An Attempt at Reconstruction with Utilization of Results and Data of Part I and Reaction constants determined in Part II

Abstract

Part I of the study on the metabolism of phosphorus in the liver of rats (17) recorded only the experimental results of our researches i. e. their descriptive systematization only), whereas Part II was concerned whit the computation of reaction constants of the metabolism in question (with quantities of phosphorus transport, the renewal periods of fractions, the specific velocities of the synthesis and those of the splitting of fractions in vivo) (18). In Part III, the present report, an attempt has been made to reconstruct — on the basis of the material of Part I and the reaction constants determined in Part II — the order of reaction in the metabolism of phosphorus in the liver of rats.

Moreover, Part III is concerned with the relation between concentration and transport viewed from the perspective of individual fractions and of the reactive arrangement as a whole. The experimental material of Part I has also been found useful in working out a methodical (theoretical) basis for the computation of reaction constants in the system of irreversible chain metabolism (14). The equations that resulted from this study (14) have made it possible to elaborate Parts II and III.

In dealing with the problematic of this Part III, three themes have been discussed. The first of these is concerned with indicators of the order of reaction: bases have been established and principles defined for criteria that can serve as indicators of the site and role of each fraction in the reactive arrangement. Special criteria have been determined concretely for each fraction. In chapter 2. a reconstruction of the order of reaction has been attempted on the basis of concrete indicators from Chapter 1. A graphic illustration of the reactive arrangement has thus been made possible. Chapter 3. describes the role of fractions in the formation of their own concentrations through the instrumentality of transport and the co-action of synthesis reactions and the splitting process. Quantitative dependence of concentration upon the inflowing transport, and, conversely, the conditionality of the outgoing transport upon concentration have also been established.

In addition to the criteria borrowed directly from Part I of the publication (percentual quantities of the renewal of fractions in the unit of time; the times of phosphorus syntheses of fractions; specific velocity of the synthesis and of the splitting of fractions), new bases

have subsequently been computed in this chapter for concrete criteria of individual fractions as, for instance, absolute quantities of phosphorus renewal and those of phosphorus transmission in the unit of time; absolute and equivalent transport quantities of individual fractions in the time of the renewal of a metabolic cycle of the whole chain. Ali bases (except those of specific velocities illustrated in Table 4 of Part II of this publication — (18) are systematically set out in Table No. 1.

In all computations the symbol 2 h stands for the time unit (this being the shortest period at which the measuring of phosphorus radioactivity in the fractions began), and 1845 m. represents the period of phosphorus renewal in the whole metabolic chain (which is identical with the period during which the fraction with the longest renewal, i. e. that of nucleoproteides, regenerates). In the sequence scheme itself, the transports have been given a period twice as long (i. e. 3690 m.), actually comprising two cycles of renewal of the chain, whereby computation with decimals has been avoided (e. g. in the ATP stable phosphorus fraction and those of adenyl acid). Absolute quantities of phosphorus transport for the renewal period of the whole chain (1845 m.) have been computed by multiplying the frequency rate of the period of renewal of the fraction by the quantity of its concentration. The absolute transport quantity of a fraction divided by the minimum of absolute transport among the fractions of the chain (in conferment’s = 19,3 mg) will give us the transport figure of the fraction in equivalent quantities. The round figures (i. e. the nearest integers except for two fractions where the equivalents represent halves between two integers) of these transport equivalents have been used as the principal basis (in addition to other criteria) for a direct reconstruction of the chain. Thus, the reconstruction of the chain has in principle been based upon the function of phosphorus transport through its fractions (combinations) in the liver of rats.

Graph 5 has been designed to facilitate comparisons of particular criteria for different factions. Ali the bases mentioned for concrete criteria are recorded in the graph as graphically comparable quantities. Thus, the task has been made considerably easier.

In chapter 2 of Part III an estimate has been made of the indicatory value of certain criteria. In order to be able to fulfil their respective roles at the fore-end of the metabolic chain, the fractions need to have the following features: a low rate of specific velocity of synthesis combined with a high rate of specific velocity of splitting; high values as regards both the absolute quantity of renewal and that of phosphorus transmission in the unit of time and for the whole period of the chain; and, lastly, a short period of time for a single renewal of a whole fraction combined with a high frequency rate of the totality of its renewal over the period of the whole chain (1845 m.). Properties opposed to those enumerated above have been made to serve as criteria that exclude the possibility of a fraction occupying a front position but, at the same time, being favourable to its being situated at the end part of the chain. This definition of the criteria ensures a frontal chain position for the fraction that reaches a great measure of transport within a short time, which, concurrently with a low specific velocity rate of synthesis provides a guarantee that a fraction with a later position in the chain shall be able to take up considerable quantities of phosphorus transport without delaying the process of transportation that goes on in it. The low underlying power for any prolonged stay of phosphorus at frontal fractions (which is conditioned by the low specific velocity of synthesis, in conjunction with simultaneous high specific velocity of splitting) is “condition sine qua non” for dealing effectually with the great volume of transport without an increase in the fractions’ own concentrations, i. e. without a long stay of phosphorus in them.

As regards some of the fractions, it has been established that all of their criteria demand the identical position to be occupied by the respective fraction in the chain of reactive arrangement. This applies specially to the group of sugar fractions (and to the labile, or the stable fraction from ATP alike, but not to the other fractions, though). On the other hand, there are many fractions certain criteria of which are mutually contradictory, and it is owing to this that some of the criteria lay it down that the fraction should take a position at the fore-end of the chain, while others demand the contrary, i. e, a position at the end part.

All these criteria have been given the rank of indicators the demands of which have to be satisfied without prejudice to other claims. As a matter of necessity, the criteria with excluding force have been given certain priority and full obligatoriness. The criteria opposed to these have been treated as facultative or auxiliary indicators that merely point to the possible position of the fraction. Contradictions among the criteria cannot be accidental, for they arise from the actual position of the fraction in the chain. Should for instance some criteria claim a frontal and others a posterior situation for a fraction, then the fraction must occupy such a position as to be simultaneously situated among the frontal and the posterior frictions. In this situation, each criterium is logical, for it denotes something real. However, if a criterium is viewed only in relation to its partner (apart from the actual position of the fraction), then two of them appear to be quite contrary.

The system of indicators, which was built up exclusively on the basis of quantitative data, in its entirety provides information about the situation and characteristic behaviour of each fraction in the order of reaction. with the help of these criteria a clear view can be had not only of the bases but also of the structure of reactive arrangement in the metabolism of phosphorus in the liver of rats. Nine criteria (eight of which are illustrated graphically) have been set up for each fraction. A tenth criterium has been discovered subsequently after the scheme of the order of reaction had been completed. This criterium is of great value, for it shoves subsequently and in a novel way that the reconstructed scheme also satisfies its own demands with regard to the arrangement of fractions in the chain.

When all indicators point to the same direction regarding the position of the fraction, it will be found quite easy to determine its situation and role in the chain. An example of this is provided first of all by the group of sugar fractions, where all the criteria without exception demand for it a position at the very front of the chain, which is in full agreement with the findings of Hevesy concerning the dependence of the amounts of organically bound phosphorus in the liver of rats upon the quantity of sugar contained in food. The frontal situation of sugar fractions also accords with our own findings to the effect that the proportions of phosphorus distribution in organic fractions of the liver of rats are almost identical with those found by Sachs (although) the absolute quantities resulting from the two analyses are strikingly different) when our results and those of Sachs are compared on the basis of phosphorus equivalence in the group of sugar fractions (13, 17).

The convincingness of our scheme as regards the organization of the order of reaction in phosphorus metabolism in the liver of rats, derives not only from the fact that the system of criteria has been drawn exclusively from quantitative data but more particularly from the finding that the experimentally determined transport quantities fit, quite equivalently (without any deficiencies or excesses except for the group of sugar fractions) into the transport chain at the place and stage assigned to the fraction by other criteria.

The frontal position of sugar fractions derives in the first place from the fact that theirs is the lowest rate of specific velocity of synthesis simultaneously with the highest rate of specific velocity of splitting, etc. These fractions possess the enumerated conditions for a frontal situation to a greater degree than any of the organic fractions. Next after the sugar fraction comes the fraction of labile phosphorus from ATP, its criteria bearing the closest similarity to those of the sugar group. Since the labile fraction receives altogether 26 equivalents of phosphorus (out of 42 equivalents transmitted by sugar), it is clear that it must have a partner for the rest of the equivalents. Now there are two partners with criteria near to those that apply to sugars and the labile phosphorus, and they are the stable phosphorus from ATP and the phospholipoids.

As regards the stable fraction, its criteria all point to one direction requiring it to occupy the particular position. On the other hand, among the criteria of the phospholipoid fraction there are some that exclude this possibility (e. g. the slow rate towards the achievement of full transport capacity, small quantity of transmission in the initial period, etc.) requiring the fraction to take its position at the end part of the chain.

The labile and the stable fractions jointly transport a total of 39 phosphorus equivalents, which is 3 equivalents less than the transport capacity of sugar (42 equivalents in 3690 minutes). The analyses in chapter 3 of Part III have shown that the calculated sugar transport exceeds the value which, according to the equation relative to concentration, should correspond to the concentration actually found. The probability therefore exists that the excess is due to insufficient reliability of the basis for calculating the rate of transport in the group of sugar fractions.

On the other hand, the excess of the trans port may well by-pass the chain in performing its function of blood sugar regulator for the needs of synthesis and splitting of glycogen. According to our criteria, the ATP transport its phosphorus at a very rapid rate, yet in such a way that the dynamics of the transport differ completely as regards the labile and the stable fraction. The stable phosphorus can only be transported from the ATP along with its organic carrier into the structure of free adenyl acid, the conferment’s and the nucleoproteids.

The consumers of labile phosphorus are of two types, (1) those that carry phosphorus into their structure, and (2) those that only use its energy. The greatest consumer of the type (1) are the phospholipoids which have all the qualifications needed for taking a position next to ATP; however, the above - mentioned exclusive indicators point to their being incapable of occupying a leading position in the principal line of transport. Nevertheless, this in no way prevents them from retaining this position in the chain, namely as a lateral branch of the chain. Thus, the demands of all contradictory criteria of phospholipoids are met, for trough situated at the end of the lateral branch, they are at the same time positioned directly behind ATP. The graph shows clearly that the phospholipoids occupy a distinct place in the chain; besides being positioned behind ATP they are also situated parallel to them, getting their amount of phosphorus out of glucose. That is to say, phosphorus as a labile fraction retrogrades to enter into the structure of glucose, from which it passes into and combines with phospholipoids in the form of glycerophosphates. Phospholipoids therefore are situated not only behind the ATP but at once parallel to them and at the lateral end of the chain. This position is reflected in the contradictory nature of the criteria themselves.

In the course of their transport the phospholipoids consume 12 equivalents of the total transport of labile phosphorus from the ATP (13 equivalents). None of the remaining fractions with an unassigned position possesses the criteria favourable enough to enable it to receive the unconsumed 13th part of labile phosphorus from the ATP. We must therefore look a likely consumer of this phosphorus elsewhere.

From the process involving the transmission of 12 equivalents of phosphorus to phosholipoids and of one equivalent of phosphorus to the unknown consumer, there resulted (through decomposition of ATP) 13 equivalents in all. It is characteristic of the fractions occupying positions at the end part of the chain that they are incapable — owing to their structures and criteria — of obtaining even one equivalent of labile phosphorus from the ADP (nor for that matter from the ATP).

The three fractions at the lower end of the chain compete as consumers of stable phosphorus. Their joint capacity of intake equals the capacity of the ADP yield. In the process, these 3 consumers merely integrate the whole of the organic complex with the stable phosphorus built in.

Three equivalents of adenyl acid originate from the ADP which passes on to a consumer three parts of labile phosphorus. Our criteria throw a slightly different light on the position and role of this fraction; consequently, our conclusions differ from current opinion. According to experiments i n v i t r o, the fraction in question could be used for the syntheses of ali those organic compounds in whose structure it is found as a bound complex. Our experiments have revealed no qualitative but only quantitative grounds for our not being able to concur with current opinion; our objection however has an exclusive force. While the ATP becomes, according to Szent-Gydrgyi, reversibly rephosphorized (from the ADP, and this in turn from the free adenyl acid) in muscles when it works, no such phenomenon is observable in the liver of rats. Thus, for instance, if the free adenyl acid were able to meet the needs of the renewal of ATP, it would have to renew itself 13 times (within 3690 minutes), whereas, actually, it does so 3 times in all.

Nor does the capacity of transport via the free adenyl acid meet the needs of regeneration of the nucleoproteids, for it could only satisfy one third of these. More interesting still is the case of conferment’s whose capacity of renewal could be amply satisfied by the free adenyl acid — with even a surplus of one equivalent of unused stable phosphorus.

However, a comparison of the initial increase of radioactivity in these two fractions showed that the conferment’s had satisfied their capacity of regeneration before the free adenyl acid \vas able to transport the required amount of phosphorus. Consequently, the regeneration in question must have proceeded through other channels. The velocity rate of the renewal of conferment’s here does not correspond with the velocity the adenyl acid would need to enable it to fulfil its role. Therefore, the general conclusion must be to the effect that free adenyl acid is not an intermediate stage for the syntheses of combinations in which it is present as a bound component. It appears that the bound and the free adds are transported by different ways.

Though the free adenyl acid cannot provide us with any information as to its role in the metabolism of the liver in rats, such knowledge might be gained from the movements of its partner from which it separates at the moment of its formation. Its partners are the three equivalents of labile phosphorus which, as shown above, cannot be consumed by the fractions situated at the end part of the chain. Consequently, the possible consumers of the three equivalents of labile phosphorus from the ADP and of the 13th equivalent of labile phosphorus from the ATP must be sought among the frontal fractions. Sugar seems to be the only likely consumer, for it is known from an earlier study of the metabolism of glucose (9, 10) and enzymologic researches (2) that glucose is able to join the process of decomposition only provided it first becomes transformed into hexozodiphosphorus acid, and in condition that it obtains for this stage both kinds of phosphorus from the labile fraction of ATP (respectively ADP). The above-mentioned transport surplus of 4 equivalents makes the conclusion inevitable that through these four equivalents only two mols of glucose can be caused to react. Moreover, studies by Ochoa and Lipmann (11, 7, 8) have shown that in complete oxidation any glucose can serve for the purpose of changing 24 new kinds of inorganic phosphorus into organically bound ones. It is obvious that two mols of activated glucose have a potential capacity high enough to affect the whole transport in the chain.

There even remain unused 6 equivalents of the potential. It seems to us that we may assume that the capacity of glucose to provide the chain with inorganic phosphorus, and the capacity of the surpluses of labile phosphorus to activate glucose in the performance of its role, is not an accidental correspondence. On the contrary, we are convinced that the two processes are in mutual relationship as partners in the performance of the same task, viz. in the regeneration of the chain of phosphorus metabolism in the liver of rats. In point of fact, approximately 10 per cent of the activated phosphorus goes back from the stage of ATP and re-enters the glucose, thus supplying it with the energy necessary to set in motion a new cycle of metabolism. From all that has been set out above there are grounds for supposing that the free adenyl acid in the liver of rats is produced as an irreversible and incidental product of the process by which the metabolic chain is provided with activated phosphorus for the needs of its auto regeneration.

Interesting is the destiny of 10 ADT equivalents the consumers of which can only be the conferment’s and the nucleoproteids, which integrate 10 equivalents of stable phosphorus. These fractions, however, lack the consumptive capacity for 10 equivalents of labile phosphorus, nor does the stable fraction enter into combination with them along the gradient of free adenyl acid. The only remaining possibility is that the labile phosphorus separates from the stable one in the act of synthesis at the moment when the ADP adenyl acid is passing over into the structure of conferment’s or nucleoproteids (making use of the energy present in its labile phosphorus).

At the end of chapter 2 it has been stated that the scheme as constructed makes possible an analysis of the order of reaction in the form of a uniform system. It can be seen that the entire transport process is accomplished in three basic stages. The first stage is affected by the group of sugar fractions transmitting 39 equivalents of phosphorus to the ATP to be built in there (two thirds of them as the labile fraction and one third as the stable one). The sugar fractions are first-class carriers, for it is they that activate phosphorus directly (inorganic into organic). The ATP is a second-class carrier because it obtains, as the on1y system at the second stage, phosphorus from the group of sugar fractions. Ali the other fractions get their supplies of phosphorus through the ATP and they are therefore third-class consumers and carriers.

The third transport stage comprises, in variuos ways, five different fractions including glucose which is, in other respects. thc “genohead” of ali first-class carriers. Glucose obtains, in retrograde way, about 10 per cent of activated phosphorus from the labile fraction, while taking approximately 90 per cent of phosphorus from the common inorganic fund. It is a feature worth noting that each of the three successive stages has the same equivalence of transport capacity as the other two (each stage obtaining and transmitting 39 equivalents at a time within a period of 3690 minutes).

In chapter 3 an analysis has been attempted of the relation between the transport and the accumulation roles of the fractions in the chain of transmittance. It is obvious that the frontal fractions with their high specific velocities of decomposition and low velocities of synthesis show a very pronounced function of transport and a minimum function of accumulation (73,2:1;-47,3:1-;—43:1). On the other hand, with reference to the fractions at the end part of the chain with specific velocities of synthesis higher than those of decomposition, the transport function only just surpasses the accumulation function of the fraction (in the case of conferment’s and phospholipoids the relevant ratio is approx. 2:1 in favour of transport) or even the other way round, the accumulation function exceeds the transport function (nucleoproteid = 2,3:1 in favour of accumulation of phosphorus in the concentration).

In this chapter, the criterium of uniformity as regards the reaction of the reactive chain has been examined and checked from another aspect. The results have shown that in conditions of equivalence among transports of particular stages the proportions of concentrations in different fractions are in the same relations as the square proportions S R

of their transports provided their fractions K K are mutually equated. This applies in much the same degree both to the cases 2here this equation actually exists and to those where the equation of the fractions S R K K bas been arrived at by arithmetical computation. As the concentrations were determined by colorimetry and the transports computed from the quantity of the rise in radioactivity during the period of their increase in the fractions, this correspondence goes to show that the reaction chain functions as a uniform system, the factor of combination resting upon the transport of phosphorus by way of the chain.

At the end of chapter 3 the equations for computation of specific velocities in the running chain have been made to conform to the fact that the quantity of transport depends for its realization on the function of time.