Medical Biochemistry I-Exam 3

Name 3 types of sphinogolipids and describe them

sphinogomyelin

 

impt membrane component esp. in nervous sytem

synthesized from 2 routes by ceramide


ceramide

 

immediate precursor of sphinogolipids

second messenger for NGF

 

globoside

 

neutral cerebroside oligosaccharides

 

gangliosides

 

acidic ceramide oligosaccharides that contain N-acetyl neuraminic acid (NANA or sialic acid)

What compounds are the precursors for sphinogosine?
serine and palmitoyl CoA

How is ceramide formed?

 

Sphingosine + acyl CoA  Ceramide + CoASH

                                       (Acyl CoA transferase)

 

How is sphingomyelin synthesized?

ceramide + CDP-choline → sphingomyelin + CMP

 

OR

 

ceramide + phosphatidyl choline → sphingomyelin + DAG

What are the 4 types of glycosphinogolipids?

cerebrosides

 

sulfatides

 

globosides

 

gangliosides

How are cerebrosides synthesized?
ceramide + UDP sugar → cerebroside + UDP
What is PAPS and what is it used for?

3’-phosphoadenosine-5’-phosphosulfate

 

It is an activated sulfate which converts galactocerebroside to sulfatide

 

What defect exists in Gaucher’s disease?
Deficiency of glucocerebrosidase, preventing degradative path of Glucocerebroside to Ceramide.  This is the most common sphingolipidoses (lipid storage disease)
What defect exists in Tay-Sachs disease?
deficiency of hexoaminidase A
What are the structural features of gangliosides?
acidic ceramide oligosaccharides that contain N-acetyl neuraminic acid (NANA or sialic acid)
What does semiconservative replication mean?
the 2 daughter double-stranded DNA molecules each contain 1 strand of parent DNA
What 3 observations have been made for all DNA polymerases?

Incoming base paring is selected by complementary base pairing with a template strand.

 

Chain growth is in the 5’ to 3’ direction

 

All DNA polymerases require a primer strand to add bases to.

 

What is the function of DNA polymerase I?

prokaryotic DNA elongation and repair

 

5′-3′ polymerization activity (synthesis)

 

5′-3′ exonuclease activity (repair and removal)

 

3′-5′ activity (proofreading)

What is the Klenow fragment?
larger subunit of DNA polymerase I that contains 5′-3′ synthesis activity and 3′-5′ proofreading activity
What is the function of DNA polymerase III?

prokaryotic DNA elongation and repair enzyme

 

complex structure; 10 subunits

 

forms a sliding clamp on the DNA strand

 

has a higher processivity than DNA polymerase I

What is the function of a topoisomerase?

enzyme that changes the topography of DNA molecules by cutting strands; allows the amount of supercoiling to be adjusted and re-ligating the strands

 

relieves supercoiling

What is the function of a helicase?
enzyme that catalyzes the unwinding of double stranded DNA by disrupting base-pair H-bonding
In replication, what is the leading strand and what is the lagging strand?

leading strand – DNA continuously synthesized in 5′-3′ direction

 

lagging strand – DNA discontinuously synthesized in 3′-5′ direction (Okazaki fragments)

How is RNA used in DNA replication?
RNA sequences serve as the primer sequences for DNA polymerase in the discontinuous synthesis of the lagging strand
What are Okazaki fragments?

pieces of DNA that are replicated on the 3’-5’ (lagging) strand that will eventually be joined by ligases to make a complete DNA replicate strand.

 

Where is E. coli DNA replication initiated and where is it terminated?

initiated at oriC

 

terminated at the tau or ter site

What are the processivities of DNA polymerases I and III?

DNA polymerase I has a low processivity (20 bases before it disassociates)

 

DNA polymerase III has a high processivity (5 million bases before it dissociates)

 

 

 

Where is DNA found in eukaryotes?
in the nucleus
How is DNA packaged in eukaryotes?
the double stranded DNA molecules are wrapped with their negatively charged phosphate groups around octamers of positively charged proteins called histones
What are some contrasts between features of prokaryotic and eukaryotic DNA replication?

Eukaryotic DNA is much longer, the synthesis is much slower and occurs in accordance with the cell cycle.

 

Eukaryotic cells use a large number of DNA polymerase molecules and begin replication at multiple sites.

 

What DNA polymerase is believed to be responsible for leading strand synthesis and which for lagging strand synthesis in eukaryotes?

leading strand synthesis – DNA polymerase delta

 

lagging strand synthesis – DNA polymerase alpha

What proteins are in nucleosomes and why do they bind to DNA?

nucleosomes contain positively charged histone proteins

 

these + charged histones are attracted to the – charged  phosphate groups of DNA

What is PCNA and what role does it play?

Proliferating cell Nuclear Antigen (PCNA) is a cyclin that forms a clamp around the template DNA strand to dramatically increase the processivity of DNA polymerase delta.

 

What is the arrangement of nucleosomes after DNA replication?

Nucleosomes are cooperatively distributed after DNA replication

 

The new histones end up along one daughter stand near each replication fork

 

The histones from the original nucleosomes are found in the nucleosomes reformed along the other new strand

 

How might nucleosomes affect eukaryotic DNA replication?

The presence of these histones (specifically the original ones, that remain associated with one strand of the replication fork) (may be why DNA polymerization is so much slower in eukaryotes)

 

What is the model used to describe DNA replication in mitochondria?
displacement loop replication
What are the overall anabolic and catabolic pathways involved in nucleotide metabolism?

anabolic – purine and pyrimidine de novo synthesis and salvage pathways

 

 

catabolic – purine and pyrimidine degradation pathways

What major difference is there between purine and pyrimidine synthesis in terms of when and how the ring is added?

purines – the ring is built as the pathway progresses.

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pyrimidines, the ring is built 1st, then added to the PRPP.

 

Both purine and pyrimidine synthesis have a molecule in common. What is that molecule?
PRPP
What molecule is used at the start of the purine synthesis pathway?
PRPP
What molecule is formed by the purine synthesis pathway?
IMP
What is the name of the enzyme that catalyzes the addition of the first component of the purine ring?
PRPP amidotransferase
What nitrogen-containing amino acid donates this first part of the ring?
glutamine
How many enzymatic steps are required to synthesize IMP from PRPP in humans?
10 steps
What is the cellular localization of all the enzymes that carry out these steps (many of the steps are carried out by enzymatic activities contained on a single polypeptide chain).
the cytosol
What carrier is utilized in two different steps to add to the ring?
N10 formyl tetrahydrofolate
What vitamin is N10 formyl THF derived from?
folate
What atom does N10 formyl THF donate? Hint- the class of enzymes known as transformylases catalyze these steps.
C atom
What are glial cells?
supporting neuronal cells that maintain the normal physiology of the nervous system
What are some of the biochemical functions of microglia, astrocytes, and oligodendocytes?

MICROGLIA

 

fight infections; release inflammatory substances that could damage neurons

 

ASTROCYTES

 

regulate molecules necessary for inter-neuron communication

 

clear glutamate in the synapse to stop the neurotransmission mediated by glutamate

 

release neural growth factors

 

take up monoamine transmitters like serotonin or dopamine

 

OLIGODENDROCYTES

 

provide fatty myelin sheaths that insulate axons in the CNS

 

multiple sclerosis results when axonal connections die off

 

Acetycholine is not metabolized by reuptake process. Explain which cells would catabolize acetylcholine after action potential?
astrocytes
Explain the differences between endogenous proteolysis (protein degradation) VS digestion related protein degradation.
What are normal blood creatinine values?
< 1.5 mg/dl
Describe the metabolism of glucogenic and ketogenic amino acids in relation to TCA cycle.

different amino acids (both ketogenic and glucogenic) synthesize different components of the TCA cycle.

 

For example, both aspartate and asparagine synthesize oxaloacetate

 

SEE PG. 71 IN DR. VENK’S LECTURE #1

Describe the lactate dehydrogenase catalyzed reaction.

(1) pyruvate→lactate (LDH M)

 

(2) lactate→pyruvate (LDH L)

 

Prolonged muscle activity results in pyruvate being converted into lactate by LDH M

 

lactate then moves from the muscle into blood and enters the liver

 

In the liver, LDH L converts lactate back into pyruvate

 

pyruvate is further metabolized to form glucose to gluconeogenesis

 

This entire process is known as Cori’s cycle.

When lactate accumulates in the blood it is a problem. Why?

accumulation of lactate in the blood leads to lactate acidosis

 

indicative of tissue hypoxia, hypoperfusion, and possible damage

Describe the catecholamine synthesis pathway.

(1) tyrosine→DOPA (tyrosine hydroxylase)

 

(2) DOPA→dopamine (DOPA decarboxylase)

 

(3) dopamine→norepinephrine (hydroxylase)

 

(4) norepinephrine→epinephrine

(methyl transferase)

 

Rate limiting step = tyrosine hydroxylase catalyzed reaction (step 1)

 

Step 1 requires molecular oxygen and tetrahydrobiopterin (THB)

 

Explain why vitamin B6 deficiency can cause brain related problem.

GABA is found in high concentrations in the brain where it serves as an inhibitory neurotransmitter.

 

glutamate→GABA (glutamate decarboxylase)

 

The enzyme requires the vitamin B6 derivate pyridoxal phosphate as a coenzyme so deficieny of vitamin B6 would impair activity of the decarboxylase and production of GABA.

Describe the biosynthesis of serotonin and melatonin.

Synthesis of Serotonin

 

1) Tryptophan→5-hydroxy tryptophan

(tryptophan hydroxylase)

 

2) 5-hydroxy tryptophan→serotonin

(decarboxylase)

 

Synthesis of Melatonin

 

1) serotonin→N-acetyl serotonin

(NAT)

 

2) N-acetyl serotonin→melatonin

(methyl transferase)

 

 

Describe the biosynthesis of histamine and its functions.

histidine→histamine

(histidine decarboxylase)

 

Functions: 1) mediate allergic and inflammatory reactions

 

2) powerful vasodilator

 

3) causes constriction of bronchioles in lungs

 

4) stimulates secretion of HCl in stomach

Describe the biosynthesis of creatine phosphate and its degradation.

Synthesis

 

1) arginine + glycine→guanido acetate + ornithine

(transamidase)

 

2) guanidino acetate→creatine

(SAM-dependent methylation)

 

3) creatine→creatine phosphate

(creatine kinase)

 

Degradation

 

1) phosphocreatine→creatinine

(non-enzymatic conversion)

 

creatinine is excreted through the urine

Which vitamin derivative is required as a cofactor for many of the decarboxylases?

pyridoxal phosphate

 

derived from vitamin B6

Describe the three nitric oxide synthases and the respective functions of the nitric oxides in different tissues.

Endothelium-derived NO

 

NO diffuses out of endothelial cells and into vascular smooth muscle cells.

 

Activates cytosolic guanylate cyclase→increases cGMP

 

Activation of cGMP dependent protein kinase→phosphorylation of smooth muscle contractile proteins and relaxation

 

Brain-derived NO

 

receptors for NO in neurons

 

stimulation of guanylate cyclase→increased cGMP synthesis

 

Proteins that are phosphorylated and activated not well understood

 

Macrophage-derived NO

 

due to infections by bacteria, NO synthesis stimulated

 

NO toxic to bacteria

Biochemical basis for Parkinson’s

degeneration of the substantia nigra leads to reduced dopamine production

 

treated by: administration of L-DOPA which is precursor of dopamine that can cross blood brain barrier

Biochemical basis for depression

Serotonin from synaptic cleft is taken up by presynaptic cells for catabolism

 

Treated with: Prozac which inhibits the serotonin reuptake process and enables prolonged serotonin presence in synaptic cleft

Biochemical basis of pheochromocytomas

tumors of chromaffin tissue that produce large amounts of catecholamines

 

leads to hypertension

Biochemical basis of Huntington’s
Now thought to be a polyglutamine trinucleotide repeat problem rather than a problem with low GABA levels
What is the definition of biopharmaceutical? What is the definition of recombinant DNA?

biopharmaceutical – drug produced in living cells

 

recombinant DNA – taking a given DNA sequence and putting it together in a different arrangment

Describe how one “recombines” DNA including cutting, hybridizing, and ligating it. What enzymes are utilized for cutting DNA?

cutting – use restriction enzymes

 

hybridizing – complementary base pairing of strands in test tube; GC and AT pairs (H bonds)

 

ligation – covalent bonds btwn base in each strand; ligases make covalent bonds; permanently put DNA strand back together; make sequence functional

 

 

What is a plasmid? Give an example of a gene found on a plasmid that is naturally occurring.

small DNA circle in bacteria that can be cut with restriction enzymes and recombinant DNA can be replicated by ligating the recombinant sequence into the bacterial chromosome and allowing the bacteria to produce many copies of the DNA sequence

 

gene on a plasmid that is naturally occurring – Bacillacis anthracis toxins in DNA

What about insulin causes difficulty in producing it be recombinant DNA means (2 things)?

not 1 polypeptide chain but 2 (A and B chains)

 

3 disulfide linkages and bacteria don’t have disulfide linkages

What are the advantages of producing insulin in bacteria?

bacteria produces more product than yeast

 

bacteria easier to set up and maintain than yeast

What are the advantages of producing insulin in yeast?

insulin is already folded and disulfide bonds are linked correctly

 

more like mammalian cells

What do we mean by calling Enbrel (etanercept) a fusion protein? Why does it involve fusing 2 proteins, i.e. what properties are due to each part? What do we mean by calling Enbrel a decoy?

block TNF alpha signal transduction pathway

 

fusion protein b/c it binds to the TNF alpha receptor with a receptor domain and a immunogammaglobulin to pull TNF alpha away from cell and float in circulation (decoy)

 

 

It might be quicker to go from the idea of inhibiting TNF to a drug by making the drug as a biopharmaceutical than it would be to go from the idea of making a conventional drug. Why? What might be an advantage of a small molecule conventional drug?

it can take a lot of time in the lab to grow small molecule drugs and see what blocks signal transduction pathway

 

This small molecule conventional drug might have a more specific effect???

What are the advantages of producing Enbrel in Chinese Hamster Ovary cells?

folding is more like a human than yeast

 

additions of sugars and other modifications are required for some proteins that can be done by the hamster ovary cells

Are drugs on the market more likely to be polyclonal antibodies, or are they more likely to be monoclonal antibodies, or are they more likely to be recombinant DNA molecules based on monoclonal antibody sequence information? Why? What is meant be humanized antibody?

recombinant DNA molecule based on moloclonal antibody sequence information b/c eliminate human immune response to mouse antibody

 

humanized antibody – monoclonal antibody exposed to  humans and those humans who don’t have an immune response have their DNA substituted for the mouse DNA to prevent an immune reaction

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