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Q.1- What is the biological advantage of secretion of proteolytic enzymes  in the zymogen form ?

Answer- Certain proteins are synthesized and secreted as inactive precursor proteins known as proproteins. The proproteins of enzymes are termed pro enzymes or zymogens. Selective proteolysis convert a proprotein by one or more successive proteolytic “clips” to a form that exhibits the characteristic activity of the mature protein, eg, its enzymatic activity. (Figure-1)

Figure-1- showing the selective cleavage of a zymogen to form an active enzyme

Examples of other Proproteins or Zymogens

Proteins synthesized as proproteins include the hormone insulin (proprotein = Proinsulin), the digestive enzymes pepsin, trypsin, and chymotrypsin (proproteins = pepsinogen, trypsinogen, and chymotrypsinogen, respectively), several factors of the blood clotting and blood clot dissolution cascades and the connective tissue protein collagen (proprotein = procollagen).

Biological advantage of having  proteolytic zymogens

The synthesis and secretion of proteases as catalytically inactive proenzymes protects the tissue of origin (e.g., the pancreas) from auto digestion, such as can occur in  Acute pancreatitis.

Physiologic processes such as digestion are intermittent but fairly regular and predictable. Enzymes needed intermittently but rapidly often are secreted in an initially inactive form since the secretion process or new synthesis of the required proteins might be insufficiently rapid for response to a pressing pathophysiologic demand . Proenzymes facilitate rapid mobilization of an activity in response to physiologic demand.

Q.2- Give a brief account of the mechanism of absorption of products of digestion of protein, highlighting the role of Glutathione in this process.

Answer- Digestive products of protein can be absorbed as amino acids, dipeptides, and tripeptides (in contrast to carbohydrates, which can only be absorbed as monosaccharides). The absorption of amino acids takes place mainly in the small intestine. There are two mechanisms for amino acid absorption-

A) Carrier protein transport system (Figure-2)

  • It is the main mechanism for amino acid absorption
  • It is an active and energy requiring process.
  • The needed energy is provided by ATP
  • There are approximately 7 carrier proteins, each specific for a group of amino acids
  • These carrier proteins are Sodium dependent symport systems
  • Each transporter has two binding sites, one for sodium and the other for an amino acid.
  • Absorption of dipeptides and tripeptides is faster than absorption of free amino acids.
  • Na+dependent cotransport of dipeptides and tripeptides also occurs in the luminal membrane.
  • After the dipeptides and tripeptides are transported into the intestinal
    cells, cytoplasmic peptidases hydrolyze them to amino acids.
  • After absorption the amino acids are transported to the portal circulation by facilitated diffusion. Na + is expelled out of the cell in exchange for K+ through the Na+ -K+ ATPase pump (Figure-2).

 Figure-2-Mechanism of absorption of amino acids, dipeptides, and tripeptides by intestinal epithelial cells. Each is absorbed by Na+-dependent co transport.

Clinical significance

1) Cystinuria- Common transporter for cystine, ornithine, arginine and lysine(COAL) is present in gut and renal tubules. Deficiency of transporter results in loss of these  amino acids in the feces and urine.

2) Hart- Nup Disease-The  transporter for tryptophan  and neutral amino acid is deficient. There is  reduced  absorption of tryptophan , tryptophan deficiency produce neurological and skin manifestation (pellagra-like rashes).Neurological symptoms are due to the fact that tryptophan is a precursor for serotonin and melatonin, while skin rashes are due to deficiency of niacin,  since niacin can be synthesized from tryptophan.

3) Food allergies– Relatively large peptides may be absorbed intact, either by uptake into mucosal epithelial cells (transcellular) or by passing between epithelial cells (paracellular). Many such peptides are large enough to stimulate antibody formation—this is the basis of allergic reactions to foods.

B) Glutathione transport system (Υ- Glutamyl cycle)- Glutathione is used to transport  neutral amino acids in intestine, brain and kidney tubules.

Glutathione. This tripeptide consists of a cysteine residue flanked by a glycine residue and a glutamate residue that is linked to cysteine by an isopeptide bond between glutamate’s side-chain carboxylate group and cysteine’s amino group.(Figure-3)


Figure-3- showing the structure of glutathione (Gamma glutamyl cysteinyl glycine)

Role of glutathione in the absorption of amino acids (Figure-4)

  • Glutathione reacts with amino acid to form gamma glutamyl amino acid.This is catalyzed by Gamma glutamyl Transferase (GGT) in the presence of Na + (figure-2) to form Υ- Glutamyl amino acid and cysteinyl glycine.
  • The Υ-Glutamyl amino acid is then cleaved to give free amino acid and 5-oxo proline.
  •  Amino acid during this process is transported inside the cell.
  • It is an energy requiring process, which is supplied by the hydrolysis of peptide bond of Glutathione.
  • 5-oxo proline in the presence of the enzyme 5-oxo prolinase and ATP forms Glutamic acid
  • Cysteinyl glycine formed in the first step is cleaved to form cysteine and glycine.
  • Glutamic acid combines with cysteine first to form glutamyl cysteine and then combines with glycine to form glutathione.
  • Glutathione is regenerated again  and that completes the Υ- Glutamyl cycle.
  • The transport of one amino acid and regeneration of Glutathione requires 3  molecules of ATP.


Figure-4- showing the role of glutathione in the absorption of amino acids

Clinical  Significance- The deficiency of 5 oxoprolinase causes oxoprolinuria

Q.3- What is nitrogen balance ? Explain its significance and enlist the conditions causing deviations in the nitrogen balance.

Answer- The state of protein nutrition can be determined by measuring the dietary intake and output of nitrogenous compounds from the body. Although nucleic acids also contain nitrogen, protein is the major dietary source of nitrogen and measurement of total nitrogen intake gives a good estimate of protein intake (mg N x 6.25 = mg protein, as N is 16% of most proteins). The output of N from the body is mainly in urea and smaller quantities of other compounds in urine, undigested protein in feces; significant amounts may also be lost in sweat and shed skin. The difference between intake and output of nitrogenous compounds is known as nitrogen balance (Figure-5)

Figure-5- Showing nitrogen balance. The intake of nitrogen is in the form of dietary proteins while the output is through urine and feces in the form of undigested proteins, urea, uric acid, creatinine, ammonia and amino acids.

States of nitrogen balance

Three states can be defined-

1)  Nitrogen equilibrium– In a healthy adult, nitrogen balance is in equilibrium, when intake equals output, and there is no change in the total body content of protein.

Intake = output : N equilibrium

2) Positive nitrogen balance– when the excretion of nitrogenous compounds is less than the dietary intake and there is net retention of nitrogen in the body as protein.

Intake > output: positive N balance

Examples- In a growing child, a pregnant woman, or a person in recovery from illness there is positive nitrogen balance.

3) Negative nitrogen balance– There is net loss of protein nitrogen from the body

In response to trauma or infection, or if the intake of protein is inadequate to meet requirements there is negative nitrogen balance.

Intake < output: negative N balance

Significance of nitrogen balance

1) Growth-The continual catabolism of tissue proteins creates the requirement for dietary protein, even in an adult who is not growing; although some of the amino acids released can be reutilized, much are used for gluconeogenesis in the fasting state.The average daily requirement is 0.6 g of protein/kg body weight (0.75 allowing for individual variation), or approximately 50 g/day. Average intakes of protein in developed countries are of the order of 80–100 g/day, ie, 14–15% of energy intake. Because growing children are increasing the protein in the body, they have a proportionally greater requirement than adults and should be in positive nitrogen balance.. In some countries, protein intake may be inadequate to meet these requirements, resulting in stunting of growth.

2) Illness and convalescence– Negative nitrogen balance is seen immediately after acute illnesses like surgery, trauma and burns.

One of the metabolic reactions to a major trauma, such as a burn, a broken limb, or surgery, is an increase in the net catabolism of tissue proteins. As much as 6–7% of the total body protein may be lost over 10 days.

Chronic illnesses like malignancy, uncontrolled diabetes mellitus and other debilitating diseases also show negative nitrogen balance

Prolonged bed rest results in considerable loss of protein because of atrophy of muscles. Protein is catabolized as normal, but without the stimulus of exercise, it is not completely replaced.

Lost protein is replaced during convalescence, when there is positive nitrogen balance. A normal diet is adequate to permit this replacement.

3) Hormones- Insulin , growth hormone and androgens promote positive nitrogen balance while corticosteroids induce negative nitrogen balance.

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