intestinal section was stripped of mesentery and fatty tissue, cut lengthwise and allowed to contact towel paper to remove excess fluid. In the inhibition studies an individual segment of tissue was incubated at 37~ with shaking in a 25-mi Erlenmeyer flask containing a gassed (O~-CO2, 95 : 5 by volume) 5-ml portion of KrebsHenseleit buffer, 0.3~ glucose, L-(I*C) amino acid (New England Nuclear Corp., Boston) and unlabeled L-amino acid(s) (Sigma Chemical Co., St. Louis; Nutritional Biochemical Corp., Cleveland). The incubation period was 1 rain. The reaction was terminated by pouring the flask contents onto a Hirsch funnel (maintained under suction). The segment was then rapidly washed with physiological saline, removed to towel paper and then weighed. Tracer amino acid was extracted with 2-5~ trichloroacetic acid. The tissue extract was clarified by centrifugation and the tracer assayed by standard liquid-scintillation counting techniques. The results are presented as percentage inhibition of amino acid transport and have been calculated from the ratio of uptake in the presence of inhibitor to that in the absence of inhibitor using paired segments of tissue from one animal (Miller et al., 1973). An incubation period of 5 sec was used to determine kinetic constants (apparent Michaelis constant, Kin; maximum velocity, Vm~x; and apparent diffusion coefficient, Ka). Tissue was incubated for 5 sec on a waterjacketed Hirsch funnel (maintained at approximately 37~ which was mounted on a vacuum flask (LaBelle et al,, 1971). The reaction was monitored with a timertimeswitch (Fisher Scientific Co,, Boston) that was coupled electronically to a relay, which controlled a vacuum line communicating with the flask. The incubation period was started by pouring the preheated (37~ 113
114
JosEPH LERNnR, PEGGY SATTELMEYERAND ROBERT RUSH
and pregassed (On-C02) 5-ml portion of glucoseenriched Krebs-Henseleit buffer, which contained substrate, onto the filter and was terminated when the relay aUowed a vacuum to evacuate substrate solution from the funnel, The tissue was simultaneously irrigated with physiological saline. RESULTS Figure 1 shows the rate of methionine uptake in 5 sec into j e j u n u m of week-old chicks as a function of methionine concentration in the bathing medium. The curvilinear function is a composite of mediated transport a n d uptake by diffusion. The linear function was generated in the presence of 40 mM leucine,
which was used to block the m e d i a t e d portion of methionine entry, a n d is interpreted to represent the rate of uptake by diffusion. The kinetic constants listed in Table 1 were derived f r o m Lineweaver & Burk plots which were generated from rates obtained when the nonmediated uptakes were subtracted from the rates found in the absence of leucine. Methionine transport is mediated at all levels of the intestinal tract in week-old chicks including the c o l o n (Table 1; Fig. 2). The mechanism in the c o l o n apparently 7.=.
0.2~
ta
0,20
160 140 0.10
120
:E 0.OS
I00 >
80 4 S S F_METHIONINE'] - I ) mM "1
--
60
Fig 2. Lineweaver & Burk plot of methionine transport in the colon. Incubation period was 5 see. Rates were corrected for entry by diffusion (see text). O, 7-15-day. old chick; t,, 90-92-week-old hen. Each value is the mean of approximately ten experiments using tissue from ten animals.
40 20
O
I
2 ~ ESTI ,mM
4
I0
5
Fig. 1. Rate of methionine uptake vs. methionine concentration in the bathing medium for transport into segments of 7-15-day-old chick jejunum. Incubation period was 5 sec. Rates were not corrected for diffusion. Upper graph represents methionine transport in the absence of leucine. Each value is the mean of approximately ten experiments using tissue from ten animals. Variabilty is given by S.E.
remains through maturation, because it is still present in hens. A carrier mechanism, however, is absent in the caecum as indicated by d a t a in Fig. 3 which show the rate of uptake to be the same when tested in the presence or absence o f leucine. In contrast to week-old chicks, the day-old animal transports methionine in the caecum b y way of two kinetically distinct processes, one with high affinity,
Table 1. Kinetic constants for methionine uptake Region
Age of bird
Km (mM)
Proximal duodenum Distal duodenum
7-15 days 7-15 days 90-92 weeks 7-15 days 7-15 days 7-15 days 90-92 weeks 7-15 days :1 day 1 day
1.5 1-8 1.4 1.2 2.2 3.6 0.8 -0.3 5'6
Jejunum Ileum Colon Caecum
V~ax (nmoles/g)* 142 200 160 100 111 166 50 -31 166
Kd (nmoles/g x raM)*
Vm~x/Ka
12.5 16.5 11.4 12 11 15 9.8 13 20 20
11 12 14 8'3 10 11 5.1 -1,5 8"3
* Incubation period 5 sec. Expcrimemal conditions are described in the text. Ka values were determined from the slope of the line in the velocity vs. substrate concentration plot for methionine uptake in the presence of 40 mM leucine. Km and Vm~x values were determined from Lineweaver & Burk plots of rates of methionine uptake corrected for diffusion by subtraction of the leucine-uninhibitablerate from the total uptake.
Methlonine influx into chicken intestine
115
100
70
t
~J oo
E
O n
7 60 np-
%
50
W Z
6O
Ld
I
Z
< l
Biochem. Physiol., 1975, Vol. 50A, pp. 113 to 120. Pergamon Press. Printed in Great Britain
KINETICS OF METHIONINE INFLUX INTO VARIOUS REGIONS OF CHICKEN INTESTINE JOSEPH LERNER~ PEGGY SATTELMEYERAND ROBERT ~USH Department of Biochemistry, University of Maine, Orono, Maine 04473, U.S.A. (Received 1 October 1973)
Abstract--1. The intestinal absorption of methionine has been studied by an 9~ vitro tissueaccumulation t~fi'hiqu~,ill ~he chicken. 2. Carrier-mediated transport for this amino acid was found in the duodenum, jejunum, ileum and colon in week-old chicks; the affinity and capacity of the small intestine for methionine absorption are relatively constant from proximal duodenum through the ileum. 3. The colon in hens can absorb methionine by a saturable process, suggesting that the large intestine functions in the adult, 4. The caecum in day-old chicks transports methionine by way of two kinetically distinct processes, one with high affinity and low capacity, and another with low affinity and high capacity. 5. By the end of the first week of life, the caecum no longer possesses the ability to transport methionine or alanine by a carrier mechanism. 6. A comparison of the specificity limits of methionine uptake at low substrate concentration in the colon and caecum revealed a contr~tng order of maximal inhibitions brought about by a series of amino acids. This information, along with the kinetic findings, suggests the presence of different mechanisms for methionine absorption in the caecum and colon.
INTRODUCTION
WE r l m e reported in depth on the various aspects of the in vitro intestinM absorption of amino acids in the chicken (Miller et al., 1973). As a matter of convenience all former studies employed sections of intestine from the region about the yolk stalk, which is to be found midway in the bowel, In contrast, in the present investigation we were concerned with elucidating kinetic characteristics of methionine influx at ~ number of different levels in the gut. In particular, we have attempted to answer questions relating to whether (a) methionine is absorbed by a carrier mechanism at every level of the intestine; (b) there is a region of focus of absorption sites; (c) the affinity of the carrier varies with location; (d) the kinetic characteristics of methionine influx are the same in select regions of chick gut and the bowel of the hen; and (e) the caecum and large intestine possess carrier sites for methionine. MATERIALS AND METHODS Male chicks (Gallus domesticus) either day-old or "week-old" (7-15 days) and female chickens 90-92 weeks old were used as a source of intestinal tissue. The tissue handling and manipulations involved in the preparation and incubation of the intestinal segments have been previously described (Miller et aL, 1973). A portion of the appropriate region of the intestine 5-15 mm in length was excised and immersed in previously gassed (O2-CO2, 95 : 5 by volume) physiological saline enriched
with 0.3~ glucose. This solution was maintained at 41~ The intestinal section was stripped of mesentery and fatty tissue, cut lengthwise and allowed to contact towel paper to remove excess fluid. In the inhibition studies an individual segment of tissue was incubated at 37~ with shaking in a 25-mi Erlenmeyer flask containing a gassed (O~-CO2, 95 : 5 by volume) 5-ml portion of KrebsHenseleit buffer, 0.3~ glucose, L-(I*C) amino acid (New England Nuclear Corp., Boston) and unlabeled L-amino acid(s) (Sigma Chemical Co., St. Louis; Nutritional Biochemical Corp., Cleveland). The incubation period was 1 rain. The reaction was terminated by pouring the flask contents onto a Hirsch funnel (maintained under suction). The segment was then rapidly washed with physiological saline, removed to towel paper and then weighed. Tracer amino acid was extracted with 2-5~ trichloroacetic acid. The tissue extract was clarified by centrifugation and the tracer assayed by standard liquid-scintillation counting techniques. The results are presented as percentage inhibition of amino acid transport and have been calculated from the ratio of uptake in the presence of inhibitor to that in the absence of inhibitor using paired segments of tissue from one animal (Miller et al., 1973). An incubation period of 5 sec was used to determine kinetic constants (apparent Michaelis constant, Kin; maximum velocity, Vm~x; and apparent diffusion coefficient, Ka). Tissue was incubated for 5 sec on a waterjacketed Hirsch funnel (maintained at approximately 37~ which was mounted on a vacuum flask (LaBelle et al,, 1971). The reaction was monitored with a timertimeswitch (Fisher Scientific Co,, Boston) that was coupled electronically to a relay, which controlled a vacuum line communicating with the flask. The incubation period was started by pouring the preheated (37~ 113
114
JosEPH LERNnR, PEGGY SATTELMEYERAND ROBERT RUSH
and pregassed (On-C02) 5-ml portion of glucoseenriched Krebs-Henseleit buffer, which contained substrate, onto the filter and was terminated when the relay aUowed a vacuum to evacuate substrate solution from the funnel, The tissue was simultaneously irrigated with physiological saline. RESULTS Figure 1 shows the rate of methionine uptake in 5 sec into j e j u n u m of week-old chicks as a function of methionine concentration in the bathing medium. The curvilinear function is a composite of mediated transport a n d uptake by diffusion. The linear function was generated in the presence of 40 mM leucine,
which was used to block the m e d i a t e d portion of methionine entry, a n d is interpreted to represent the rate of uptake by diffusion. The kinetic constants listed in Table 1 were derived f r o m Lineweaver & Burk plots which were generated from rates obtained when the nonmediated uptakes were subtracted from the rates found in the absence of leucine. Methionine transport is mediated at all levels of the intestinal tract in week-old chicks including the c o l o n (Table 1; Fig. 2). The mechanism in the c o l o n apparently 7.=.
0.2~
ta
0,20
160 140 0.10
120
:E 0.OS
I00 >
80 4 S S F_METHIONINE'] - I ) mM "1
--
60
Fig 2. Lineweaver & Burk plot of methionine transport in the colon. Incubation period was 5 see. Rates were corrected for entry by diffusion (see text). O, 7-15-day. old chick; t,, 90-92-week-old hen. Each value is the mean of approximately ten experiments using tissue from ten animals.
40 20
O
I
2 ~ ESTI ,mM
4
I0
5
Fig. 1. Rate of methionine uptake vs. methionine concentration in the bathing medium for transport into segments of 7-15-day-old chick jejunum. Incubation period was 5 sec. Rates were not corrected for diffusion. Upper graph represents methionine transport in the absence of leucine. Each value is the mean of approximately ten experiments using tissue from ten animals. Variabilty is given by S.E.
remains through maturation, because it is still present in hens. A carrier mechanism, however, is absent in the caecum as indicated by d a t a in Fig. 3 which show the rate of uptake to be the same when tested in the presence or absence o f leucine. In contrast to week-old chicks, the day-old animal transports methionine in the caecum b y way of two kinetically distinct processes, one with high affinity,
Table 1. Kinetic constants for methionine uptake Region
Age of bird
Km (mM)
Proximal duodenum Distal duodenum
7-15 days 7-15 days 90-92 weeks 7-15 days 7-15 days 7-15 days 90-92 weeks 7-15 days :1 day 1 day
1.5 1-8 1.4 1.2 2.2 3.6 0.8 -0.3 5'6
Jejunum Ileum Colon Caecum
V~ax (nmoles/g)* 142 200 160 100 111 166 50 -31 166
Kd (nmoles/g x raM)*
Vm~x/Ka
12.5 16.5 11.4 12 11 15 9.8 13 20 20
11 12 14 8'3 10 11 5.1 -1,5 8"3
* Incubation period 5 sec. Expcrimemal conditions are described in the text. Ka values were determined from the slope of the line in the velocity vs. substrate concentration plot for methionine uptake in the presence of 40 mM leucine. Km and Vm~x values were determined from Lineweaver & Burk plots of rates of methionine uptake corrected for diffusion by subtraction of the leucine-uninhibitablerate from the total uptake.
Methlonine influx into chicken intestine
115
100
70
t
~J oo
E
O n
7 60 np-
%
50
W Z
6O
Ld
I
Z
< l
