Constituents of salivary supernatant responsible for stimulation of oxygen uptake by the bacteria in human salivary sediment

Archs oral Bid. Vol. 35, No. 2, pp. 145-152, 1990 Printed in Great Britain. All rights reserved

Copyright 0

000379969/90 $3.00 + 0.00 1990 Pergamon Press plc

CONSTITUENTS OF SALIVARY SUPERNATANT RESPONSIBLE FOR STIMULATION OF OXYGEN UPTAKE BY THE BACTERIA IN HUMAN SALIVARY SEDIMENT M. KORAYEM,G. Department

of

WESTBAY

and I.

KLEINBERG*

Oral Biology and Pathology, State University of New York, Stony Brook, NY 11794, U.S.A. (Received 13 April 1989; accepted 25 August 1989)

Summary-The 10,OOOgsupernatant of wax-stimulated whole saliva was fractionated by gel filtration and its components were tested along with amino acids, small peptides and urea for their ability to stimulate this oxygen uptake, and for their effects on pH. Fractions containing the larger components, the proteins and large peptides, stimulated much less oxygen uptake than unfractionated supematant, and caused a small decrease. in pH. Analysis with anthrone indicated that both these effects were due mainly to the carbohydrate associated with these constituents. In contrast, fractions containing the remaining lower molecular-weight components stimulated substantial oxygen uptake and a rise in pH; both effects were like those seen WIth whole saliva supernatant. The oxygen effects were attributed mainly to certain amino acids and small peptides in the small molecular-weight fractions. Ornithine, arginine, proline and glutamic acid consistently stimulated oxygen uptake by the oral microflora in a test of 23 amino acids with the sediments of 13 subjects. Ornithine and arginine at the same time stimulated a significant rise in pH, whereas the other two amino acids showed no such effect. Variable and sometimes significant oxygen uptake was seen with alanine, aspartic acid, asparagine, glutamine and cysteine in 4-7 of the subjects; infrequent or no effects were seen with the remainder of the amino acids tested. There was some evidence to suggest that amino acid stimulation of oxygen uptake may be inducible. Urea had no effect on uptake but did contribu1.e significantly to the pH rise. Small peptides containing those amino acids that could stimulate oxygen uptake also stimulated such uptake; peptides without such acids did not. It was

concluded that certain amino acids, mainly present as residues of hydrolysable peptides and proteins, are the source of most of the oxidizable substrate in saliva. The remainder appears to be due to the carbohydrate

associated with certain salivary proteins and large peptides.

Key words: salivary bacteria, saliva, oxygen uptake, saliva pH, salivary sediment

INTILODUCI’ION

The supernatant of stimulated whole saliva contains substrates that are rapidly oxidized by the mixed bacteria in salivary sediment (Hartles and McDonald, 1950; Burnett, 1954; Eggers-Lura, 1956; Szabo et al., 1960; Hartles, 1963; Hartles and Wasdell, 1955a, b). Most of the oxidizable substrates appear to be associated with the amino acid, peptide and protein components (Wasdell, 1962; Guggenheim, 1966; Molan and Hartles, 1971). The remainder are associated mostly with the carbohydrate attached to salivary protein (Molan and Hartles, 1971; Caldwell and Pigman, 1966; Ellison, 1979). When amino acids were examined for their ability to stimulate oxygen uptake (Molan and Hartles, 1971), the concentrations needed to achieve full activity were several times greater than those normally present in whole saliva (Kleinberg, Kanapka and Craw, 1976). This indicated that the oxygen uptake activity arising from the free amino acids in saliva is less than that potentially available from the amino acids present as constituents of salivary peptides and proteins. As these studies had not identified the salivary components specifically responsible for stimulation of *To whom correspondence

should be addressed.

oxygen uptake by the bacteria in the sediment, we have now attempted to do so. Salivary supernatant was fractionated in a similar manner to that used by Kleinberg et al. (1979). Salivary constituents were also assessed for their ability to produce changes in pH in order to determine whether any constituents other than carbohydrate, specifically protein, peptides and amino acids, could affect both oxygen uptake and acid-base functions of the sediment bacteria (Kleinberg, 1970a, b). MATERIALS

AND METHODS

Preparation of salivary sediment mixtures and fractions of salivary supernatant

Wax-stimulated whole saliva was collected from subjects who had not eaten or carried out oral hygiene for at least 12 h. The saliva was centrifuged at 10,OOOg for 30 min at 4°C. The sediment was washed three times and then suspended in distilled water at a concentration of 50% (v/v). The supernatant was divided into two portions; one was concentrated 10 times by lyophilization to dryness and subsequent resuspension in distilled water; the other was used to obtain various fractions for testing (see Kleinberg et al., 1979 for details of fractionation and fraction composition). 145

M. KORAYEM et al.

146

In brief, 4 ml samples of salivary supernatant were applied to a column (1 x 7 cm) containing Bio-Gel P-4 (Bio-Rad Laboratories, Richmond, Calif., U.S.A.) and eluted with distilled water (operating pressure 145 cm; flow rate 300pl/min). Fractions (300 ~1) were collected and their optical density determined at 280 nm. Large (A). intermediate (B, and B?) and low (C) molecular-weight fractions [Fig. l(a)] were each pooled and concentrated by lyophihzation to 10 times their normal salivary concentrations. A portion of fraction A obtained from several supernatant fractionations was re-dissolved in distilled water at its original concentration in saliva and 2 ml was applied to a column (1 x 37 cm) containing Bio-Gel P-200 (Bio-Rad Laboratories). After elution with distilled water (flow rate 120 pI/min; operating pressure 20cm), the optical density of the eluate fractions was determined at 280 nm. Fractions corresponding to regions A,, AZ and A, in Fig. l(b) were each pooled, freeze-dried and then re-dissolved in distilled water at a concentration 10 times that of the original saliva. The carbohydrate content of each pooled fraction was determined with anthrone (Sandham and Kleinberg, 1969). Fraction C, which has a high 280 nm absorbance because of its uric acid component, was analysed for and shown to contain urea. Urea was measured calorimetrically by the method of Coulombe and Favreau (1963) as modified by Biswas and Kleinberg (1971). Amino acid, smaN peptide and urea solutions Amino acids and small peptides are end-products of the hydrolysis of salivary protein and large pep-

:,u 0.6 (a)

C

1

0

20

O-0

40 60 80 Fraction Number

100

120

Fraction Number

Fig. 1. (a) Fractionation of salivary supernatant by gel filtration. Fraction B was divided into B, and Br by pooling eluate fractions within the first and second halves of B, respectively. (b) Subfractionation of fraction A by gel filtration. The carbohydrate contents of the sub-fractions A,, A, and A, are shown in the inset.

Table 1. Composition of the mixture used to simulate the free amino acid composition of 10,OOOgsalivary supernatant Type of ammo actd Alanine Arginine Aspartic acid Cystine Glutamrc acid Glycine Histidine Isoleucine Leucine Lysme Methionme Phenylalanine Prohne Serme Threonine Tyrosine Valine

Concentration (PM) 51.1 1.8 27 2 18 0 95.2 75.9 9.7 7.8 9.3 53.0 3.7 Il.5 19.3 19.7 21.9 11.1 23.5

tides by the oral bacteria and it is these compounds that are taken up by the micro-organisms to meet their biochemical needs (Payne, 1976; Kleinberg et al., 1979). The amino acids found free in 10,OOOg supernatant of wax-stimulated whole saliva and their respective concentrations are shown in Table 1 (Kleinberg et al., 1976). In some experiments, these amino acids were combined at the concentrations shown and at 10 times these concentrations; each mixture was then tested for their effects on both oxygen uptake and pH. Amino acids were examined individually for their ability to stimulate oxygen uptake; this mainly involved testing 23 amino acids with sediments from each of 13 subjects (Table 2). Each amino acid was tested sequentially at a final concentration of 0.33 mM, except for tyrosine, which was tested at 0.2 mM because of its lower solubility. To accommodate this number of amino acids, sediment from each subject had to be collected and the tests done on two or three separate occasions. In response to the observation that the initial rate of oxygen uptake with a given amino acid could vary in some subjects from day to day, some experiments were carried out in which individual amino acids were provided at a higher concentration (3.4mM). It was possible that this might induce oxygen uptake activity and thus oxygen uptake when the provision of a test amino acid was repeated. Preliminary experiments had indicated that prolonged exposure to an amino acid (achievable by using a higher addition concentration) could indeed stimulate a more rapid uptake of oxygen upon re-challenge with the same amino acid. Urea was tested at 5.4 and 27.7mM; the lower concentration is within the normal range for saliva (Jenkins, 1978); the higher is much more than normal and included for comparison. The following peptides (obtained from Bachem, Torrence, Calif., U.S.A.) were each examined at a final concentration of 0.33 mM for their effects on oxygen uptake and pH: glutamylproline, prolylproline, arginylserine, methionylserine, glycylornithine, lysylargmine, leucylarginine and glycylserine. An

147

Saliva components and sediment oxygen use Table 2. Amino acid slimultion of oxygen uptake activity of the salivary sediment bacteria in different individuals Subject Amino acid Alanine Arginine Asparagine Aspartic acid Cltrulline Cysteine y-NH? butyric acid Glutamlc acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionme Ornithine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

RW

RS

LD

GW

HK

IK

CC

TD

L

HM

MC

MT

RC

+ _ _ _ _ + _ _ _ _ _ + _ + _ _ _ _ _

_ + _ + _ + + _ _ _ _ + _ + _ _ _

+ _ + + _ _ _ + + + _ _ _

+ + + _ + _ _ _ _ _ + + + _

+ + + + + _ _ + + _ _ _ _ _ _ + + _ _ _

+ _ + + _ + + _ _ _ _ + _ + _ _ _

+ _ _ + + _ + _ _ _

+ + + + + _ + _ _ + -

+ + + + _ + _ _ _ + _ + _ _ -

_ + + + _ _ + + + _ + _ + _ _ -

+ + + _ + _ + _ + _ + _ + _ + _

+ + + + _ + _ + + _ + _ + _ + + _ _

+ + + _ + _ + _ _ _ _ + _ + _ _

additional group of peptides, which included arginylglycine, arginylserine, arginylisoleucine, arginylvaline, aspartylarginine, glycylornithine, methionylserine, glycylserine and glutamylproline, were tested for oxygen uptake at a constant pH of 7.0, using a pH-stat to control the pH (Model TTTIC, Radiometer, Copenhagen, Denmark). Before use, the purity of all pepl:ides was examined by twodimensional paper chromatography (Kleinberg et al., 1979).

Procedurefor testing the efiects on oxvgen uptake of salivary supernatant, its various fractions, individual amino acids, small peprides and urea Distilled water (500 ,ul) was pipetted into an incubation chamber constructed from Perspex, as shown in Fig. 2. The chamber was maintained at 37°C with water circulated from a constant temperature bath (Heto water bath, London Company, Cleveland, Ohio, U.S.A.). The contents were stirred at 200 rev/min with a magnetic stirrer (Micro-V Model, Cole-Parmer, Chicago, Ill., U.S.A.) and Tefloncoated magnetic bar (2 x 7 mm). The PO, and the perspex

reaction chamber

I

pH were simultaneously measured with oxygen and glass pH electrodes respectively; both electrodes were in continuous contact with the chamber contents throughout each incubation. After equilibration with atmospheric oxygen and the attainment of a constant temperature (37YJ), 250 ~1 of a 50% (v/v) aqueous suspension of salivary sediment were added to the chamber. This resulted in a short period of rapid oxygen consumption as was evident from the rapid decrease in the PO, and its duration below baseline. A fall in the pH was usually seen at the same time. Both the oxygen and pH effects are attributable to the small amount of sediment carbohydrate used during this time (Sandham and Kleinberg, 1969) and, in the case of oxygen uptake, possibly to some endogenous nitrogenous substrates as well. Once the PO2 and the pH were stable, a portion (30 ~1) of either salivary supernatant, supernatant fraction, urea, amino acid mixture, individual amino acid or peptide was added and the PO, and pH were simultaneously measured. As indicated above, the concentration of supernatant and its fractions in this added sample was 10 times that originally present in unfractionated saliva. The effect of combining various supernatant fractions was also tested. Each fraction was concentrated to 20 times its normal salivary concentration. Equal volumes were then combined so that constituents could be added at 10 times their concentration in saliva supernatant. As before, 30 ~1 of each combination was tested.

mupnelk stirrer

RESULTS

magnetic mixw --

Fig. 2. Diagram of the apparatus used for simultaneously measuring the oxygen uptake and pH of incubating salivary sediment mixtures.

Effects

of salivary supernatant and its various fractions

on PO, and pH The addition of 30 ~1 of salivary supernatant to a salivary sediment mixture resulted in an immediate

M.

148

KORAYEMet

al.

6

6

i

hi,i23 Time

::

(h)

Fig. 3. Effects of salivary supernatant and either fractions A, B or C on oxygen uptake and the pH. Shown in (a) are the effects of unfractionated salivary supernatant; in (b), the effects of fraction A: in (c), the effects of fraction B; and in (d), the effects of fraction C. 0

and substantial decrease in PO, and a progressive rise in pH [Fig. 3(a)]. The PO? rapidly reached a constant value that was sustained for about 30min before rising sharply, indicating completion or near completion of use of the readily oxidizable constituents. The addition of fraction A stimulated a decrease in the PO,. but this was substantially less than with unfractionated salivary supernatant [cf. Fig. 3(a) and 3(b)]. Fraction A was accompanied by a fall rather than the rise in pH seen with salivary supernatant [Fig. 3(b)]. At the moment the PO, began to rise, the pH stopped decreasing and also began to rise. The addition of fraction B [B, plus B,; Fig. 3(c)] stimulated a fall in the PO,, almost as much as that observed with unfractionated salivary supernatant [cf. Fig. 3(a) and 3(c)]. At the same time, the pH showed a rapid and significant rise. When tested alone, fractions B, and B2 both stimulated a PO, decrease, fraction B, more so than B, ; only B, caused a change in pH, a rise similar to that seen with fraction B. Fraction C, the last component eluted from the Bio-Gel column, produced a very slight decrease in PO, and a distinct rise in pH [Fig. 3(d)]. Combining fractions A and B, B and C, and A and C gave POZ and pH readings that were more or less as if the effects seen in Fig. 3 for combination members had been averaged. Effects of sub-fractions

I 0

oj(c) I

0

6

I 1

1 2

1

I

2

1

I

3

6

I

3

Time(h)

Fig. 4. Effects of fractions A,, A? and A, on oxygen uptake and the pH. In (a) and (b) are the effects of fractions A, and A,. respectively; in (c) are the effects of fraction A,. small PO, response was observed that lasted for approx. IO min (Fig. 5). Addition of salivary supernatant thereafter resulted in a rapid and substantial drop in the PO,, which lasted for 30 min. A second addition of amino acid mixture resulted in a PO* response similar to that observed on the first occasion. Addition of the same amino acids at concentrations 10 times those normally present in salivary supernatant resulted in a PO, response similar in magnitude to that seen with unfractionated salivary supernatant. Supernatant (which contains urea) favoured a rise in pH; the amino acid mixture did not. E#ect of individuaI amino acids The ability of the 23 amino acids to stimulate oxygen uptake in the sediment microflora of the 13 subjects is summarized in Table 2. Figure 6 shows an example of the PO, tracings from which these results were compiled. All subjects showed oxygen utilization (a PO, decrease) with ornithine, arginine, proline and glutamic acid. Variable oxygen uptake (in 4-7 of

A,, A, and A,

Sub-fractions A, and A, of fraction A had little or no effect on the PO, [Fig. 4(a) and (b)] or pH. On the other hand, sub-fraction A, [Fig. 4(c)] produced a decrease in the PO,. slightly less than occurred with fraction A [cf. Figs 3(b) and 4(c)], and a small decrease in pH. Analysis with anthrone showed A, to contain the largest amount of carbohydrate [Fig. I(b); compare to Kleinberg et al., 19791. Effect of urea Addition of urea at either 5.4 or 27.7 mM had no effect on the PO,, but both concentrations resulted in a substantial increase in pH. Effect of mixtures

a

o-(tb)

of amino acids

With amino acids at concentrations those found free in salivary supernatant

similar to (Table l), a

‘oh

4 Time

lh)

Fig. 5. Comparison of the changes in PO2 and pH of a sediment incubation mixture after the addition of amino acids (a.a.) and salivary supernatant. The amino acids in the mixture are those shown in Table 1. The first and second addition (30 ~1) yielded a final concentration of each amino acid in the mixture similar to that in salivary supernatant and in Table 1. The third amino acid addition (60 ~1 of a higher concentration amino acid solution) yielded a final concentration of each amino acid 10 times that normally present in supernatant.

149

Saliva components and sediment oxygen use (a)

o (bl 0

2

I

3

6 4

0

2 Time

4

(h)

Fig. 7. Changes in the PO, and pH of salivary sediment mixtures in response to added proline. In (a), 30~1 of 90 mM proline was added initially and again after 3.5 h. In (b), a proline addition similar to those in (a) was made. Note that it caused no delay in PO,. The pH stopped rising in both experiments (a) and (b) when proline was added but began to rise once proline was completely oxidized. The final proline concentration was 3.4 mM.

5

6

Fig. 6. Changes

hmem

in the PO,

8

9

with different

amino

acids.

the 13 subjects) was seen with alanine, aspartic acid, asparagine and glutamine, and with cysteine but less so. For the remainder of the amino acids, oxygen uptake was seen in only &2 of the subjects and any response was generally poor. The amino acids often showed a delayed oxygen uptake response, i.e. less uptake at first than later (see, for example, the effects of proline, glutamic acid, arginine and ornithine in Fig. 6). However, when re-examined using higher amino acid levels than in Table 2 to produce a longer exposure time, the slower initial oxygen response was usually overcome. For example, 30~1 of a 90mM prohne stock solution added to an incubating sediment suspension resulted in a slow, gradual increase in the POZ response [Fig. 7(a)], taking nearly 3 h for the oxygen uptake to reach the maximum rate possible for the system conditions. A subsequent similar addition of proline gave an immediate maximum response [Fig. 7(a)]. On other occasions, in the same individual proline gave the rapid and maximum response immediately [Fig. 7(b)]. Similar, progressively greater uptake was seen with glutamic acid when tested at 3.4 mM, and, on occasion, with tyrosine, methionine and histidine, which normally show no or poor oxygen uptake.

lation of oxygen uptake activity. On the other hand, for peptides such as glutamylproline, whose constituent amino acids could both stimulate significant oxygen uptake, less uptake occurred than with either of their constituents alone. In general, peptides with one or more oxygen-stimulatiing amino acids were less effective stimulants than their constituent amino acids or acids alone. DISCUSSION

Fractionation fractions

of salivary supernatant and testing of its

Our fractionation experiments showed that a substantial amount of the oxidizable substrate in salivary supernatant is contained in fraction B, especially the B, portion. This fraction contains most of the amino acids of the supernatant along with many small peptides (Kleinberg et al., 1979). Because the free amino acids had to be added at 10 times their normal concentration in order to stimulate sediment oxygen

Effects of the peptides

The amino acids that either did or did not stimulate oxygen uptake also produced such responses as constituents of small peptides. This can be seen in the representative experiments shown in Fig. 8, where arginylserine and glut aminylserine showed considerable oxygen uptake, which their amino acid controls indicated was due to arginine. When peptides such as methionylserine and glycylserine were tested they, like their constituent amino acids, showed no stimu-

Fig. 8. Changes

in PO, and pH with glutaminylarginine.

arginylserine

and

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M. KORAYEMer al.

uptake to a level similar to that of unfractionated supernatant, it is evident that the free amino acids in saliva (Table 1; Kleinberg et al., 1976) can only be responsible for a small part of the total oxygen uptake activity of the supernatant (Fig. 5). This observation is in complete agreement with that of Molan and Hartles (1971). who found that the concentration of ammo acids needed to stimulate as much oxygen uptake activity as that of salivary supernatant was considerably higher than that found free in saliva. Consequently, amino acids from readily transported and hydrolysed small peptides (Payne, 1976: Kleinberg et al., 1979) and amino acids from more slowly hydrolysed larger salivary peptides and proteins (Kleinberg et al., 1979) would have to make up the discrepancy. This conclusion is supported by the earlier observation that hydrolysis of peptides associated with the B fraction is rapid, and that the amino acids they contain and release in largest quantities as free compounds are proline. glutamic acid, glycine and lysine, with lesser amounts of arginine, serine and aspartic acid (Kleinberg et al., 1979). Of these, we now show that proline. glutamic acid and arginine can stimulate oxygen uptake markedly (Table 2). Thus, the ease of hydrolysis of the BZ fraction peptides would explain the rapid oxygen uptake that occurs when unfractionated salivary supernatant is added to salivary sediment, whereas slower hydrolysis of the larger salivary peptides and proteins found in fraction A and the intermediate size peptides found in fraction B, would explain the much slower and prolonged uptake seen thereafter with unfractionated supernatant (Fig. 3). Molan and Hartles (1971) estimated that the carbohydrate in salivary supernatant IS responsible for approx. 20% of the oxidizable substrate. Our results are in agreement with this observation as fraction A was responsible for a similar percentage of the total supernatant oxygen uptake and this uptake can be attributed largely to the metabolism of its carbohydrate. Anthrone analysis indicated that fraction A contains virtually all of the supernatant carbohydrate (Kleinberg et al., 1979) and most of this carbohydrate is in sub-fraction A, (Fig. 1). When tested (Fig. 4), sub-fraction A3 stimulated oxygen uptake and a fall in pH like that observed with unfractionated fraction A (cf. Figs 4 and 3). The carbohydrate involved is probably part of salivary glycoprotein (Caldwell and Pigman, 1966), which the oral bacteria can easily hydrolyse and metabolize (Leach and Critchley, 1966). Little or no oxygen uptake was observed with fraction C (Fig. 3); however, the concomitant rise in pH can be attributed to base formation arising from the use of the urea that is usually found in this fraction (Kleinberg et al., 1979). This is supported by the observation that the addition of urea also caused the pH to rise and, like fraction C, had no effect on oxygen uptake. It is thus evident that certain amino acids contribute more than others to the stimulation of sediment oxygen-uptake activity. Arginine, ornithine, proline and glutamic acid contribute the most; a usually substantial increase in oxygen consumption occurred in all 13 subjects when tested with these (Table 2 and Fig. 6). Alanine, aspartic acid. as-

paragine. glutamme and cysteine were less umversal in their activities; they stimulated oxygen uptake in the sediment microflora in only 4-7 of the 13 subjects. The first four of this group of amino acids showed much greater uptake than did cysteine. The remainder of the 23 amino acids showed generally small effects on only O-2 of the subjects. Amino acid and small peptide oxidation Because the metabolic pathways involved in amino acid intermediary metabolism by the sediment bacteria are largely unknown, one can only speculate as to the possible metabolic sites where the observed uptake effects might have occurred. The arginine effect on uptake presumably takes place through ornithine because arginme m the sediment bacteria is catabolized first to citrulline and then to ornithine via the arginine deiminase pathway (Kleinberg et al.. 1979; Kanapka and Kleinberg, 1983). Neither of these two steps involves oxygen uptake, but subsequent degradation of ormthine may lead not only to putrescine and carbon dioxide (Kanapka and Kleinberg, 1983) but also glutamate and, as we found, to substantial oxygen uptake (Fig. 6). Proline, the other of the four most prominent oxygen-stimulating amino acids, can also lead to glutamate (Stryer. 1987). If so, then catabolism of glutamate would be the key to the degradation of the four most consistent stimulants of oxygen uptake by the sediment bacteria. Glutamate degradation by sediment has not been explored. However, upon de-amination. glutamate would yield a-ketoglutarate; from there, succinate is a possibility and this is an end-product in radioautographs used to trace arginine degradation (Kanapka and Kleinberg, 1983), although the amounts produced are small. On the other hand, gas chromatography has shown that substantial amounts of acetic acid are formed when glutamate is degraded (Wickham and Kleinberg, 1979) but the metabolic pathway(s) involved has not been traced. Some oxidative-reductive interactions may be involved with both proline and glutamate; proline when it produces 6 -aminovalerate (Costilow and Laycock, 1968) and the glutamate when it is involved in glutathtone-facilitated uptake of amino acids (Stryer, 1987). Alanine after de-amination and cysteine after deamination and loss of its thiol group are likely to feed into pyruvate (Stryer. 1987). from whence the pyruvate with the aid of (1) pyruvate dehydrogenase and phosphateacetyl transferase or (ii) pyruvate oxidase can stimulate oxygen use and yield acetic acid and carbon dioxide (Murphy et al., 1985: Condon, 1987). Degradation of aspartate. another of the oxygenstimulating amino acids, has not been studied in sediment (or plaque). However, upon de-amination, it yields oxalacetate (Stryer, 1987); from there, production of succinate (Hammen, 1969) and/or propionate and carbon dioxide (Wickham and Kleinberg, 1979) are possibilities. Asparagine and glutamine, two more amino acids that stimulated significant oxygen uptake in some subjects, can upon deamination yield aspartate and glutamate respectively; from there, aspartate and glutamate degradation could stimulate oxygen uptake, as suggested above. Whatever some of these pathways of degradation

Saliva components and sediment oxygen use

prove to be, and whichever of the bacteria in the sediment flora predominate in these reactions, amino acid stimulation of oxygen uptake generally involves oxygen use during de-(amination aided by amino acid oxidases, and oxygen use when the organic acids produced by the de-amination process enter carbohydrate pathways that involve oxidation of metabolic intermediates (Stryer, 1987). It has also been shown that ornithine can yield a hitherto unknown am ino acid, Ns-( 1 carboxyethyl)ornithine (Thompson, Curtis and Millar, 1986). This compound has been found in high concentration in the cytosol of Streptococcus Iactis (Thompson et al., 1986) and its formation requires pyruvate and NADH. Accordingly. its synthesis and subsequent degradation could be linked through NADH and appropriate respiratory enzymes to oxygen uptake, but whether this is so is not known. We found that arginine and ornithine stimulated significant oxygen uplake, whereas very little uptake was stimulated by citlulline (Table 2), the intermediate in arginine to ornit hme conversion by the arginine deiminase pathway (Kanapka and Kleinberg, 1983). This could be because citrulline in our test was provided extracellularly and thus, unlike that originating as a metabolic intermediate from arginine, it may not be readily transported. Arginine and ornithine can enter cells easily, possibly by an antiport transport system where both amino acids use the same membrane carrier (Thompson, 1986). When a variety of small peptides were tested, those containing residues of arginine, ornithine, glutamate and proline generally produced oxygen-stimulating effects but not quantitatively similar to those of their constituent amino acids (Fig. 8). For reasons yet to be determined, less oxygen uptake was observed with peptides than with their constituent oxygenstimulating amino acids. Peptides with amino acids that stimulated little or no oxygen uptake activity also showed minimal oxygen uptake effects. Basically, these findings were consistent with the view of peptldes undergoing hydrolysis upon or following their transport into the bacteria in the sediment, where peptidases present in association with bacterial cell membranes and cell walls could catalyse their hydrolyses (Payne and Gilvard, 1973; Kleinberg et al., 1979; Rogers et al., 1986, 1988). Induction

of oxidatrve activity

An interesting observation was that prior exposure of the sediment microflora to some amino acids would, if the acid was provided at a high enough level, result in enhanced oxygen uptake upon subsequent re-exposure to it (Fig. 7). In Fig. 7, the initial addition of proline to the incubation mixture resulted in a slow, gradual rise in oxgyen uptake, which took almost 3 h to reach the maximum rate, whereas a second addition resulted in the appearance of a maximum rate within the first 5 min. On other days and with sediment from the same subject [Fig. 7(b)], the first addition of proline caused a response similar to that observed before with the second proline challenge. These findings and the gradual improvement in oxygen uptake sometimes seen with other amino acids suggest an induction effect, which might include

151

an increase in membrane transport, production of necessary co-factors or intermediates, or even the emergence of bacteria able to use the amino acids supplied at more efficient rates. With regard to this last supposition, however, the composition of the major sediment microflora does not appear to change appreciably under these incubation conditions (Kleinberg et al., 1981). Relationship

to pH-rise factors

Hartles and Wasdell (1955a, b) observed, upon dialysis of stimulated whole saliva, that the dialysate contains a heat-stable factor which stimulates oxygen uptake and enhances glycolysis in the salivary bacteria. Other investigators then tried, but with limited success, to identify the salivary constituents responsible (Hay and Hartles, 1965; Guggenheim, 1966; Mishiro, Kirimura and Ishihara, 1966; Ishihara, 1967; Molan and Hartles, 1969; Kirimura, Morita and Mishiro, 1970; Holbrook and Molan, 1975). The reason for this became clear when Kleinberg et aI. (1976) and Kanapka and Kleinberg (1983) found that small arginine peptides are able to stimulate glycolysis; we have now shown that such peptides also stimulate oxygen uptake (Fig. 8). As a number of salivary constituents contain arginine (Kleinberg et al., 1979), each could be a stimulant of both glycolysis and oxygen uptake. Of these, small arginine peptides are dialysable and heat-stable; consequently, such compounds would satisfy the search initiated by Hartles and Wasdell (1955a, b) for a dialysable, heatstable compound or compounds in saliva with both glycolysis-enhancement and oxygen-uptake activities. Free arginine would not qualify as that unknown compound because there is generally little of it in saliva (Kleinberg et al., 1976) and its effect on glycolysis enhancement, except through its effect on the pH, may be poor (Kanapka and Kleinberg, 1983). One needs to keep in mind also that arginine and small arginine peptides both stimulate formation of base by the bacteria in salivary sediment and this enhances their ability to raise the pH (Kleinberg et al., 1976, 1979). This could stimulate both oxygen uptake and glycolysis in uncontrolled systems should the pH rise from acidity to neutrality and above (Kleinberg et al., 1979). Small peptides without arginine, but containing proline, glutamic acid or some of the other oxygenstimulating amino acids, have the potential to show the oxygen-uptake function but not glycolysis enhancement or pH-rise activity. Should such peptides in saliva fractionation experiments overlap with the urea normally present in saliva dialysates (Kleinberg et al., 1976), a false indication of a single oxygen uptake- and glycolysis-stimulating compound could result because urea, although it does not have oxygen-stimulating capability, does have a significant pH-rise function. One can conclude from this discussion that because saliva contains peptides with different amino acid compositions and thus with the potential to stimulate oxygen uptake, enhance glycolysis and/or affect the pH, many active fractions carrying one or more of these functions are possible. With such a range of possibilities, it is easy to understand why earlier attempts to identify the components in saliva respon-

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for

and/or

its oxygen

uptake,

glycolysis

enhancement

pH-rise activities have had limited success