Poisonous mushrooms Bill Indge The material in this presentation is designed to support the article ‘Edible and poisonous mushrooms’ by Chris McInerny in the April 2015 issue of Biological Sciences Review (pp. 26–29). It concentrates on one of the toxins, -amanitin, produced by one of the most poisonous of all fungi: the deathcap, Amanita phalloides.

Physiological systems Bringing it all together Molecules We will also be looking at the way in which Amanita phalloides affects the liver. In order to understand this we will be drawing on information from a range of different topic areas. These are summarised in the diagram. In this case the central topic that we are going to consider is poisonous mushrooms and, in particular, the effect of -amanitin. We will return to this diagram at the end. Ecology Cells Poisonous mushrooms Genes and genetics Physiological systems

Amino acids and peptide bonds R R The starting point for this exercise is a secure understanding of amino acids and the way in which they join by condensation to produce a polypeptide. The next four slides illustrate the principles underpinning Question 1. This slide shows the basic structure of an amino acid. The molecular detail shown here is sufficient to enable students to understand how peptide bonds are formed. O O H H N C C N C C H OH H OH H H

Amino acids and peptide bonds R R The next two slides in the sequence show the elimination of a molecule of water and the formation of the bond. O O H H N C C N C C H OH H OH H H

Amino acids and peptide bonds R R O O H H N C C N C C H Peptide bond OH H H OH H

1 (c) In the simplest cyclic polypeptides, the amino acids bind together to form a ring. How many peptide bonds would there be in a cyclic polypeptide containing eight amino acids? Explain how you arrived at your answer. (2 marks) A simple diagram such as that shown should enable students to answer the question successfully. The marking scheme is shown below. (c) 8; There is a peptide bond between the amino acids forming the polypeptide plus an additional bond where the two end amino acids bind to form a ring; (2 marks) Amino acid Peptide bond joining two amino acids

Digesting peptides and polypeptides Endopeptidases break peptide bonds in the middle of a polypeptide chain producing smaller polypeptides Although -amanitin is a polypeptide it is not digested in the gut. The next two questions look at one of the reasons why this is so. This slide looks at the two main types of protein-digesting enzyme secreted by the gut. Exopeptidases break peptide bonds in the end of a polypeptide chain releasing amino acids

Exopeptidases break peptide bonds in the end of a polypeptide chain releasing amino acids Endopeptidases break peptide bonds in the middle of a polypeptide chain producing smaller polypeptides Cyclic polypeptide so no smaller polypeptides produced; No free amino/ carboxylic acid groups; Will not bind with enzyme; This slide relates this information to question 2 and provides the answer. -amanitin is not broken down by endopeptidases. Use this information to suggest why exopeptidases do not digest -amanitin. (2 marks) 

Bile salts and their circulation intestine Bile salts and their circulation Bile salts are produced in the liver and enter the duodenum Most of the bile salts are reabsorbed in the lower part of the small intestine These bile salts are returned to the liver in the blood liver The next two slides provide a simple summary of enterohepatic circulation of bile salts. The questions involve no more than basic recall. (Stored in gall bladder) then through bile duct 3 (a) Describe the route by which bile salts produced in the liver enter the duodenum. (1 mark) 

Bile salts and their circulation intestine Bile salts and their circulation Bile salts are produced in the liver and enter the duodenum Most of the bile salts are reabsorbed in the lower part of the small intestine These bile salts are returned to the liver in the blood liver Hepatic portal vein 3 (b) Name the blood vessel through which bile salts are returned to the liver. (1 mark) 

4 There is a delay of 2–6 days between eating fungi containing -amanitin and the development of symptoms associated with liver damage. (a) Explain why this delay might suggest that it would be inappropriate to treat patients showing symptoms with substances that remove poisons from the gut. (2 marks) In Question 4, students are required to apply this basic understanding of enterohepatic circulation. They have been told the -amanitin follows the same pathway. Part (a) is linked to the time taken for food to pass through the gut. Takes less than 2 days/short time for substances in gut to be absorbed; Will pass out as faeces; So would not expect any -amanitin to still be in gut

(b) In spite of this delay, patients are often given oral doses of activated charcoal. Activated charcoal absorbs poisonous substances in the gut which are then safely removed from the body in faeces. Use information provided in this question to explain why giving activated charcoal might still be a useful treatment for a person who has eaten fungi containing -amanitin. (2 marks)   Part (b) involves an understanding of the importance of enterohepatic circulation -amanitin is recycled/passes back into the gut; Activated charcoal may remove the -amanitin that re-enters the gut; So would not reach such high concentrations in the liver;

Getting into liver cells Concentration of -amanitin/µmol dm–3 Rate of uptake of -amanitin/ µmol min–1 mg–1 protein 50 1.0 100 2.2 150 3.0 200 3.8 400 4.4 600 5.0 800 The next sequence of slides relates to an investigation into the way in which -amanitin enters liver cells. The first part of the question is based on a table showing the effect of -amanitin concentration on its rate of uptake.

Make sure that you understand the data Read the stem It is suggested that -amanitin enters cells by facilitated diffusion, making use of the same transport protein as bile salts.   Scientists investigated factors affecting the rate of uptake of -amanitin through liver cell membranes. In the first experiment they looked at the effect of the concentration of -amanitin on its rate of uptake. Their results are shown in the table. A reminder that data need understanding. Start by reading the stem. There are some important clues here that will help in answering the questions that follow.

Check the table headings in the stem Concentration should be clear enough Concentration of -amanitin/ µmol dm–3 Rate of uptake of -amanitin/ µmol min–1 mg–1 protein 50 1.0 100 2.2 150 3.0 200 3.8 400 4.4 600 5.0 800 Check the table headings. Concentration of -amanitin should be clear enough and ought not to require further explanation.

Check the table headings in the stem This is rate of uptake, not the total amount of -amanitin taken up Concentration of -amanitin/ µmol dm–3 Rate of uptake of -amanitin/ µmol min–1 mg–1 protein 50 1.0 100 2.2 150 3.0 200 3.8 400 4.4 600 5.0 800 Care needs to be taken with the second column heading. It is rate of uptake, not total amount taken up. This is a common misinterpretation with data such as these.

Make sure that you know what the table as a whole is telling you Put the information in this box into words Concentration of -amanitin/ µmol dm–3 Rate of uptake of -amanitin/ µmol min–1 mg–1 protein 50 1.0 100 2.2 150 3.0 200 3.8 400 4.4 600 5.0 800 The final step is to make sure that the table as a whole has been understood. A simple way of doing this is to take one of the boxes in the second column and put it into words.

Concentration of -amanitin/ µmol dm–3 Rate of uptake of -amanitin/ µmol min–1 mg–1 protein 50 1.0 100 2.2 150 3.0 200 3.8 400 4.4 600 5.0 800 The first question requires a description of the general trend or pattern revealed in the table. The following slide ought to remind students of what they need to do to gain credit. (a) Describe the effect of -amanitin concentration on its uptake by liver cell membranes. (2 marks)

 Describe Follow these guidelines Look for the overall trend or pattern Describe this in terms of the column headings Quote values where they peak or the gradient changes and 2 marks are guaranteed.  A recipe for success when it comes to answering questions that require a description of data. Sometimes it helps to sketch a simple graph

Concentration of -amanitin/ µmol dm–3 Rate of uptake of -amanitin/ µmol min–1 mg–1 protein 50 1.0 100 2.2 150 3.0 200 3.8 400 4.4 600 5.0 800 The answer to the question. (a) Describe the effect of -amanitin concentration on its uptake by liver cell membranes. (2 marks) The rate of uptake increases and then levels out (as the concentration of -amanitin increases); Between 400 and 600 µmol dm–3;

Concentration of -amanitin/ µmol dm–3 Rate of uptake of -amanitin/ µmol min–1 mg–1 protein 50 1.0 100 2.2 150 3.0 200 3.8 400 4.4 600 5.0 800 Part (b) requires an explanation of these changes. (b) Explain the change in the rate of uptake at concentrations of -amanitin between 400 and 800 µmol dm–3. (2 marks)

Explain Follow these guidelines: Explain means ‘Give a reason why’. A description, however good, will not gain marks. Start your answer with the word ‘Because’. If you are asked to use a table, or a graph or diagram, make sure you do. It is a good idea to include a phrase such as ‘in the table’ or ‘the graph shows’. This will show your examiner that you are using the relevant information. Describe this in terms of the column headings. Quote values where they peak or the gradient changes. If you do all this, 2 marks are guaranteed. The command word ‘explain’ creates problems for many candidates. Answers require a reason to be given. The points listed on the slide should always be taken into consideration in questions of this sort.

Concentration of -amanitin/ µmol dm–3 Rate of uptake of -amanitin/ µmol min–1 mg–1 protein 50 1.0 100 2.2 150 3.0 200 3.8 400 4.4 600 5.0 800 The answer to part (b), starting with ‘Because’. (b) Explain the change in the rate of uptake at concentrations of -amanitin between 400 and 800 µmol dm–3. (2 marks) Because -amanitin is taken up by facilitated diffusion; At concentrations above 400 µmol dm–3 the carriers are saturated with -amanitin;

Getting into liver cells Don’t forget Stem Axes Graph as a whole Getting into liver cells Rate of uptake of amanitin/ µmol min–1 mg–1 protein 800 600 400 200 The next part of this question looks at data presented as a graph. The same basic rules apply to ensure a thorough understanding of the graph before attempting the questions. Amanitin only Amanitin + bile salts 0 30 60 90 120 Time/s

Getting into liver cells 6 (a) Explain why the units for -amanitin uptake are given per gram of protein. (2 marks) (b) Explain how the results shown in the graph support the suggestion that -amanitin enters cells by facilitated diffusion, making use of the same transport protein as bile salts. (2 marks) Getting into liver cells Rate of uptake of amanitin / µmol min–1 mg–1 protein 800 600 400 200 The questions are shown on this slide, but to ensure room for the answer, they are repeated with the answers on the next slide. Amanitin only Amanitin + bile salts 0 30 60 90 120 Time/s

6 (a) Explain why the units for -amanitin uptake are given per gram of protein. (2 marks) Because this allows the results to be compared; As uptake depends on the number of carrier proteins present/ the number of carrier proteins may vary from membrane to membrane; 6 (b) Explain how the results shown in the graph support the suggestion that -amanitin enters cells by facilitated diffusion, making use of the same transport protein as bile salts. (2 marks) Because the graph shows that the rate of uptake is lower when bile salts are present; -amanitin and bile salts are competing for the same transport protein;

This question requires you to apply knowledge to a new situation. 6 (c) The fetus is not harmed if a pregnant women eats fungi containing -amanitin. Suggest why the fetus is not poisoned by -amanitin. (2 marks) This question requires you to apply knowledge to a new situation. The question will be based on information you should know. You will be given all other information you need. This slide shows the last part of the question. It requires students to apply their knowledge to new situations. This slide shows the question, some advice and the answer. -amanitin cannot pass through the placental membranes; No -amanitin/bile salt carrier proteins present; All you needed to know was that substances reach a developing fetus by way of the placenta.

Facilitated diffusion Physiological systems Bringing it all together Molecules Protein structure Returning to the diagram with which we started this presentation, you can see how an understanding of the way in which -amanitin affects the body requires an understanding of molecules, cells, physiological systems and genetics. Ecology Cells Facilitated diffusion Poisonous mushrooms Genes and genetics Physiological systems Protein synthesis The gut and digestion