Chapter 19, Problem 25DAP

(a)

Summary Introduction

To explain: The effects of DNP $\left(\text{2,4-dinitrophenol}\right)$ on inhibited mitochondrial preparation.

Introduction:

The knowledge of the steps of respiratory chain and the pathway through which ATP (adenosine triphosphate) is produced comes from dissecting the whole process by using uncouplers and inhibitors.

(b)

Summary Introduction

To describe: The process that is directly affected by DCCD.

Introduction:

DCCD (dicyclohexylcarbodiimide) reacts with carboxyl side chains of acidic amino acids and its addition to actively respiring mitochondria decreases the consumption of oxygen and ATP production rate. Addition of DNP revert oxygen consumption; however, production of ATP remains inhibited.

(c)

Summary Introduction

To explain: Fillingame assay ATPase activity instead of ATP synthase activity.

Introduction:

The oxidative phosphorylation machinery in Escherichia coli and mammals are remarkably similar. If DCCD is added to a culture of wild-type Escherichia coli strain then further growth is blocked.

(d)

Summary Introduction

To explain: Whether DCCD-binding protein missing in mutant strain or it is just altered.

Introduction:

Fillingame selected DCCD-resistant mutant in which presence of DCCD diminishes slight amount of aerobic growth. To test whether the DCCD component is ATP synthase and or ATPase, he isolated membrane fraction from wild-type and mutant strain.

(e)

Summary Introduction

To explain: The expected result if DCCD-binding protein were in stripped membrane and if it were in soluble fraction.

Introduction:

To know DCCD-sensitive protein is an integral part of membrane or it is a part of solubilized fraction, Fillingame prepared stripped membrane and soluble ATPase using dithiothreitol.

(f)

Summary Introduction

To describe: The way through which Fillingame knew that which of the labeled proteins were of interest.

Introduction:

Fillingame exposed intact membrane of mutant and wild-type strain to $\text{C}\text{ DCCP}$ to identify DCCD-sensitive protein. Then he separated protein using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

(g)

Summary Introduction

To explain: The reason behind obtaining specifically labeled same protein in wild-type fraction.

Introduction:

Fillingame exposed intact membrane of mutant and wild-type strain to $\text{C}\text{ DCCP}$ to identify DCCD-sensitive protein. Then he separated protein using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

(h)

Summary Introduction

To explain: The deduction of structure, topology, and function of protein of interest.

Introduction:

On examining the protein of interest, it was found that M_r of protein of interest was about $9$ kDa (kilodalton). It was also found that protein was soluble in nonpolar solvent such as methanol and chloroform.

(i)

Summary Introduction

To explain: The reason behind mutant in which serine residue is substituted with alanine is more sensitive to DCCD.

Introduction:

In later experiments, it was found that aspartate at 61 position react which DCCD. However, if serine residue in mutant strain is replaced with alanine at 21 position, then it becomes less sensitive to inhibition by DCCD more than wild-type.

(j)

Summary Introduction

To explain: The role of DCCD in oxidative phosphorylation.

Introduction:

It was found that DCCD-inhibited protein is a central part of F₀F₁ ATP synthase of prokaryotes, plants, and animals.