tained whale tissues from Alaska and penguin 

 and seal tissues from Antarctica. From our 

 experience, however, we feel confident that we 

 are looking at essentially unaltered LDH pat- 

 terns in frozen tissue. 



CANTINO: Does it ever happen that you 

 get a change in pattern? 



MASSARO: If you mean, "can the pattern 

 be changed experimentally?," there is some 

 evidence in the affirmative. Kaplan's group 

 appears to have done this in tissue culture by 

 varying oxygen tensions. 



PAPACONSTANTINOU: The point with oxy- 

 gen tensions was that they didn't get any changes 

 unless they used abnormally high oxygen con- 

 centration. You never would find that in living 

 tissue. Isn't that about right? 



MASSARO: That is true. 



LOVETT: May lask a rather naive question, 

 perhaps? Has anyone looked carefully at some 

 of these purified isozymes from the point of 

 view of small molecules that might be function- 

 ing in a regulatory sense, at different stages 

 differently? For example, in an embryo as 

 compared with an adult? 



MASSARO: Not that I know of. 



LOVETT: There might be something like 

 intermediates of other pathways, or some other 

 coordinating system. I' m thinking about function. 

 For example, how rapidly can it turn over? 



MASSARO: To my knowledge, nothing has 

 been done on this particular aspect of the 

 problem. 



GRUN: I had the impression that the idea 

 was that there were two genes and that the 

 tetramers you got were random combinations 

 of polymers of these two genes. If A were 

 active, you'd expect to find only, or mostly, 

 LDH-5. If B were active, you'd expect the re- 

 verse of this. Some of the patterns that were 

 on your figures made it look as though there 

 wasn't a maximum at one end tapering off to the 

 other end, as though there was something 

 missing. 



MASSARO: Well, you have to keep one thing 

 in mind when you work with these zymograms. 

 Each of these isozymes, LDH-1, LDH-5, and 

 -2, -3, and -4, has different kinetic properties. 

 When they are placed together into a reaction 

 mixture which has a certain level of substrate 

 and allowed to react, those having a higher turn- 

 over rate are going to show up as bigger, 

 heavier blobs than those which have a very 

 slow turnover rate. 



PAPACONSTANTINOU: How much varia- 

 tion in turnover rate is there? 



MASSARO: There is about a two-fold dif- 

 ference between beef LDH-1 and -5. 



GRUN: Are -2,-3 and -4 intermediate? 



MASSARO: Present evidence suggests that 

 the properties of these heteropolymers reflect 

 the proportions of the parental monomers which 

 they contain. 



DEERING: Do you always find that you get 

 the proper ratios of isozymes if you know the 

 relative amounts of A and B? Take a situation 

 in which the amounts of A and B are not equal; 

 you wouldn't expect a 1:4:6:4:1 ratio in that 

 case. If you get the actual amounts of A and 

 B you can always predict the amounts of 1, 2, 

 3, 4, and 5, or is there the possibility of some 

 active mechanism which skews this in one direc- 

 tion or another? 



MASSARO: Theoretically you can predict 

 the distribution of isozymes, all things being 

 equal. 



TS'O: At least, Kaplan thinks so. 



DEERING: It's merely a function of the con- 

 centration of the two? 



MASSARO: It looks that way. However, 

 in vitro the distribution can be skewed by un- 

 known factors. 



PAPACONSTANTINOU: Under all situa- 

 tions the recombination seems to follow the 

 binomial theorem. The reason you get the dif- 

 ferent combinations may be because one is 

 turning over a thousand times more rapidly than 

 the other. At least, my impression from Markert 

 was that they never had any conditions under 

 which they didn't follow recombination explain- 

 able by the binominal theorem. 



MASSARO: I don't really think that this is 

 worth pursuing to any great length. 



GROSS: You look at a zymogram and you 

 see a gene product; your conclusions about 

 the amounts of these gene products depend on 

 the rate of the reaction in the gel. Has anyone 

 ever measured how much LDH-1, LDH-2, etc., 

 are present in a homogenate? The question 

 that's implied by this is, does the difference 

 that you see in an isozymic pattern really re- 

 flect the difference in quantity? 



MASSARO: If we know the turnover rates 

 of the isozymes under our conditions, it does. 

 This is the big problem. Of course, one way to 

 find out is to resolve the isozyme mixture by 

 electrophoresis, cut out the individual isozymes 

 and measure the quantities and turnover number 

 of each. If you get good recoveries for each iso- 

 zyme you have the answer. 



Now, we have done this. Unfortvinately, this 

 kind of analysis is usually unsatisfactory if 



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