73.
You are studying the binding of proteins to the cytoplasmic face of cultured liver cells and have
found a method that gives a good yield of inside-out vesicles from the plasma membrane.
Unfortunately, your preparations are contaminated with variable amounts of right-side-out
vesicles. Nothing you have tried avoids this contamination. Somebody suggests that you pass
the vesicles over an affinity column made of lectin coupled to Sepharose beads.
What is the rationale of this suggestion?

• (A) Right-side-out vesicles will be lysed by lectin coupled to Sepharose beads.
• (B) Right-side-out vesicles will simply bind to the lectin coupled Sepharose beads.
• (C) Lectin will bind to the carbohydrate residues present only on the inside out vesicles.
• (D) Lectin will bind to only glycoproteins and glycolipids present on the inside out vesicles.

  Lectin-Sepharose selectively binds right-side-out vesicles due to exposed glycoproteins on their external surface, allowing inside-out vesicles to pass through uncontaminated.

  Correct Answer

  B. Right-side-out vesicles will simply bind to the lectin coupled Sepharose beads. This exploits plasma membrane asymmetry where carbohydrates face outward.

  Option Breakdown

  • A. Right-side-out vesicles will be lysed by lectin coupled to Sepharose beads: Incorrect. Lectins bind carbohydrates non-destructively; they agglutinate or adsorb vesicles without lysis.​

  • B. Right-side-out vesicles will simply bind to the lectin coupled Sepharose beads: Correct. External glycoproteins (exposed in right-side-out) bind lectins like concanavalin A; inside-out vesicles lack accessible glycans.​

  • C. Lectin will bind to the carbohydrate residues present only on the inside out vesicles: Incorrect. Opposite—carbohydrates are extracellular; inside-out hides them inside.​

  • D. Lectin will bind to only glycoproteins and glycolipids present on the inside out vesicles: Incorrect. Inside-out vesicles expose cytoplasmic face (no glycans); binding targets extracellular components.​

  Right-side-out vesicles lectin sepharose purification leverages membrane glycoprotein asymmetry for clean inside-out vesicle isolation from liver cell plasma membranes.​

  Plasma membranes orient carbohydrates extracellularly. Right-side-out vesicles expose these lectin-binding sites; inside-out vesicles expose cytoplasm (glycan-free). Passing over lectin-Sepharose removes contaminants in flow-through.​

  Technique Comparison

  Option	Mechanism	Correct?	Rationale
  A. Lysis	Destructive	No	Lectins agglutinate, don’t lyse ​
  B. Binding	Affinity adsorption	Yes	External glycans bind ConA ​
  C. Inside-out binding	Wrong orientation	No	Cytoplasmic face glycan-free
  D. Inside-out glycoproteins	Wrong surface	No	Glycans extracellular only

  Answer B—key for vesicle polarity studies.​

  Exam Applications

  GATE tests membrane asymmetry concepts. Wheat germ agglutinin or concanavalin A selectively remove >90% right-side-out vesicles. Flow-through yields pure inside-out for binding assays.​