Biochemistry OneNote Red terms

Electrophoresis:

negatively charged side:

positively charged side:

Electrophoresis Native-PAGE: Analyzes protein in native states *diff proteins may have the same migration b/c diff mass-to-charge, mass-to-size ratios 


SDS-PAGE Uses sodium dodecyl sulfate, a detergent that denatures covalent interactions Separates proteins by _________ only (b/c SDS binds to proteins and creates large chains w/large negative charge, overcoming the protein's charge)

Anion exchange chromatography uses a + charged resin and therefore anions would be attracted to it and cations (+) would elute quicker.


cation exchange chromatography uses a - charged resin and therefore cations would be attracted and anions (-) would elute quicker.

anion exchange chromotography, _____ ions will be retained longer and move slower and ____ ions with move quicker and elute first

Column Chromatography = "like dissolves like" Column is filled with silica (polar beads) or alumina beads as adsorbent Gravity moves solvent and compounds down column Size & Polarity determine migration: Less polar compounds move ______, polar compounds are attracted to polar beads Useful for macromolecules (other than protein) like DNA

Size-Exclusion Chromatography DOESN'T DEPEND ON CHARGE AFFINITY FOR STATIONARY V. MOBILE PHASE! Depends on size


______ compounds move faster

______ compounds move slower

Affinity Chromatography:

Customizing columns to bind a protein of interest - i.e. coating beats w/receptor for a target protein; immunoprecipitation; enzyme substrate analogues Protein is retained, then eluted w/ (antibody or free receptor) solute to free protein from column

4 chiral carbon = how many stereoisomers?

While an _______ is a stereoisomer that differs in configuration at any single stereogenic center,


an ______ is a cyclic saccharide and differs in configuration, specifically at the hemiacetal/acetal carbon, also called the anomeric carbon.

stability:

Chair --> boat -- > planar


energy:

planar --> boat --> chair

Deoxyribose --> pentose in DNA, has an H instead of the OH on _____ carbon

type of chromatin:

_______ = dark under scope; compact; transcriptionally inactive. 

_______ = light; less compact; genetically active in transcription during S phase.

Removal of RNA primers (eukaroyte or prokaryote?)

DNA Polymerase I (5' > 3' endonuclease):

RNase H (5'>3' exonuclease):

Prokaryotic cells or Eukaryotic cells ?

Origin of replication: 

One per chromosome: 

Multiple per chromosome:

DNA replication:

*______ is necessary (creating an RNA primer) because DNA polymerase isn't able to start elongation w/o primer **Unwinding of DNA by ______ creates supercoils.

_______ relieve torsional stress by introducing negative supercoils (via single- or double-stranded nicks and resealing)

Direction it's read:

DNA replication: ______
Transcription: ______
Translation: ______


Direction it's synthesized:

DNA replication: _____
Transcription: ______
Translation: ______

Transcription Enzymes involved: Helicase & topoisomerase - to unwind and de-supercoil the ds-DNA 

RNA Pol I = in nucleolus, makes rRNA 

RNA Pol II = in nucleus, make hnRNA (heterogenous nuclear RNA; pre-mRNA) and some snRNA 

RNA Pol III = in nucleus, makes tRNA and some rRNA spliceosome (snRNA and snRNPs, small nuclear ribonucleoproteins)

Recall that euchromatin is accessible and genes are active; heterochromatin is inaccessible 

Histone acetylation - acetylates lysine residues on histone tails, which decreases + charge and weakens histone association w/chromosomes (euchromatin)

Histone deacetylases -  involved in gene silencing (heterochromatin)

DNA methylation - add methyl groups to cytosine and adenine nts  Linked w/gene silencing. Heterochromatin is more methylated.

Translation:

Initiation: 

In prokaryotes, the small ribosome 30s subunit binds to the ______ in 5' UTR of mRNA. 

In eukaryotes, small 40s subunit binds to the ______. 

Initiator tRNA (Met for eukaryotes, fMet for prokaryotes) binds to AUG start codon at ____ site.

Translation:



Termination When stop codon (UAA, UGA, UAG) enter ______, a release factor binds, causing water to be added to peptide chain. Peptidyl transferase and termination factors hydrolyze chain from final tRNA.

Three binding sites on ribosome = A.P.E.

 A site = aminoacyl site Holds incoming charged aa-tRNA complex of next aa, as determined by the mRNA codon at that site. 

P site = peptidyl site Holds the tRNA attached to the growing peptide chain (before transferred to new aa) Where Met is added first at start codon Peptidyl transferase on ribosome catalyzes the transfer of the nascent polypeptide from P to A site with the addition of a peptide bond 

E site = exit site Uncharged/inactivated tRNA passes before exiting ribosome and unbinding from mRNA

Triglyceride formation is by nucleophilic attack by the glycerol OH group at the FA carboxylic acid. This is a dehydration/dehydration rxn where H2O is removed

Phosphoglycerides= polar head group (thru phosphodiester links) + glycerol + FA's (thru ester links) Also called glycerophospholipid, phospholipid Glycerol backbone whose 2 OH's form ester linkages with Fatty Acids, and the third is a phosphodiester linkage with a polar head group.

*________ = base-promoted ester hydrolysis of triacylglycerols (triglycerides) 

Lye = NaOH 

Ester hydrolysis is nucleophilic substitution (SN1) and gives cleavage of the TAG and formation of sodium salt of the fatty acid and glycerol

_______ - aligning pores of connexin subunits; permits water and some solutes to pass

_________ - prevents leaking of solutes in between cells Not used for intercellular transport; prevents paracellular transport (in the intercellular space) Found in epithelial cells and act as physical link for a layer of tissue Can limit permeability enough to create a transepithelial voltage difference i.e. in renal tubule lining - restricts solutes and water

_________ - bind adjacent cells by anchoring cytoskeletons Thru interactions between transmembrane proteins associated with filaments inside adjacent cells Found at interface between 2 layers of epithelial tissue

what type of solution is the cell in?





[solutes] inside cell is higher than outside. Water will rush in, lysing cell.

what type of solution is the cell in?



[solutes] outside cell is higher than inside. Water will move out. (shrivel)

what type of transport?



_______ transport --> delta G is negative diffusion of a solute or water depends ONLY on the electrochemical gradient of the compound of interest, and membrane permeability. Generally increase as T increases (bc depends mainly on increase in entropy S) 



_______ transport --> delta G is positive May or may not be affected by T increase, depending on enthalpy (delta H)  Movement of solute against its conc gradient

________ Substrates move down their concentration (chemical) gradient as per the potential energy

______ Simple diffusion of water - moving from low to high solute conc (i.e. high to low water conc), trying to bring solute concentrations to equimolarity

_________ - down concentration gradient When large, polar, or charged molecules pass thru membrane with help from a transporter or channel Via channels (can be open or closed; analogous to tunnel) or carriers (slower kinetics; analogous to revolving door)

Movement of solute against its conc gradient: 

Primary active transport - uses ATP Coupling with ATP hydrolysis to move particle up its gradient - uses transmembrane ATPase 

Secondary active transport - uses an electrochemical gradient to power transport. Coupling a particle moving up its conc gradient to one moving down its gradient. No direct coupling with ATP hydrolysis.

Symporter v. antiporter

Overall Rxns:
 Glycolysis (ANAEROBIC) Glucose + 2 ADP + 2Pi + 2 NAD+ --> 2 Pyruvate + 2 ATP + 2 NADH +2H2O 

Lactic acid fermentation (pyruvate reduction) by Lactate Dehydrogenase: (ANAEROBIC) Pyruvate + NADH + H+ <--> Lactate + NAD+ 

Pyruvate Dehydrogenase (pyruvate oxidation for downstream Krebs and ETC):  

Pyruvate + CoA + NAD+ --> acetyl-CoA + CO2 + NADH + H+ (irreversible) 

Gluconeogenesis: 
2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 6 H2O --> Glucose + 4 ADP + 2 GDP + 6 Pi + 2NAD+ + 2H+ 

Pentose Phosphate: Glucose-6-Phosphate + 2 NADP+ --> Ribose-5-Phosphate + 2 NADPH 

 

*Pyruvate Dehydrogenase (PDH complex),  pyruvate carboxylase (gluconeogenesis),  and enzymes of Citric Acid Cycle (TCA cycle) are found in mitochondrial matrix.

Which GLUT transporter?

_______ - transporter in hepatocytes and pancreatic cells 

Glucose in absorptive state will carry glucose to liver via hepatic portal vein  

Km is high of this GLUT; thus it has low affinity for glucose (conc of substrate when enzyme is active with half of its Vmax. The lower the Km, the higher the affinity for the substrate) Liver will pick up glucose in proportion to its conc in blood (first-order kinetics) - i.e. preferentially after a meal when [glucose] is high this GLUT (and glucokinase) serves as glucose sensor for insulin release in the beta-islet cells of pancreas

What GLUT transporter is this?

_______ - transporter in adipose and muscle tissue; respond to glucose concentration in peripheral blood Low Km = high affinity (i.e. transporters will be saturated when [glucose] is just a bit higher than normal) 

Zero-order kinetics - transporters will transmit a constant rate of glucose influx, even during absorptive high [glucose] phase, because saturated Cells respond to insulin - increase or decrease # of transporters on cell surface 

Insulin increases GLUT expression in cells: 

Muscles: store excess glucose as glycogen (as does liver) 

Adipose: uses glucose to form DHAP (dihydroxyacetone phosphate), to be converted to alpha-glycerol phosphate, to store FA's as triacylglycerols (triglycerides) 

 Pancreatic amylase – breaks down polysach to disaccharides; brush border peptidases break down disacch to monosaccharides 

SGLT symporter moves glucose and Na+ into intestinal epithelial cells; GLUT moves fructose transports fructose into epithelial cells and then into blood (interstitial fluid)

Rate limiting step of glycolysis?

Fermentation: reversible 

Lactate dehydrogenase:

  reduces pyruvate to lactate, and oxidizes NADH --> NAD+, which replenishes the NAD+ for the _______ step in glycolysis

PENTOSE PHOSPHATE PATHWAY 


*unique b/c no ______ is produced or used 

2 functions: Produce 2 NADPH (per glucose-6-phosphate) Provide ribose-5-phosphate for ________ 





Glucose-6-phosphate dehydrogenase (G6PD): oxidizes glucose-6-phosphate to 6-phosphoglucanate, and reduces NADP+ to NADPH.  *rate-limiting step and Is stimulated by ______ and NADP+; inhibited by NADPH.

Gluconeogenesis: (anabolic / liver/ kidney)

Inhibited by: _____

Stimulated by: ___, ___, ___, ____




Acetyl-coA shifts metabolism of pyruvate: High acetyl-coA inhibits Pyruvate Dehydrogenase (step that makes acetyl-CoA before citric acid cycle), and stimulates Pyruvate Carboxylase (of gluconeogenesis) Net effect is shift from burning pyruvate for citric acid cycle, to gluconeogenesis Mainly from beta-oxidation (NOT glycolysis) b/c body is in post-absorptive, low-blood-glucose state

________ *Rate-limiting step of gluconeogenesis

Gluconeogenesis needs high levels of Acetyl-coA to occur and for it to be from ______ and not glycolysis because if it occurred from glycolysis it would burn the glucose it creates. This will inhibit pyruvate dehydrogenase so that pyruvate can be converted to oxaloacetate via pyruvate carboxylase and then oxaloacetate can be converted to PEP via PEP caboxykinase

Gluconeogenesis needs high levels of acetyl-CoA because that will inhibit ______ and switch to ______ so the source of Acetyl-Coa can't be from glycolysis because that would burn new glucose made via gluconeogenesis and source of acetyl-Coa must come from ________

Also, where does liver get energy during gluconeogenesis? (FASTING STATE)  = from FA's! glucose produced by hepatic gluconeogenesis is not a source of energy for liver (would defeat the point) Gluconeogenesis requires ATP, which is provided by FA beta-oxidation Thus hepatic gluconeogenesis is dependent on beta-oxidation in liver: when blood sugar is low, adipose break down TAG's to glycerol (which can then be used in gluconeogenesis) and free FA's Acetyl-coA from FA's can be converted to ketones (the transportable acetyl-CoA) as alternative fuel for other tissues during starvation states

Pyruvate = 2C 

Acetyl-CoA = 2C 

Oxaloacetate = 4C 

Citrate = 6C 

Succinate= 4C F

umarate= 4C 

Malate= 4C

Ways to get Acetyl-coA:

_____ and ____ can be converted to pyruvate which can lead to acetyl-coA via pyruvate dehydrogenase


_____, _____, _____ can be converted to acetyl-coA directly

Coenzyme Q is a ubiquinone 

 *Quinones are _____ due to a conjugated ring system (but not necessarily aromatic)

Fatty acid synthesis occurs in the ____ and Fatty acid oxidation occurs in the ____ and they're mutually exclusive.

Rate-limiting step of FA synthesis = acetyl-CoA Carboxylase (activates substrate so that synthesis coupled to favorable energetics) 

Rate-limiting of FA oxidation = Carnitine Acetyltransferase (only FA bonded to carnitine can go to matrix, the site of oxidation, so this controls the amnt entering oxidation cycle)

*rough versus smooth ER: Rough ER (RER) is involved in some protein production, protein folding, quality control and dispatch. It is called ‘rough’ because it is studded with ribosomes 


Smooth ER (SER) is associated with the production and metabolism of fats and steroid hormones. It is ‘smooth’ because it is not studded with ribosomes and is associated with smooth slippery fats.

what type of fat, cis or trans?

double bonds / unsaturated / liquid:

single bonds/ saturated/ solid:

contribute to arterial disease:

Fatty acid synthesis:

Acetyl coA --> (enzyme) ---> (intermediate) -- > (enzyme) -- > (final product)

Fatty acid synthesis questions:

Acetyl coA --> (acetyl-coA carboxylase) ---> (Malonyl coA) -- > (fatty acid synthase) -- > (Fatty acid Palmitate)

What's the rate limiting enzyme for fatty acid synthesis? 

What hormone stimulates both enzymes in fatty acid synthesis?

The NADPH that is used in the fatty acid synthase enzyme, comes from ___ and ___

Acetyl-coA can't enter the cytoplasm straight from the mitochondria so _____ enters the cytoplasm through the ____ and is split into oxaloacetate and acetyl CoA in the cytoplasm via _______


NADPH is required because need to reduce acetyl groups that are added to FA chain and comes from citrate shuttle

Before the actual beta oxidation:

1- Fatty acid + coenzyme A ---- (acyl-coA synthase /ATP) ----> Fatty acyl-coA

2- 

FA-entry into Mitochondria Short chain FA's freely diffuse into mitochondrial matrix 

Long-chain FA's are transported by carnitine shuttle into mitochondrial matrix 

Activated FA in Fatty acyl-CoA is transferred to carnitine (transesterification), which is transported thru the inner mitochondrial membrane by carnitine-acyltransferase I (RATE-LIMITING for beta-oxidation!!) Inhibited by malonyl-CoA (activated acetyl-CoA of FA biosynthesis)

Ketogenic amino acids - 

glucogenic amino acids-

both keto and glucogenic amino acids -

When Q=K, the system is at equilibrium and there is no shift to either the left or the right.  

When Q<K, there are more reactants than products. As a result, some of the reactants will become products, causing the reaction to shift to the right. 

When Q>K, there are more products than reactants. To decrease the amount of products, the reaction will shift to the left and produce more reactants.

______ is used to fuel energetically unfavorable rxns, or activate other molecules

glucagon, epinephrine, cortisol, growth hormone


glycogenolysis: 



gluconeogenesis: 


lipolysis: 


inhibit glucose uptake by muscle and adipose cells:

U = Q - W

Adiabatic:

Isothermal:

Isochoric/volumetric: